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Marine bacteria: wall composition and osmotic fragility

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Marine bacteria: wall composition and osmotic fragility
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Marine bacteria: wall composition and osmotic fragility
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Sud, Inder Jit,
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Gainesville FL
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University of Florida
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Amino acids ( jstor )
Bacteria ( jstor )
Cell walls ( jstor )
Cells ( jstor )
Enzymes ( jstor )
Hexosamines ( jstor )
Sugars ( jstor )
Teichoic acids ( jstor )
Washing ( jstor )
Water distillation ( jstor )
City of Gainesville ( local )

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University of Florida
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University of Florida
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Copyright Inder Jit Sud. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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MARINE BACTERIA: WALL COMPOSITION

AND OSMOTIC FRAGILITY












By

INDER JIT SUD


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF
THE UNIVERSITY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY










UNIVERSITY OF FLORIDA
April, 1963

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Plate 2 Electron ricrographs of 11.3, 29 and M.B. 98 showing
flagellation.

Fig. 4 M.B. 29 (x 17,000). Fig- 5 M.B. 98 (x 8,000).

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ACKNONTLDGMENT


The author wishes to express his appreciation to
Drv Max E. Tyler and Dr, Darrel B. Pratt for the guidance during this study, This w6rk was supported in part by a NSF research grant (no 021471) and by a Public Health Service training grant (no 2G-869) from the General MIedical Science Division, Public Health Service*

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TABLE OF CONTENTS


Page
LIST OF TABLES. .iv LIST OF PLATES ANDFIGURES.. v INTRODUCTION***#. 0 4.0. . .

LITERATURE REI .4

MATERIALS AID METHODS#. . .21 EXPERIMENTAL RESULTS... ov*9vo35 DISCUSSION, .. o.. a . . .a. .71

SUN YtAR . a.aa.a. a. 80


iii

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LIST OF TABLES


TABLE Page
1. CARBOHYDRATE METABOLISM OF THE M-ARINE BACTERIA 41

2. PHYSIOLOGICAL CHARACTERISTICS OF THE MARINE BACTERIA .e.,. 43
3. SENSITIVITY OF MARINE BACTERIA TO ANTIBIOTICS 44
4. LYTIC PROPERTIES OF MARINE BACTERIA 45

5. COMPOSITION OF CELL WALLS OF MARINE BACTERIA AND P. AERUGINOSA .,. 64

6. SUBSTANCES IDENTIFIED IN CELL WALL HYDROLYSATES OF MARINE BACTERIA AND P, AERUGINOSA .,.,. 66

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LIST OF PLATES ANM FIGURES


Page
PLATE I. Phase contrast micrographs of B. 29,
M. B. 65, and M.1B. 37
PLATE 2. Electron micrographs of M, B. 29 and
M. B. 98 showing flagellation .,.,. 40
PLATE 3. Electron micrographs of M. B. 29 after
lysing and washing with various procedures .,. 49
PLATE 4. Electron micrographs of cells of M., B. 29
after lysis in water and treatment with
various enzymes ".52

PLATE 5. Electron micrographs of cell walls of M. B. 29
prepared by mechanical disintegration and
washing with various procedures .,., 56

PLATE 6. Electron micrographs of cell walls of M. B. 29
obtained by mechanical disintegration and.
action of various enzymes 58

PLATE 7. Electron micrographs of cell walls of 1. B. 29,
M. B. 65, 14, B, 98, and P. aeruinosa .,.o. 60

FIGURE 23. Disaggregation of cell walls of M. B. 29
and P, aerudinosa by sodium dodecyl sulfate .,. 62
PLATE 8, Chromatograms of ninhydrin positive substances
in cell wall hydroiysates of marine bacteria
and P. aeruginosa o 70

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INTRODUCTION


A major function of the bacterial cell wall is to maintain cellular integrity in adverse external environments. Thus, most

bacteria can survive in media of low osmotic pressure, an ability dependent upon the presence of a cell wall rigid enough to prevent

osmotic swelling and bursting of the bacterial protoplasm. The cell walls of Gram-positive organisms can withstand high internal osmotic pressures, e.a. 20 to 25 atmospheres in Staphylococcus aureus (Mitchell and Mobyle, 1956). In contrast, this pressure in Gram-negative organisms is low, between 2 and 3 atmospheres as in Escherichia coli (Mitchell and Ployle, 1956), but it has been shown that these cells can be groim in salt-rich media and remain intact when placed in distilled water (Doudoroff, 1940).

This protective function of the cell wall is apparently absent in the marine bacteria which have been isolated and studied. Their

cells lyse when put in distilled water, the cell wall rupturing allowing the cellular contents to escape (Johnson et al., 1943). The occurrence of lysis can be shown by a number of determinations such as decrease in turbidity and viability and release of protein and nucleic acids into the suspending medium. A marine bacterium has been estimated to possess an internal osmotic pressure of about 20 atmospheres (Johnson and Iarvey, 1937; Johnson, Zworykin, and Warren, 1943) and the lysis

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was the result of a critical difference in the osmotic pressure inside and outside the cells.

The seat of osmotic fragility has been shown (Boring, 1961) to be the weakness of the cell wall; whole cells and penicillin-induced spheroplasts of a marine bacterium were found to be nearly alike ii osmotic fragility. It was suggested that the penicillin-sensitive component of the cell wall, while conferring a characteristic shape to the organism, was unable to protect it from osmotic lysis. In non-marine bacteria, it is this component which is believed to give the cell wall its rigidity and mechanical strength.

The cause of weakness in the cell walls of marine bacteria is not known but suggestions have been made that it is due to thinness, or to difference from ordinary cell walls in chemical composition or physical structure (Salton, 1956). A low hexosamine content in the cell wall of a marine bacterium has been implicated in osmotic fragility (Brown, 1960). This compound has been shown to be a component of the mucopeptide layer of the cell walls of E. coli (Weidel, Frank, and Martin, 1960), a layer responsible for the rigidity of the cell walls.

Some strains have been found to be less fragile osmotically than others (Tyler, Bielling, and Pratt, 1960; MacLeod and Matula, 1962) indicating the existence of a spectrum of osmotic fragility in these bacteria. If the weakness of the cell walls were due to a low hexosamine content, then it might be possible to correlate the wall hexosamine content with the degree of osmotic fragility exhibited by the marine organism.

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This investigation .ras undertaken to establish and compare the cell wall compositions of three marine bacteria selected on the basis of their lytic behaviour in various test media, the extent of lysis being taken as an indication of their degree of osmotic fragility. Of the three organisms, M.B. 65 was the least and H.B. 98 the most susceptible to lysis while M.B. 29 was intermediate. The cell walls of a non-marine pseudomonad, Pseudomonas aeruginosa, were also isolated and analysed as earlier studies had indicated that the selected marine bacteria might be pseudomonads.

After considerable difficulty, a method was devised which gave clean cell walls of the marine bacteria. The fragile nature of the cell walls, especially those of M.B. 98 and M.B. 29, was evident from the electron micrographs. The analytical results indicated that the cell walls were predominantly lipoprotein in nature. They wmre completely soluble in phenol and were extensively disaggregated by sodium-dodecyl-sulfate. bo sugars, except glucosamine, were detected; the hexosamine values were low as compared to those of P. aerurinosa and other Gram-negative bacteria.

The data suggested that the hexosaine content of the cell wall influenced the degree of osmotic fragility. From the electron micrographs, it was considered possible that thinness of the wall

ras partly responsible for the w eak nature of the cell walls of the marine bacteria.

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LITERATURE REVID4


The literature, pertinent to the subject of this thesis, has been reviewed under two sections; (a) bacterial cell walls and (b) osmolysis of marine bacteria.



Bacterial Cell Walls


The external structures of bacterial cells, responsible for the rigidity and integrity of cells, are generally referred to as the "cell walls." In Gram-positive bacteria, the existence of a wall as a separate and distinct entity has been demonstrated in a variety of ways. This has not been possible in Gram-negative bacteria; whether they possess a wall distinct from the cytoplasmic membrane is uncertain.

Apart from their obvious mechanical function, very little is known about their biochemical activities. It is generally agreed that the isolated cell walls of Gram-positive bacteria are devoid of any enzymatic activity. The situation with the cell walls of Gram-negative bacteria is less certain; several reports on the enzymatic activities associated with the "envelope" preparations of such bacteria have appeared (14arr, 1960; Hunt, Rodgers, and Hughes, 1959; Salton, 1961a).

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The removal of the cell wall, partially or completely by the action of agents such as lysozyme and inhibition of its synthesis by the action of penicillin on whole cells, results in the loss of the protective function and spherical bodies are produced. These are osmotically fragile in contrast to whole cells and have also lost the characteristic shape if they happened to be derived from rod-shaped cells, The bodies from Gram-positive bacteria, produced by the action of lytic enzymes, have been shown to be free from wall components (Freimer, Krause, and McCarty, 1959; Vennes and Gerhardt, 1959) and are called protoplasts. Gramnegative bacteria, on the other hand, give rise to bodies which retain some of the wall constituents (Shafa and Salton, 1958; Salton, 1958) and their membranes react positively with cell wall antibodies (Ifolme et al., 1960). The term spheroplast was suggested to describe such bodies.

Both protoplasts and spheroplasts are reasonably stable in media of appropriate osmotic pressure. They resemble whole cells in permeability properties and in their ability to carry out biosynthetic activities such as protein synthesis, indicating that the cell wall is not essential for such activities as long as protection from osmotic effects is provided. The presence of a cell wall, however, seems to be necessary for multiplication since these bodies have been found to be unable to reproduce. Cell walls may also act as reservoirs for certain metabolites; Gerhardt (1959) and Butler et al. (1958) obtained evidence suggesting that certain amino acids may be stored in the walls.

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The rigidity and the mechanical strength of the cell wall is believed to be due to its mucopeptide component. Salton (1958) demonstrated this with the isolated cell walls of Rhodospirillum rubrum; treatment of the walls with lysozyme resulted in their becoming spherical in shape and the mucopeptide components were released. The formation of spherical bodies, by the action of lysozyme or penicillin on whole cells, is due to the disorganisation of the mucopeptide of the wall; lysozyme acts by degrading the mucopeptide enzymatically whereas penicillin interferes with its synthesis. In both the cases, the rigid part of the wall is

weakened and the wall is unable to perform its mechanical function.

Attempts at isolating the cell walls were first made in 1887 when Vincenzi obtained what he believed to be the walls of Bacillus subtilis by extraction with 0.5 per cent NaOH. It is now known that treatment with alkali and acids leads to the degradation of part of the wall structure (Abrams, 1958; Baddiley et al., 1958). Cell walls can be obtained by mechanical disintegration of cells followed by differential centrifugation (Salton and Home, 1951a, b). The use of mechanical methods for disruption of bacterial cells is by no means of recent origin; as early as 1901, NacFadyen and Rowland had used agitation with fine sand for disrupting the typhoid bacillus. King and Alexander (1948) refined this method by using small glass beads. Dawson (1949) showed that the cells of Staphylococcus aureus,

on vigorous shaking with glass beads, gave a preparation containing

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cell walls. He used the Mickel tissue disintegrator (Mickel, 194S) for providing rapid and vigorous shaking of the cell suspension.

Salton (1956) and Cunwins and Harris (1956a) have clearly shown that the residues from mechanically disrupted cells consist primarily of cell walls and this method is now widely used for obtaining bacterial cell walls. It is preferred since it avoids drastic chemical alteration of the walls. The method, as described by Salton and Home (1951b), involves shaking of heavy cell suspensions with an equal volume of glass beads (0-15-0.20 mm in diameter) in a Nickel tissue disintegrator. The cell walls are separated from the suspension by differential centrifugation. They are then either washed repeatedly with appropriate solutions or treated with enzymes

for the removal of cytoplasmic material from them.

Various solutions have been used for the cleaning of cell walls. ashing with 1 M NaCl or with phosphate buffer was found to be more effective than washing with water alone (Salton, 1953). Holdsworth (1952) used washing with sodium acetate solution followed by extraction Aith 90 per cent phenol for cleaning the cell walls of Cornebacterium diphtheriae. The cell walls of Gram-negative bacteria,

however, have been found to be less amenable to cleaning by washing.

In their studies of bacterial cell walls, Cummins and Harris

(1956b; 1958) employed enzymes for removing cytoplasmic material from cell walls. Since then, the use of proteolytic enzymes, ribonuclease, and deoxyribonuclease has become wzidespread. It is now knoimn that

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bacterial cell walls are not altered in physical appearance by these enzymes but it is possible that surface components are removed. Salton (1953) reported that trypsin removed the M protein from streptococcal cell walls and that the amino acid composition was simpler after trypsin treatment, sulfur-containing and aromatic amino acids being eliminated. The removal of surface protein antigen by the action of pepsin on Corynebacteriim (Cummins, 1954) and solubilisation of about 40 per cent of isolated cell walls of Streptococcus by the action of trypsin (Barkulis and Jones, 1957) have been reported Similarly Knox and Bandesen (1962) found that trypsin released a number of low molecular-weight peptides from isolated cell walls of Lactobacillus casei. Salton has recommended that the use of enzymes, especially crude enzymes which may contain wall degrading enzymes as well as other insoluble protein material, should be carefully controlled.

Other methods for obtaining bacterial cell walls are also available. Those involving disintegration of cells with sonic and ultrasonic vibrations, decompression rupture, and pressure cell disintegration suffer from the disadvantage of fragmentation and solubilisation of the wall (Slade and Vetter, 1956; Marr and Cota-Robles, 195). Foster, Cowan, and 1m!aag (1962) have recently described a device for rupturing of bacteria, under controlled conditions, by explosive decompression in a closed system. Autolysis and osmotic lysis of whole cells also yield cell walls; Weidel (1951) used toluene for autolysing the cells of E. coli and a lytic principle, associated with the cultures of Bacillus cereus, was found to digest cell contents of a number

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of Bacillus spp. (Norris, 1957) giving clean cell walls. These methods

have not been widely used because of the risk of degrading the wall enzymically.

The most widely used criterion for the purity of cell wall preparations is the absence of cytoplasmic material as determined with the electron microscope. Although it is not a very satisfactory criterion, wall preparations free from nucleic acids, electron-dense cytoplasmic material, and intracellular pigments can be obtained by

careful control of the procedure employed. In certain cases, the purity of a wall preparation can be determined by using any special property of the cell walls under examination. Thus, with Micrococcus lysodeikticus, Sarcina lutea, or Bacillus megaterium, the purity can be determined by dissolving the walls with lysozyme and weighing the lysozymeinsoluble residue.

Isolated bacterial cell walls have been repeatedly examined by electron microscopy. They generally retain the shape and outline of the organism from which they had been derived The walls of many Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus

faecalis, have a homogeneous appearance, although the walls of Bacillus megaterium give a vague impression of being fibrous (Salton and Williams, 1954). In the walls of some bacteria, such as Rhodospirillum rubrum and Halobacterium halobium, a spherical macromolecular type of fine structure has been observed (Salton and Williams, 1954; Houwink, 1956). In some bacteria, such as E. coli, the isolated walls appear

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homogeneous by the usual method of examination in the electron microscope, but thin sections of cells have clearly established the multilayered nature of the wall. The walls of E. coli appear to have three layers visible in the electron microscope (Kellenberger and Ryter, 1958). The existence of these layers has been confirmed by their separation by chemical methods; Weidel et al. (1960) were able to separate an outer lipoprotein layer soluble in phenol, an inner insoluble and rigid layer containing the mucopeptide constituents, and a middle lipopolysaccharide layer.

The thickness of bacterial walls has been estimated from

measurements on thin sections of isolated walls or cells and on the shadows cast by the walls during electron microscopy. It varies from
0
100 to 200 A, the walls of Gram-positive bacteria being thicker than those of Gram-negative organisms (Birch-Andersen and 14aaloe, 1953; Kellenberger and Ryter, 1958). The cell wall accounts for about 20 per cent of the dry weight of the cell (Mitchell and Moyle, 1951; Cummins, 1956; Salton, 1956), but this value may vary depending upon the phase of growth or cultural conditions as in Streptococcus faecalis (Shockman et al., 1958; Toennies and Shockman, 1959).

Chromatography of cell wall digests has proved to be an invaluable tool in studies on the chemical composition of bacterial cell walls. Such studies have revealed a number of unusual components in the walls and the list is steadily growing. Most of the information available concerns the Gram-positive bacteria because of the relative

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simplicity of their wall composition as well as structure and the ease with which they can be prepared. The cell walls of Gramnegative bacteria are more complex in nature, both chemically and structurally. Some excellent reviews on this subject have appeared within the last few years, the more recent being those of Work (1961) and Salton (1961a, b, 1962).

Bacterial cell walls have been found to contain a variety of chemical components: amino acids, lipids, carbohydrates, and phosphorus, but no nucleic acids, purines, and pyrimidines. A more complete picture of the chemistry of bacterial walls became available with the discovery of diaminopimelic acid (DAP) by Work (1951) and its detection in the cell walls of various bacteria, the isolation of muramic acid by Strange and Dark (1956) and its presence in the walls of all bacterial species so far examined (Salton, 1957; Cummins and Harris, 1956a; Work, 1957), the detection of D-amino acids (Salton, 1957; Snell, Radin, and Ikawa, 1955; Ikawa and Snell, 1956; Park, 1958), and the discovery of teichoic acids (Baddiley et al., 1958, Abrams, 1958) and teichuronic acids (Janczura et al., 1960, 1961).
The cell walls of Gram-positive bacteria differ noticeably

from those of Gram-negative bacteria; the former contain a limited variety of amino acids, a small amount of lipid material, and a high amino sugar content, whereas the latter contain proteins with the usual variety of amino acids, a high amount of lipid, and a

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small content of amino sugar. The cell walls of a large number of Gram-positive bacteria were examined by Cummins and Harris (1956a, b; 1958). The main components, invariably found, were glucosamine, muramic acid, glutamic acid, alanine, lysine, and diaminopimelic acid; in some cases there were also up to five sugars, one or two other amino acids, or galactosamine. A recurring type of "basal

unit" was soon recognised (Work, 1957) and the term "mucopeptide" was proposed by Mandelstam and Rogers (1959) to describe this unit. It is a complex of amino acids and amino sugars and is now recognised as the structural "backbone" common to the cell walls of Grampositive bacteria.

In addition to these components, some other polymeric substances have been isolated from walls and partially or fully characterised. These include oligosaccharides, polysaccharides, teichoic acids, and teichuronic acid. These polymers are less widely distributed and teichuronic acid has been reported in walls of Bacillus subtilis (Janczura et al., 1960, 1961) only. The chemical structure of teichoic and teichuronic acids has been established. The name teichoic acid refers to the polymers of glycerophosphate and ribitol phosphate; the former type of polymer was first detected in the walls of Staphylococcus aureus (Mitchell and Moyle, 1951) and later on was found in the cell walls of other bacteria. Ribitol phosphate polymers were detected in the cell walls of Bacillus subtilis and Lactobacillus arabinosus (Baddiley et al., 1958). The telchoic acids

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of B. subtilis (Armstrong et al., 1961) and S. aureus 11 (Baddiley,

Buchanan, Martin, and Rajbhandhary, 1962) contain ribitol units joined by phosphodiester linkages and most of the ribitol units carry ester linked D- alanine (Baddiley, Buchanan, Rajbhandhary, and Sanderson, 1961) and glucosyl residues. Teichuronic acid, isolated from the walls of B, subtilis, is composed entirely of Nacetyl-galactosamine and glucuronic acid (Janczura et al., 1960, 1961).

Diaminopimelic acid (DAP) and mramic acid are two important constituents of bacterial cell walls which are generally absent in other types of organisms (Rhuland, 1960). DAP is present in most bacteria rith the exception of some Gram-positive cocci and lactobacilli, (Work, 1951; Hoare and Work, 1957). Muramic acid, first isolated from a product obtained from the exudates of germinating spores of Bacillus meaterium (Strange and Dark, 1956), is glucosamine carrying ether-linked lactic acid at the 3-position (Strange and Kent, 1959) and probably originates from glucosamine (Zillikin, 1959, Richmond and Perkins, 1960). The key role of this amino acid in the structure of the bacterial wall mucopeptides has been recognised and a broad outline of the mucopeptide structure has been established from the studies of products isolated from walls and mucopeptides after digestion with lysozyme and streptomyces amidase (Salton, 1956, 1957 Ghuysen and Salton, 1960; Chuysen, 1961; Primosigh et A)16, 1961).

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There is little doubt now that the mucopeptide forms the

rigid backbone component composed of covalently bonded amino acids and amino sugars. The relation of other wall compounds to the mucopeptide component is less certain although it seems likely that they are attached to the mucopeptide by weak linkages. Evidence for this is provided by the extractibility of teichoic acids (Archibald etal., 1961) and teichuronic acid (Janczura et al., 1961) with trichloroacetic acid in cold and the removal of oligosaccharide and polysaccharide residues with both picric acid (loldmzorth, 1952) and formamide (Krause and McCarty, 1961). In all the cases the wall

polymers have been obtained in solution leaving behind insoluble racopeptide residues still possessing the structural rigidity and the appearance of the original cell walls as seen in the electron microscope (Archibald et al., 1961; Krause and McCarty, 1961).

The walls of Gram-negative bacteria show more complexity in

chemical composition and structure than those of Gram-positive bacteria (Salton, 1961b). The protein, lipid, and polysaccharide complexes form part of the cell wall and, in addition, the specific mucopeptide constituents are also present. The existence of a rigid mucopeptide layer has been clearly demonstrated by the studies of Weidel et al. (1960) on the cell walls of Es coli, although the overall concentration of mucopeptide components is lower than in the walls of Gram-positive bacteria. Thus, there is now enough evidence that the walls of some Gram-negative bacteria possess a

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mcopeptide of similar comosition to that found in the cell walls of Gram-positive bacteria and it is the mucopeptide component which represents the "basal structure" of the walls of most bacteria.

The bacterial cell walls are disaggregated by the action of

surface-active agents such as sodium-dodecyl-sulfate (SDS). The hemolytic action of SDS has long been known and the mechanism has been explained in terms of the "collapse" of oriented lipid and cholesterol layers in the red cell membrane (Schulman et al., 1955). The Gram-negative bacteria are generally resistant to the bactericidal action of certain surface-active, anionic compounds (Baker et al., 1941) but there have been reports of killing by high concentrations (0.2 per cent) of SDS (Lominsky and Lendrum, 1942). As the walls of these bacteria contain appreciable amounts of lipid, it is likely that the disaggregation of cell walls on exposure to SDS involves a physico-chemical change in the wall structure (Shafa and Salton, 1960). Complete disaggregation of isolated bacterial cell walls has been reported and it has been suggested that the mucopeptide complex of the alls forms a network extending across the multilayered wall rather than a continuous* separate layer (Shafa and Salton, 1960)o However, Weidel et al, (1960) used SDS (0.4 per cent) during the isolation of the mucopeptide layer from the cell walls of E. coli, the detergent removing some of the protein and lipid from the outermost layers, a process which was completed by the action of phenol leaving behind the rigid mucopeptide layer.

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The taxonomic importance of the cell wall composition has

been stressed by some workers (Cummins and Harris, 1956a, b; Cummins, 1956, 1962). Each bacterial genus and even each species often has a particular pattern of amino acids, amino sugars, and sugars superimposed on the basal =mcopeptide unit. Such patterns may prove useful in bacterial classification, although in some cases considerable variation in wall composition of a particular species has been reported (Slade and Slamp, 1962).



Osmolysis of Marine Bacteria


The internal osmotic pressure of one marine bacterium has

been estimated to be about 20 atmospheres (itchell and Moyle, 1956) which approximately counterbalances that of the sea water. In media of low osmotic pressure, the cell walls of these bacteria are unable to withstand the internal pressure and the cells undergo lysis. In contrast, the terrestrial bacteria, though living in environments of low osmotic pressure, possess an internal pressure which may be as high as 20 to 25 atmospheres as in the case of Gram-positive bacteria (itchell and Moyle, 1956). In Gram-negative bacteria this pressure is low, between 2 and 3 atmospheres (11itchell and Moyle, 1956). Their cells, after having been growm in media of high osmotic pressure, are protected against osmotic shock when

transferred to distilled water (Doudloroff, 1940). While growing

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in such media, the cells probably develop a high internal osmotic pressure (Christian and Ingrau, 1959). Thus, the cell walls of terrestrial bacteria are strong enough to resist large differences in external and internal osmotic pressures.

Early studies on the osmolysis of marine bacteria were carried out with two luminous species, Photobacterium fisheri and Photobacterium harveyi. In 1915, Harvey made the original observation of the cytolysis of a marine bacterium while studying the phenomenon of biological luminescence. He demonstrated the dependence of luminescence on cellular integrity by showing that a dense cell suspension gave a bright light in oxygenated sea water, but no

light in oxygenated tap water. He suggested that the cells were lysed in tap water, thus disrupting the system responsible for luminescence.

The osmolysis of P. fisheri was investigated by Hill (1929). He estimated lysis by measuring the disappearance of luminescence in diluted sea water and salt solutions. In distilled water containing 6 per cent sea water and in 0.0312 MI NaCl, the luminescence disappeared within a few minutes but appropriate concentration of sucrose was found to protect it. He suggested that the disappearance of luminescence was an osmotic effect. The observation, that microscopic examination revealed little change in the appearance of

cells which had ceased to give off light in diluted sea water, led him to suggest that Ithe cells were surrounded by a rigid envelope

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which did not swell but ruptured at a critical difference in osmotic pressure between the cells and the medium.

Korr (1935) pointed out that the disappearance of luminescence in hypotonic solutions might not indicate complete lysis and loss of viability. He found that some cells remained viable for several hours in distilled water. A majority of cells, however, undergo lysis in hypotonic media; Johnson and Harvey (1937) found that when dense cell suspensions were diluted with distilled water, the suspensions became clear and foamy, the motility and luminescence ceased, and the optical density and viability decreased considerably. They (1938) also made quantitative measurements of viability, respiration, and luminescence during cytolysis of P.

harveyi. Their results, in general, showed that the above three functions showed a gradual decrease with progressive dilution of sea water with distilled water. Some salts and sucrose, at certain concentrations, showed protective effects.

Direct visible evidence of lysis of marine bacteria was furnished by Johnson et al. (1943). Electron micrographs of marine cells, which had earlier been placed in distilled water, showed evidence of lysis; the cell wall was found to be ruptured resulting in exudation of intracellular contents. That the cell walls are

involved in osmotic fragility of marine bacteria is indicated by the recent work of Boring (1961). He compared the lytic patterns of whole cells and penicillin-induced spheroplasts of a marine

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bacterium by estimating the degree of lysis in a series of graded concentrations of aC1. The whole cells were protected from lysis in 0.06 to 0.08 m ilaC1 solutions, while the spheroplasts required 008 to 0.10 M solutions. These values indicated that as far as the osmotic fragility was concerned, whole cells and spheroplasts were almost equally fragile and the rigid, penicillin-sensitive component of the cell wall did not confer any added protection to the cells against osmotic lysis.

Brown (1960), in his studies on a marine bacterium, found that the hexosamxine content of the isolated cell walls was lower than that reported for non-marine, Gram-negative bacteria. He suggested that the weakness of the walls of marine bacteria was due to the low content of amino sugar. In this connection, it is interesting to note that the cell walls of a non-marine bacterium, Vibrio metschnikovil, have been found to be low in amino sugar content (Shafa and Salton, 1953) and the cells of this organism are subject to osmotic lysis in distilled water. Amino sugars have been shown to be components of the mucopeptide layer of bacterial cell walls (Weidel et al., 1960) mid a low amount of such components can result in a weakened mucopeptide layer.

In the present study, three marine bacteria were selected to represent a spectrum of susceptibility to osmotic lysis. Such differences in fragility in marine bacteria have been reported (Riley, 1955; MacLeod and Matula, 1962). It was hoped that a comparison

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20


of the chemical compositions of the cell walls of selected marine bacteria might indicate some relationship between osmotic fragility andwall composition.

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HATEIALS AND METHODS


The methods, used in this study, are presented in the following sections, each section corresponding with a particular phase of the investigation.



General Methods


Artificial sea water (=J) was used in cultivating the three marine organisms used in this study. It was composed as follows: NaCl, 23.5 g; HgS04.7120, 6.2 g; MgC12.6112O, 5.1 g; Cl, 0.75 g; distilled water, 1,000 ml. One per cent trypticase (B.B.L.) in ASW was used as a source of nutrients for the organisms. Stock cultures were maintained on trypticase-ASW slants containing 2 per cent agar. The same amount of agar was added to the basal medium whenever solid medium was used, unless otherwise stated. Nutrient broth was used for the cultivation of Pseudomonas aeruAinosa. Two per cent agar was added to nutrient broth for preparing solid medium. All media were sterilised at 15 pounds pressure and 121 C for 15 min.

All glassware was initially cleaned in chromous acid solution; subsequent to any use, it was then cleaned with Haemo-sol (Meinecke and Company, Inc.) and rinsed several times with tap

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water and three times with distilled water. For nitrogen estimation, the glassware was further rinsed with deionised water* All chemicals used were of reagent or chemically pure grade.


Organisms


The three marine organisms came from a collection of

marine bacteria isolated by Bielling (1958) from coastal Atlantic waters off Florida. The isolation was based on the ability of the

isolates to grow in media containing sea water or sea water salts, but not in media lacking in them. The organisms, designated as M.B. 29, M.B. 65, and M.B. 98, were selected mainly on the basis of their lytic properties.

A loopful from each of the three stock cultures, kept under oil, was inoculated into trypticase-ASW broth. The growth was streaked on solid medium in Petri dishes and well-isolated colonies were picked and transferred to slants. The inability of these cultures to develop in trypticase-distilled water medium was checked before starting work.

P. aeruginosa, used in the comparative study, was isolated by W. S. Silver in the department.


Growth Conditions and Harvesting


The marine bacteria were routinely cultivated in 1 per cent trypticase-AW broth. For obtaining small quantities of cells, Erlenmeyer flasks (250 ml) containing 50 ml of the liquid medium

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were inoculated from starter broth cultures and shaken at 30 C;

4 to 6 hr old cultures were used.

For obtaining large crops of cells to be used for cell wall preparations, several Erlenmeyer flasks (1,000 ml) containing 200 ml of the trypticase-ASW medium were inoculated with starter cultures and the flasks were shaken at 30 C for 12 hr. The amount of inoculum was so adjusted that the culture was ready for harvesting when it had just reached the stationary stage,

An essentially similar procedure was used for the cultivation of P. aeruginosa with the exception that nutrient broth was used in place of trypticase-ASW medium and the temperature of incubation was 37 C instead of 30 C.

For centrifuging large volumes of cultures, a continuous

flow centrifuge (Servall SS-1, type KSA-1) was used. For preparing the cell walls, 6 liters of a culture were handled at a time and the cells were used directly without washing.


Methods for Characterisation of M.B. 29, M.B. 65, and M.B. 98

MorphologY.- The morphology of the marine organisms was studied under phase optics and wet mounts of young broth cultures were examined for motility under the light microscope. For determining the type of flagellation, the cells were fixed by adding

..









2 drops of a I per cent solution of osmic acid to I ml of a young broth Culture. After standing for 5, min, the cells were centrifug6d and resuspended in a few drops 'of distilled water. The suspension was sprayed on copper grids previously coated with a collodion film; the grids were allowed to dry and then examined with an electron microscope,

Ehylioloa.t- Certain physiological properties of the marine organisms were wca:ri-ned. The method of Iugh and Leifson (1953) was used for determining the type of carbohydrate metabolism. The medium was slightly modified to suit the organisms; tryptioase,

0.2 per cent, was substituted for peptone and AMI for NaCI. Duplicate tubes of, the medium were inoculated by stabbing; one tube was sealed with a layer of petrolatum and designated as the "closed tube." The tubes were incubated at 30 C and examined periodically over, a period of one month.
The action of the following antibiotics on these bacteria was studied: chlortetracycline, chloramphenicol, erythro--Wcin, penicillin, dihydrostreptnrycin, triple sulfa, oxytetracycline, tetracycline, and the vibriostatic agent 0/129 (diamino-di-isopropylpteridine, supplied by Dr. Jo M-. Shewan),. One drop of a young broth culture was spread over the surface of solid medium in a Petri dish and the inoculum was allowed to dry for a short time. Sensitivity discs were then placed on the surface of the agar and the plates were incubated at 30 0. For testing the vibriostatic

..









agent 0/129, a saturated solution was put in a small cavity in the agar surface on which the organism had already been spread. These plates were incubated in an upright condition. The various plates were examined after 24 hr and the presence of a zone of inhibition of growth around the disc or the cavity was taken as an indication of sensitivity.

The oxidation of tetramethyl-p-phenylene-diamine (oxidase test) was determined by Kovac's method (1956).

Some other physiological activities of these marine bacteria

had previously been studied by Tyler et al. (1960) and some of the data have been included in this thesis to give a more complete picture of the physiology of these bacteria.

Lytic susceptibilitv*- The lytic susceptibilities of the

marine organisms were examined according to the method of Tyler et al. (1960). The test solutions, in which the extent of lysis was determined, were distilled water, tSld, and 0.5 M and 0.05 M concentrations of KC1, IlaC, MgC12.61120, and potassium phosphate buffer of pH 7.0. The test solutions were dispensed, in 10 ml amounts, in optically matched test tubes. Cells from a shaken broth culture were collected by centrifugation and were resuspended in sea water. The suspension was adjusted to give an O.D. of 0.80 at 500 mu when diluted 1:100; 0.1 ml was added to each tube. After mixing by inversion, the tubes were incubated at 37 C, and their optical densities were read at 500 ml, after 15 min, in

..








a "Spectronic 20" spectrophotometer (Bausch and Lomb), The per cent residual turbidities were calculated by taking the optical density of the control (ASJi suspension) as 100 per cent residual

turbidity.


Preparation of Cell Walls


The method finally adopted for preparing the cell walls of the marine bacteria was as follows. The cells were suspended in

0.5 M potassium phosphate buffer, pH 7,0, to give a heavy suspension. Equal volumes of the suspension (3 ml) and "Ballotini" glass beads, approximately 0*007 mn in diameter (C.A. Brinka- Co., New York), were shaken for 20 min at maximum oscillation in a Nickel tissue disintegrator. The disrupted and viscous cell suspension was transferred to a beaker; the cuvettes were washed with M/15 phosphate buffer, pH 7,0, and the washings were added to the beaker. The contents of the beaker were thoroughly mixed and then left standing, after addition of a drop of DNA-ase solution in baffer (0.05 mg per ml), for 20 vran. The now watery suspension was then carefully decanted and the beads were washed 3 times with 14/15 buffer, the washings being added to the supernate. The supernate was centrifuged at 1,100 x g for 10 min to remove unbroken cells and coarse debris. The crude cell wall fraction was removed and centrifuged at high speed and the cell wall residue was then washed 10 times with dilute buffer. After the final washing, the

..









cell wall suspension was again centrifuged at 1,100 x g for 10 min to remove any remaining cell debris. The supernate, which contained the cell walls, was examined by electron microscopy and the preparations, which were sufficiently free of cytoplasmic contamination, were pooled and centrifuged at high speed. The cell wall residues were resuspended in a small amount of suspending medium to give about 20 mg dry weight of cell walls per ml.

The cell walls of M,B. 29 were kept suspended in 14/15 buffer. Electron microscopy showed evidence of disintegration of the walls when they were washed with distilled water. This was not observed in the case of the cell walls of :1.B. 65 and M.B. 98; hence, after

the final washing with dilute buffer, the cell walls were washed 4 times with distilled water and finally suspended in distilled water. The cell wall preparations were stored in tightly stoppered

tubes at 0 C.
The cell walls of P. aeruainosa were prepared as follows. The cells, after harvesting, were suspended in distilled water to give a heavy suspension. Equal volumes of the suspension and "Ballotini" glass beads were shaken in a Mickel tissue disintegrator for 40 min. The contents of the cuvettes were transferred to a beaker, diluted with distilled water, and stirred.

No increase in viscosity due to the release of intracellular DNA was observed. Presumably, the organism produces an extracellular DNA-ase; several strains of P. aeruginosa have been found to possess this property (Streitfold et al,, 1962).

..








The suspension was decanted from the glass beads and centrifuged at 1,100 x g for 10 min to remove the coarse debris. The supernate was poured off carefully and centrifuged at high speed; the residue was then washed 4 times with 1 II NaCl solution and 4 times with distilled water., The complete removal of the chloride ions was checked by adding a AgNO3 solution to a small quantity of the suspension. The final suspension in distilled water was again centrifuged at 1,100 x g to remove any remaining coarse debris and the supernate was then centrifuged at high speed. The residue was resuspended in distilled water and examined with an electron microscope. It was stored at 0 C until used.


Electron 'iacroscopy of Cell Walls

The cell wall preparations were mounted on copper grids previously covered by a collodion film. The preparations were shadomd with chromium at an angle of 25 degrees and then examined

-th a Phillips E14-100 electron microscope. The electron microscopy was performed by Mr. T. Carlisle and Hr. E. J. Jenkins, Physics Department, University of Florida.


Disaggregation of Cell Walls With Detergent


The disaggregation of the isolated cell walls in sodiumdodecyl-sulfate (SDS) was studied by adding 0.1 ml of a cell wall suspension to 10 ml of a 0.1 per cent solution of SDS. The contents

..









were mixed quickly by inverting the tubes and the optical densities were read at 500 m1, in a "Spectronic 20" spectrophotometer. The tubes were again read after specified intervals upto a period of 30 min. The per cent residual turbidity was calculated for each reading, taking the optical density in distilled water as 100 per cent residual turbidity.


Action of Phenol on isolated Cell Walls


It was shown by Weidel et al, (1960) that phenol can solubilise the lipoprotein and lipopolysaccharide components of the walls of E. coli, thus exposing the rigid mucopeptide layer of the wall. A similar reaction was tried with the cell walls of M.B. 29, About 200 mg cell walls, dry weight, were transferred to a 250 ml Erlenmeyer flask and 10 ml of 95 per cent phenol were added and the flask was shaken to suspend the walls homogeneously. An additional 90 ml of the phenol solution were added and the flask was put on a rotary shaker at 37 C. The cell walls were completely solubilised giving a clear solution. Water was gradually added to see if there was any precipitation. The mixture became milky and two layers were found to separate after standing while a white precipitate accumulated at the interface. The top layer was decanted and the remaining liquid was filtered. The residue was scraped from the filter paper and washed several times with distilled

..









water. After dialysis against water for 24 hr, the material was dried first on filter paper pads and then in a previously weighed dish to a constant weight in an oven at 100 Co

The weight of the recovered material was determined and weighed amounts were used for estimating protein, lipid, hexos. amine, and reducing substance using the methods described in the following section.


Chemical Anayvsis of Cell Walls


All quantitative analyses were done in duplicates with cell wall samples prepared at different times.

Dry weights.- Aliquots of cell wall samples were dried, in weighing dishes, in an oven at 110 C. The dishes were weighed at

intervals until they reached a constant weight.

Total nitrogen.- Nitrogen was estimated colorimetrically by Nessler's reaction. One to 2 mg of cell walls (dry weight)

were digested with 04 ml of 25 per cent (v/v) %2S04 in Kjeldahl flasks. Digestion was continued until the mixture became brown" The flasks were then cooled, a drop of 30 per cent 11202 was added and the heating was resumed until the solutions became colorless, The flasks were allowed to cool and the following solutions were then added to each flask in the order given: I ml of 6 per cent sodium citrate, 3.5 ml of I N NaOU, 20 ml of deionised water, and I ml of Nessler's reagent. After the last addition, the contents

..









were mixed quickly and the optical densities were read immediately at 505 ni in a "Spectronic 20" spectrophotometer.

The Nessler's reagent was made from the commercial Folin

and Wu reagent according to the directions given. A standard cuive was prepared with known amounts of glycine and cell wall nitrogen was estimated by reference to this curve. Two samples, containing known amounts of glycine, were always run with the cell wall batches to serve as a check of the procedure.

Total phosphorus.- This was estimated by using a colorimetric method described by Fiske and Subbarow (1925). Between 2 and 3 mg cell walls (dry weight) were used for digestion with concentrated H2SO4. The amount of phosphorus was estimated by reference to a standard curve of KfH2PO4 solutions.

Total lipid.- Lipid material was determined as described by Salton (1953) with a slight modification. The ethereal extract of the cell wall hydrolysates was washed 4 times with distilled water to remove the non-lipid material which was found to have been picked up during ether extraction of the hydrolysates. The lipid material was estimated by evaporating the solvent in a hood at room temperature and weighing,.

Reducing substance.- The cell walls were hydrolysed as described by Salton (1953) and the hydrolysates were analysed colorimetrically for reducing substance using the anthrone reagent.

..









Glucose was used to determine a standard curve which served as a reference. The reducing substance was expressed in terms of glucose*

IHexosamine4- This was estimated by using the Elson and M organ reaction as described by Kabat and Mlayer (1948) Knom quantities of glucosamine hydrochloride were used to prepare a standard curve and the results were expressed in terms of glucosamine4

Protein. Protein was estimated by the method of Lowry et al. (1951) using the Folin-Ciocalten reagent* About 4 mg of cell walls (dry weight) were suspended in 1 11 NaOTI and the suspension was incubated at 37 C overnight, A standard curve, using crystalline bovine alburin, was prepared and used as a reference.
Paper chromatog.aphCi.- Detection of arino acids and carbohydrates was done by paper chromatography. Ihatrtmn no I filter paper was used throughouti Glass msemi jars were used for ascending and a chromatocab (Research Equipment Corp*, Oaklad, California.) for descending chromatography.

Amino acids and amino sugars.- Cell walls (20 mg dry weight) were hydrolysed i 6 ml of 6 N IICIin a sealed tube for 16 hr at 120 Ci The hydrolysate was decolorised with activated charcoal, filtered, and then dried on a steam bath The residue was dissolved in a small amount of distilled water and then redried in a vacuum desiccator over NaOII pellets and concentrated I{ SO4. The final

residue was dissolved in 0.5 ml of distilled water,

..









A platinum wire, with a small loop (2 mm diameter) at one end, was used for spotting on the chromatogram paper. The amino acids and amino sugars were separated by two dimensional chromatography. The first solvent used was n-butanol:acetic acid:water (60:15:25, v/v) and the second solvent was phenol:ammonia (I ml anmmonia added to 200 ml of phenol-water solution; 1 lb phenol + 113 ml water). After development, the solvents were removed by evaporation at room temperature in a chemical hood. For descending chromatography, the spotted paper was first equilibrated with the solvent for 10 to 15 hr before development.
The amino acids were detected by reaction with 0.25 per cent solution of ninhydrin in acetone. After spraying the reagent, the paper was allowed to dry and then heated at 100 C for 10 min. The amino acids were identified by position or color and by comparison with chromatograms of known amino acids. For detecting the hexosa-ines, the Elson and Morgan reaction (Partridge and Westall, 1943) was used on "the paper chromatograms.

Sugars.- Cell wall samples were hydrolysed in sealed tubes with 6 ml of 2 N H2SO4 for 2 hr at 100 C. The hydrolysates were neutralized with a solution of Ba(0i)2 to pu 6.5; the precipitate of DaS04 was filtered off and the filtrate was evaporated in a vacuum jar containing Na0H pellets and concentrated H2SO4. The residue was dissolved in a few drops of distilled water.

The chromatogram papers were spotted using the platinum

..









wire loop and the solvent used was isopropanol:water (60:40). Multiple ascending development was used for obtaining a greater separation of sugars,

The reducing sugars were detected by spraying with anilinehydrogen-phthalate reagent (Partridge, 1949). Other reagents such as naphthoresorcinol and phloroglucinol were used for the detection of ketopentoses (Smith, 1960). The colorimetric reaction of Dische (1953) was employed to detect any heptoses; the reaction was carried out with the extracts of cell walls prepared for the sugar chromatography.

..












EPERIITAL RESULTS


This study involved the preparation and analysis of the

cell walls of three selected marine bacteria. It was extended to include the isolation and analysis of the cell walls of a nonmarine pseudomonad as well as the examination of some of the morphological and physiological properties of the marine organisms.

The marine bacteria, M.B. 29, M.B. 65, and M.B. 98, developed rapidly in shaken cultures at 30 C in trypticase-ASW broth and maximum growth could be obtained in 12 hr, The growth in stationary cultures, though less rapid, attained approximately the same level. A heavy pellicle was observed on the surface of the stationary cultures, especially in the case of 1.4. 29 and M.B. 98. An abundant growth was obtained on trypticase-ASW slants in 24 hr. Twenty-four hr old colonies of 14,B. 29 and M.B. 98 were about 2 mm in diameter while those of M.B. 65 were smaller. The colonies were smooth, entire, circular, and slightly raised; they were cream colored in the case of M.B. 29 and M.B. 98, and golden-yellow in the case of M.B. 65.

Shaken broth cultures, 4 to 6 hr old, were examined under phase optics (Plate 1). The marine organisms were pleomorphic in nature, from straight to slightly curved rods, The cells of M.B. 65 35

..






















Plate I Phase contrast micrographs of M.B 29, M.B 65, and
1.B. 98 (x 2,4OO).

Fig. i M.B. 29 Fig. 2 M.B. 65 Fig. 3 1,.103 98

..

















PLATE I


















0 qw44

v pop






oil

Fig. 1 Fig
















doe qb

4w Fig-


o 2

..









were thinner and longer (1.5 to 3.0 microns long) than those of M.B. 29 and M.B. 93 (1.0 to 2,0 microns long). Some hemispherical and less phase-dense areas were observed in the case of MB. 29; they appeared to be protruding from the cells. Their significance was not investigated.

The marine bacteria were found to be Gram-negative. Wet mounts of broth cultures of M.B. 29 and M.1. 93 sh-owed the cells to be motile and the electron micrographs of their cells, fixed with osmic acid, showed the flagellation to be polar and monotrichous (Plate 2). Broth and slant cultures of M.D. 65 were examined at different stages of growth but no motility was observed.

The Hugh and Leifson technique (1953) was used for determining the type of carbohydrate metabolism (Table 1), 1.1B. 29 and M.D. 93 produced acid from so-e of the carbohydrates in the open tubes; no reaction was observed in any of the closed

tubes. All the positive tests were visible within 24 hr and prolonged incubation revealed no adaptive response. i.D. 65 did not produce acid from any of the carbohydrates tested, in either the open or the closed tubes. The marine organisms gave a positive oxidase test, i.e. the oxidation of tetra-methyl-p-phenylenediamine with the formation of a blue spot on the filter paper. The control tests with P. aeruginosa and E. coli were positive

..





4o





PLATE 2


Fig. 4


Figo

..









TABLE i


CARBOIHYDRATE WETABOLISM OF TIE I RINE BACTERIA1


Carbohydrate Open tube Closed tube
H. B. 4. B, M.,B. M*B. M.B, T.B.

29 65 98 29 65 98


X y l o s e 2.
Arabinose-, .
Glucose A A
Mannose .
Galactose A A
Mannitol A A
Sorbitol
Rhamnose .
Sucrose A A
Lactose A A
Maltose A A
Raffinose .




1 Incubated at 30 C for 72 hr.
2 = alkaline or no reaction.
3 A = acid production.

..








and negative respectively. Some other physiological properties of these marine organisms had been previously examined by Tyler et al. (960), Their data, along with the properties examined above, are given in Table 2.

The sensitivity of these organisms to antibiotics was
studied and the data are given in Table 3. Chloramphenicol and erythromycin inhibited all three organisms, whereas triple sulfa inhibited M.B, 29 and M.B. 98 but not M.B. 65. The latter was inhibited by chlortetracycline and oxytetracycline. The vibriostatic compound 0/129, considered to be a specific inhibitor of vibrios, inhibited 11.B. 65 but not .B. 29 and M.B. 98*


Ltic ProDerties of Cells


The lytic properties of the marine organisms in various
test solutions were examined by a procedure similar to that used by Tyler at al. (i960). The extent of lysis in test solutions, as measured by the per cent residual optical density, was taken as an indication of the degree of osmotic fragility. The results are presented in Table 4. They indicated that M.B. 65 was more resistant to lysis than M.B. 29 and M.B, 98. The solutions of

0.05 X K-phosphate buffer were most effective in lysing the cells, the per cent residual turbidities being 9 per cent and 8 per cent for M.B. 29 and M.B. 98 respectively as compared to 65 per cent for MOB. 65. In distilled water, M.B. 65 underwent very little

..





43



TABLE 2 PIHYSIOLOGICAL CHARACTERISTICS OF THE MARINE BACTERIA


Property M.B. 29 M.B. 65 M.B. 98
goldenPigment cream cream yellow

utility +1 +

Oxidase test + + +

Starch hydrolysis* + +

Gelatin liquifaction* + + +

Nitrite from nitrate*

H2S production'

Indol production*


*data from Tyler et al, (1960).
+ = positive test. 2_ = negative test.

..









TABLE 3

SENSITIVITY OF TWRINE BACTERIA TO ANTIBIOTICS


Zone of Inhibition
Antibiotic

Amount/disc M.B. 29 M.B. 65 M-B. 98


Chlortetrcycline 5ug ++
Chloramphenicol 5u+g 1 4+ ++

Erythro-rcin 2ug -+H +4+ +.

Penicillin 2 units

Dihydrostreptocin 2ug

Triple sulpha 50ug 1+ +

Oxtetracycline 5ug +

Tetracycline 5ug

Vibriostatic
compound 0/i29* ++


+$++9+++l = relative degree of

a uninhibited.
a saturated solution
in the agar.


inhibition.



was put in a small cavity

..







TABLE 4

LYTIC PROPERTIES OF MARINE BACTERIA


Per cent residual turbidityl
Test mediumM.B 29 m3. 65 M.B. 98

Distilled Water 62 95 36
0.05 M NaC! 73 95 63
0.5 M NaC1 98 95 95
0,05I'MKC 43 90 36
0.5 11 KOI 88 92 77
0.05 M MGCY 2 112 109 10
0.5 14 1C2 115 110 102
0.05 14 K-phosphate
buffer2 9 65 8
0.5 H K.,phosphate
buffe 91 100 82


tPer cent residual turbidity
S2.D.' in suspending medium x Ioo O.D. in ASW 2H7.0

..









or no lysis as compared to 62 and'36 per cent residual turbidities of M.B. 29 and M.B. 98 respectively. In both concentrations of MgC12, there was no decrease in optical density; in fact, residual optical densities of more than 100 per cent were observed. The results of this experiment showed that M.B. 65 was the least susceptible to lysis with 90 per cent or more residual turbidities in all test media except 0.05 M K-phosphate buffer. M.B. 98 was found to be the most susceptible of the three organisms with M.B. 29 showing intermediate susceptibility.


Preparation of Cell Walls


Considerable difficulty was encountered in obtaining satisfactory preparations of the cell walls of the marine bacteria. Various methods for the disintegration of cells and for the removal of cytoplasmic material from cell walls were tried before a suitable method could be devised. The earlier attempts were confined to M.B. 29, and once the final method was adopted it was used without change for the preparation of the cell walls of the other two marine bacteria. No difficulty, however, was encountered

in obtaining clean cell walls of P. aeruginosa.

The methods tried for the isolation of the cell walls generally consisted of the following steps: (a) the disruption of cells,

(b) differential centrifugation to remove the coarse debris, and

(c) the treatment of the residue, obtained from the supernate, by

..









different washing and enzymatic treatments, The cells of M.B. 29 were disrupted by lysis in cold or hot water, in I per cent aqueous n-butanol, in 0.1 per cent sodium-dodecyl-sulfate, and also by the treatment of cells with 5 per cent trichloroacetic acid (TCA)* A large majority of the cells were disrupted as observed with the electron microscope. In the case of n-butanol, sodium-dodecylsulfate, and TCA, there was considerable fragmentation of the treated cells (Figure 6); hence these methods were discarded. In other cases, partly empty cell walls could be observed with considerable cytoplasmic material -dhering to them.

Lysis of cells with water was carried out by a 20-fold dilution into distilled water of'a heavy cell suspension in ASNt. After removal of the coarse debris by centrifugation at 1,100 x g for 10 min, the supernate was examined by an electron microscope (Figure 7). Almost all the cells appeared to have lysed and in most cases the cell wall was clearly seen to have broken allowing the cellular contents to escape. The cell walls were, however, heavily contaminated with cytoplasmic material. These cell walls were then subjected to various treatments. Further washing with I M NaCI and/or distiled water did not have any effect; after washing 4 times with NaC1, there was some evidence of fragmentation into smaller, irregularly-shaped fragments which were flat in appearance and free of cytoplasmic material (Figure 8). Of particular interest was the presence of small circular discs which were, later on, observed in

..




















Plate 3 Electron micrographs of M. B7, 29 after lysing and washing
with various procedures.

Fig. 6 An AST' suspension of cells was diluted 20-fold rith 0.1 per-cent SDS; the suspension was centrifuged at 14100 x g for 10 min. The supernate was centrifuged at high speed
and residue was washed 4 times with water (x 9,000).
Fig. 7 An ASW suspension of cells was diluted 20-fold with
distilled water and the coarse debris was removed as above;
the supernate was examined (x 9,000).
Fig. 8 Cells were lysed and the coarse debris was removed as
above. The residue from the supernate was washed 4 times with 1 1 NaCl and 4 times with distilled water
(x 8,000).

..






49





PLATE 3


Fig. 6 Fig.


P ig. 8

..








almost every preparation. These discs were of rather uniform size and flat in nature. Such discs have been observed in the cell wall preparations of Salmonella typhimurium (Herzberg, personal communication) obtained in this department. Their nature remained doubtful.

The adhering cytoplasmic material appeared to be protein

in nature. Ultraviolet absorption spectra of TCA extracts did not show any appreciable absorption at the 260 mu wavelength at which nucleic acid shows absorption. In an effort to remove adherent matter, various enzymes (RtA-ase, trypsin, ficin, pepsin, bromelin), singly and in combination, were tried# All enzymatic treatments were carried out at room temperature vrith the material suspended in appropriate potassium phosphate buffer solutions of M/15 concentration. The time of incubation was usually 2 hr. When the material was to be treated ith a second enzyme, the hydrogen-ion concentration of the suspension was adjusted and the second enzyme added, The suspensions were then centrifuged, washed 3 times with 1M15 buffer, pH 7.0, and the final residues, still suspended in buffer, were examined by electron microscope.

The results did not show any detectable decrease in the amount

of cytoplasmic material. With trypsin alone ex-tensive fragmentation of the cell walls was observed (Figure 9). Most of the fragments

were free from cytoplasric contamination. With trypsin and RNA-ase together, the effect of trypsin was even less than when it was used alone; the cytoplasmic material was even more pronounced (Figure 10).

..





















Plate 4 Electron micrographs of cells of MB. 29 after lysis
in water and treatment with various enzymes.

Fig. 9 Cells were lysed by dilution ith water and coarse
debris was removed by centrifugation at 1,100 x g
for 10 win; the residue from supernate was suspended
in 11/15 buffer, PH 8,0, and trypsin (2mg per ml of suspension) was added. 70-xture was incubated
for 2 hr at room temperature; suspension was
centrifuged and residue washed 3 times with r4/15
buff er, p11 7.0 (x 6,500).
Fig. 10 The procedure used was same as above, except that RNA-ase (0.05 mg per ml of suspension) was added
with'trypsin (x 13,500),
Fig. 11 The procedure used was same as above, except that only ficin (2 mg per ml of suspension) was used
(x 9,000).

..












PLATE 4























Fig. Fig. 10


Fig. 11

..








The same kind of results was Obtained with ficin, alone or in combination with RN-ase (Figure 11), and other enzymes.
In an attempt to improve the cleanliness of the wall I proparations, the conventional method of breaking the cells by vigorous shaking with glass beads in a M4ickel tissue disintegrator was employed. Here also a variety of conditions were tried. For suspending the cells during disintegration, 3 media were tried. These were AST1, I H NaCi, and 0,5 IN K-phosphate buffer, pH 7,0. Washing procedures tried involved I 1 NaCI, IV/15 K-phosphate buffer, pH 7.0, and distilled water, Along rith these, various enzymes were tried under the conditions described earlier.
Equal volumes of a heavy cell suspension and "Ballotinil glass beads (0#007 ir- diameter) ware shaken 'in the IMickel tissue disintegrator for 20 min. The length of time used was arbitrary but later results show d it to be satisfactory. The increased viscosity was reduced by the addition of a trace of DIIA-aso and, after removal of the beads, the suspension wascentrifured ,9 1,100 x g for 10 min The supernate was then centrifuged at high speed and the residue was subjected to various treatments. Disintegration in sea water suspension followed by 4 washings with

1 II NaCI solution gave a heavily contaminated preparation (Figure 12). Similar results were obtained when I M NaCI or 0.5 14 buffer was used as the suspending medium and 1 14 NaCl solution was used for washing (Figure 13). However, better results were. obtained.

..









when the suspending medium was 0.5 M buffer and the cell walls were washed with M/15 buffer (Figure 14). The cell walls were stll contaminated but there was practically no fragmentation. The cell walls, obtained after this treatment, were used for further trials. Washing with water (Figure 15) and the use of various enzymes did not remove the cytoplasmic material. With JUTA-ase and trypsin, there was considerable fragentation of the cell walls as was observed earlier (Figure 16). Ficin, alone or in combination with RNA-ase, gave similar results (Figure 17), as did pepsin (Figure 13).

Since the results obtained by disintegration in the presence of 0.5 M buffer followed by washing ith M/15 buffer were the best so far, further washing of the cell walls with the dilute buffer was tried. A gradual decrease in the amount of cytoplasmic conta.ination was observed and, after 10 washings, the suspension was found to contain cell walls which were practically free from cytoplasmic material (Figure 19). The preparation was considered to be satisfactory for chemical analysis, despite some fragmentation of the cell walls.

The same procedure was then used for preparing the cell

walls of P.B. 65 and I.1.B. 93 and the appearance of the preparations obtained is shown in Figures 20 and 21 respectively. The cell

walls of P. aeruginosa were prepared as describe-d in Materials and M Iethods. Clean cell walls were obtained in the first trial (Figure 22).

..



















Plate 5 Electron nicrographs of cell walls of M.B. 29 prepared by mechanical disintegration and washing
%ith various procedures.

Fig. 12 Cells suspended in ASt and disrupted in lackel
tissue disintegrator; coarse debris was removed
by centrifugation -at 1,100 x g for 10 min Cell
ualls were washed 4 times vith i 11 NaC! and 3
times with distilled water (x 7,500).
Fig, 13 Procedure used was the same as used above, except
that cells were' suspended in 0.5 M buffer, pH 7.0,
during disruption (X 7,500).
Fig. 14 Procedure used was same as for Fig. 13, except
that M/15 buffer, pH 7.0, was used for washing
in place of NaCl solution and distilled water
(x 4,500).
Fig. 15 Procedure used was, same as for Fig. 14, except
that distilled water was used after washing with
11/15 buffer (x i4,500).

..













PLVI P, 5


Figo 13


Fig, 12


Fig. 14


Fig. 15

..




















Plate 6 Electron micrographs of cell walls of 14.B. 29 obtained by mechanical disintegration and action of
various enzymes.

Fig. 16 Cells were suspended in 0.5 it buffer, p'I 7.0,
and disrupted in nickel tissue disintegrator.
Coarse debris was removed by centrifugation at
1,100 x t'or 10 min; residue from supernate was
suspend~l'in 11/15 buffer, pH 7.0, and IMA-ase
(0.05 rg per ml of suspension) and trypsin (2 mg
per ml of suspension) were added; mixture was
incubated for 2 hr at room temperature. txture
was centrifuged and residue was washed 4 times
with 11/15 buffer, pH 7.0 (x 10,000).
Fig. 17 Procedure used was same as above except that ficin (2 mg per ml of suspensionS only was
used (x 4,500).
Fig. 18 Procedure used was same as above, except that pepsin (2 mg per ml of suspension) was used and
suspending buffer had a pH of, 1.5 (x 5,500).

..












PLATZ- 6








JWL -auk



01 0 ,ig. 16 P i -z 17


Fig. 10

..























P1ate 7 Electron micrographs of cell walls of H.B. 29, M.B. 65,
M.B. 98, and P. aerugixosa.
Fig. 19 Cell walls of N.B. 29 (x 4,500).
Fig. 20 Cell valls of M.B. 65 (x. 5,500).
Fig. 21 Cell walls of M.D. 93 (x 4,500).
Fig. 22 Cell walls of P. aeraginosa (x 11,500).

..















T? *2?TJ


zz 0 21a


6T IOTJ


oz a 2,tq.


Z ZT*VrId

..









It might be ephasized here that the method finally adopted

for preparing the cell walls of the marine bacteria and P. aeruginosa did not involve the use of any enzyme, except DNA-ase which was used to reduce the viscosity produced during the breakage of the cells of the marine bacteria.

From the electron micrographs, the walls of M.B. 29 and M.B. 98 appeared to be thin and fragile in nature as compared to those of M.B. 65 and P. aeruginosa. The cell walls of M.B. 65 a speared to be thicker than those of the other two marine bacteria but thinner than those of P. aeruginosa.


Disaggregation of Isolated Cell lialls. by Sodiurm-dodecyl sulfate


The extent- of disaggregation of the isolated cell walls of the marine bacteria and Po aeruginosa was examined in 0.1 per cent solution of the anionic detergent. The maiiha cell walls were disaggregated to a greater extent than the cell walls of the nonmarine pseudomonad, Thus, the per cent residual turbidity for '1.B. 29 was 15 as compared to 55 per cent for P. aeruginosa (Figure 23). The values for M.B. 65 and 14.B. 98 were 10 and 14 per cent respectively. The greater sensitivity of the cell walls of the marine bacteria to SDS was also evident during the attempts to prepare the cell

walls of 1M.B. 29, when extensive fragmentation of the walls was observed by treating the cells with the detergent (Figure 6).

..












DISAGGREGATION


OF CELL WALLS OF M.B. 29 AND P. AERUGINOSA BY SODIU1M DODECYL SULFATE

















P. aeruginosa


M.B. 29


.5 10


Time (minutes)


Figure 23

..









Action of Phenol on Isolated Cell Walls of M.B. 29


The isolated cell walls of M.B. 29 were shaken with phenol

in an attempt to remove the lipoprotein component of the cell walls. It was hoped that an analysis of the insoluble material left behind might indicate the presence of the components detected in the rigid layer of the cell walls of E. coli. On shaking with phenol, the cell walls of M.B. 29 were completely dissolved by the solvent. The material, precipitated after the addition of water, was analysed. The data obtained were: protein, 75 per cent, lipid, 18.5 per cent, hexosamine (as Clucosamine), 2.01 per cent, reducing value (as glucose), 0.97 per cent. This composition of the precipitate was found to be very similar to that of the cell walls of M,3. 29.

The cell walls of M,B. 65 and M.B. 98 were also dissolved

when shaken with phenol. However, the material precipitated after the addition of water was not enough for quantitative studies.


Chemical Analysis of Cell Walls


Quantitative data.- The values for the quantitative analytical data have been expressed as per cent dry eight of the cell walls, The results are shovnm in Table 5.

The nitrogen contents of the cell walls of the marine bacteria ranged from 12.2 to 12.8 per cent as compared to 8.4 per cent

..









TABLE 5


COMPOSITION OF CELL WALLS OF MARINE BACTERIA
AND PSEUDOMONAS AERUGINOSA


Chemical Per cent dry weight cell wall
constituents .N. ae.
X.B. M.B. 111B. Po aeru29 65 98


Nitrogen 12.5 1242 12.8 8.4
Phosphorus 0.9 1.2 1.1 1.2
Protein 76.0 74#8 76.2 653
Lipid 19.4 18.5 18.2 19.3
Reducing
substance* 1.8 1.5 1.0 10.0
Hexosamine** 1.9 1.7 0.9 2.6


expressed as glucose
expressed as glucosamine

..









for the cell walls of P. aeruginosa. The protein contents of the marine cell walls were also higher, ranging from 74.8 to 76.2 per cent, in contrast to 65,3 per cent for the walls of P. aeruginosa, The lipid contents of the walls varied from 18.2 to 19,4 per cent

which were about the same found in the walls of P. aeruginosa. However, in the marine bacteria the lipid and protein together comprised 93 to 96 per cent of the total dry weight of the cell walls, These values are considerably higher than 85 per cent for the walls of P. aeruginosa.

The amount of hexose-mdne in the walls of the marine bacteria ranged from 0.9 to 1.9 per cent as compared to 2.6 per cent for the walls of P. aeru-ginosa. The reducing values for the walls of the marine bacteria were close to the respoctivc hexosamine values. Since no sugars wrere detected in their cell alls, the reducing values were a reflection of the hexosamine contents. The reducing

value for the walls of P. aeruginosa was 10 per cent; the large difference between this and the hexosamine value was explained by the detection of glucose and rhamnose in the cell walls.

Chromatographic analysis.- The presence of amino acids,

amino sugars, and sugars in the cell walls was detected by paper chromatography of the wall hydrolysates. The various substances identified are given in Table 6. They were identified by comparison with chromatograms of known compounds and from their Rf values. No attempt was made to quantitate any of the constituents; however,

..





66



TABLE 6


SUBSTANCE$ IDENTIFIED IN CELL WALL IYDROLYSATES OF MARINE
BACTERIA AND PSEUDOMONAS AERUGINOSA



Substance *M B11#44,O e u
29 65 98 rinosa


Sugars
Glucose +
Rharanose +
Glucosamine + + + +
Nuramic acid* +

Amino acids
Aspartic acid + + + +
Arginine + + +
Diaminopimelic acid + + + +
Glutamic acid + + + +
Glycine + + +
Serinie + + +
Proline + + +
Hydroxyproline +
Leucine + + + +
Isoleucine + + +
Phenylaanine + + +
Alanine + +
Threonine + + +
Tyrosine + + +
Valino + + +
Cysteic acid +
Methionine + + +


Identification based on R, value.

..









the size of the spots and the depth of color with ninhydrin gave an approximate idea about the relative concentrations of some of these constituents.

On paper chromatograms, only hexosamine was detected in the walls of the marine bacteria. It gave a positive test with the Elson and Morgan spray reagent and was identified as glucosamine from its R value (Smith, 1960). Glucose and rhamnose were found in the walls of P. aeruginosa. The spot of rhamnose was quite weak as compared to that of glucose. The presence of two hexosamines was also detected; their spots were close to each other and the one with the R value of 64.2 was identified as glucosamine. The lower spot, with the Rg value of 61, did not correspond with any of the known amino sugars that were used as references. From

its Rg value, it was believed to be muramic acid (Smith, 1960). This substance was not available for direct comparison.

The hydrolysis of the walls appeared to be complete since discrete spots were obtained by spraying with ninhydrin. However, poor separation of methionine and valine, and of leucine, isoleucine, and phenylalanine was obtained. The extent of migration of these two groups of amino acids is nearly the same in various



distance substance travels from origin
distance glucose travels from origin

..









solvents. The spots of these amino acids, obtained with the unknowns, were roughly comparable in size and shape to those found on the chromatograms of known amino acids.

Most of the amino acids, generally found in the hydrolysates of proteins, were detected in the cell walls of the organisms studied (Plate 8). Of the sulfur-containing amino acids, cysteic acid and methionine were found in P. aeruginosa; methionine was also detected in the walls of the marine bacteria. Proline and

hydroxyproline were both present in M.B. 98; the former was absent in M.B. 29 and the latter in M.B. 65 and P. aeruginosa. Diaminopimelic acid was detected in the walls of all the organisms studied. Spots identified as due to glucosamine were detected in the case of M.B. 65, I'l.B. 98, and P. aeruginosa but not in M.B. 29, although it was found in the latter during sugar chromatography. From the size of the spots and the depth of color with ninhydrin, arginine, lysine, alanine, aspartic acid, and glutamic acid appeared to be present in greater concentrations than the rest of the amino acids; this was found to be true in the case of all the cell wall hydrolysates examined.

..

























Plate 8 Chromatograms of ninhydrin positive substances in
cell wall hydrolysates of marine bacteria and P.
aeruginosa,
*daminoplmelic acid was identified by one dimensional chro-matography in each solvent system.

..


















PLATE C,



119 0 19ca
12 P 19



50 C>01450 0114







QN. 29 20 0 6 M.B. 65



10 10
,,7 '',C::), ,11 C 0 17(Z:,
15 19 (I .15 19

160 --6

9 400 )1 0 Q Ot4

!P O
13 1




2 (:::) ()6 .H. 98 3 ( P~? (1) .___i = alanine
10 2 = aspartic acid
17e 19 3 = cyateic acid a
17 4 = diaminopinelic acid'
5 = glucosanine
6 = glutamic acid
16 glycine
8 series
9 = hydroxyproline
50 31 10 proline
11 arginine
12 lysine
13 = threon5ne
14 tyrosi~ne
15 ohinn
16 valine
17 phenylalanine
known 18 leucine
amino acds 19 isoleuoine



Z TAIOL, i AM= ACD i WAER

..












DISCUSSION


The marine bacteria examined in this study can be tentatively placed in the family Pseudomonadaceae on the basis of the properties studied. These organisms occur both as straight and curved rods but, as noted by Hayes and Burkholder (Bergey's Manual, 1957, p. 90), the borderline between the straight rods found in Pseudomonas and curved rods found in Vibrio is not sharp. According to Shewan, Hodgkiss, and Liston (1954), a sharper differentiation between pseudomonads and vibrios can be made by the use of a vibriostatic agent reported to be a specific inhibitor of vibrios. Since M.B. 29 and M.B. 98 were insensitive to this agent as well as penicillin, were polarly flagellated, and metabolised carbohydrates oxidatively, they could be placed in the genus Pseudomonas.

If the salt requirements of the genus Halobacterium were less rigidly defined, M.B. 65 could be placed in this genus on the basis of production of a golden-yellow pigment (presumably carotenoid), its inability to produce acid from carbohydrates, and its pleomorphic nature. This genus includes species requiring at least 12 per cent salt for growth, a property not possessed by M.B. 65, The genus Pseudomonas has been placed in 71

..









the family Pseudomonadaceae together with bacteria which "attack glucose and other sugars either oxidatively or fermentatively." However, the M'anual includes in this genus species (Pseudomonas

-elatica, Pseudomonas nigrifaciens) which are without apparent action on sugars. With this precedent, and the fact that this genus includes some non-motile species, M.D. 65 could also be placed in this genus. These facts exemplify difficulties caused by delineating genera on the basis of a single physico-chemical property.

In some marine bacteria, the presence of a cell wall does not offer protection against osmotic lysis (Boring, 1961). If the osmotic fragility of these bacteria is due to the chemical composition of the walls (Brown, 1960), then it was considered possible that some relationship between the degree of osmotic fragility and wall composition might exist. The marine bacteria, selected for this study, differed in lytic susceptibility; such differences have been reported during studies on these bacteria (Tyler et al., 1960; MacLeod and Matula, 1962).

The difficulties encountered during the attempts to prepare clean cell walls of the marine bacteria were two-fold; the fragmentation of the cell walls which was observed during the various washing and enzymatic treatments, and the failure to remove the cytoplasmic material from the walls. The cell walls of Gramnegative bacteria are generally less amenable to various washing

..









methods which usually succeed in the case of Gram-positive bacteria. In the present study, methods which have been used for the preparation of cell walls of other Gram-negative bacteria were found to

be unsuitable. Washing with NaCI and the use of various enzymes resulted in the fragmentation of the walls without the removal of the cytoplasmic material. The fragile nature of the cell walls was evident from these observations.

The proteolytic enzymes, such as trypsin, generally do not affect the integrity of isolated cell walls although they may remove some surface components such as the M protein in streptococcal cell walls. The fragmentation of the cell walls of M.B. 29 by the action of trypsin and other enzymes may be indicative of a nature different from that of other bacteria. It is possible that the predominantly lipoprotein nature of the cell walls of the marine bacteria, together with other features such as the absence of sugars, renders them susceptible to degradation by such enzymes.

The failure of enzymes to remove the adhering cytoplasmic material from the walls could not be explained unless it was assumed that either the material was somehow inaccessible to the enzymes, or it was of a nature not susceptible to such agents. From the electron micrographs, it was difficult to determine whether the material was trapped inside the walls or attached on the outside.

The method finally adopted was the simplest of the various

methods tried. It did not involve the use of any enzyme except

..









DNA-ase which was used to reduce the viscosity produced during the breakage of cells. The method was found to be satisfactory for the other two marine bacteria, especially in the case of M.B. 65.

Some differences in the appearance of the cell walls of the organisms were noticed in the electron micrographs. The walls of I,. 65 appeared to be thicker than those of M.B. 29 and M.B. 98 but thinner than those of P. aeruginosa. The cell walls of the latter two marine organisms were thin and fragile looking. Since M.B. 65 was found to be the least susceptible to osmotic lysis, it appeared possible that the thickness of the walls was partly responsible for resistance to lysis. From ultrathin sections of cell walls and whole cells, it has been possible

to measure the thickness of the walls of some bacteria. Similar studies on marine bacteria night give some useful information in this regard.

The analytical data revealed a close resemblance in the gross chemical compositions of the walls of the marine bacteria. Their nitrogen contents were considerably higher than those found in the cell walls of P. aeruginosa and other Gram-negative bacteria. However, the cell walls of certain halophilic bacteria have a high nitrogen content (Gibbons et al., 1955), in which respect they resemble the marine bacteria. The lipid and phosphorus contents of the walls of the marine bacteria and P. aeruginosa were comparable to those reported for other Gramnegative bacteria (Salton, 1953). Lipid and protein together comprised

..









*about 94 to 96 per cent of the dry weights of the walls of the marine bacteria. Similar data on protein contents of walls of other Gram-negative bacteria are not available but, from their reported nitrogen contents, the amount of lipoprotein in their walls should be much like that of P. aeruginosa (85 per cent).

Some minor differences in the amino acid complement of the walls were observed but these may not be significant. Although no

attempt was made to quantitate the amino acids, some of them appeared to be present in greater amounts than others; these were

arginine, lysine, alanine, aspartic acid, and glutamic acid, Nuramic acid, a characteristic component of the wall mucopeptide, was not detected in the walls of the marine bacteria though it was found in those of P. aeru ,-nosa, it is possible that it was present in amounts too small to be detected by the methods used in this study. The formation of spheroplasts from marine bacteria (Boring, 1961) by the action of penicillin on whole cells points towards the existence of a penicillin-sensitive component in the cell walls. The effect of penicillin, in the case of the cells of E. coli, is believed to involve an inhibition of the synthesis of the mucopeptide and its incorporation into the walls of the bacterium (Park and Stroringer, 1957, Weidel et al., 1960). By this analogy, presence of a mucocomplex in the walls of the marine bacteria is indicated. Moreover, the detection of diaminopimelic acid (DAP)

..









in the marine cell walls indicated the presence of such a complex since this amino acid can be considered as an indicator for the presence or absence of the R-layer links (Weidel et ali, 1960).

The cell walls of the marine bacteria were found to be soluble in phenol and they were e-tensively disaggregated on treatment with sodium-dodecyl-sulfate (SDS). Both these chemicals were used by eidel et ai. (1960) for the isolation of the R-layer of the cell walls of E. coli. In a paper published after the conclusion

of this study, W.eidel, Frank, and Leutgeb (1963) have pointed out that autolytic enzymes can damage the mucopeptide layer (R-layer) if suitable precautions against their action are not taken during

the preparation of cell waIlls. They showed that the cell walls of Salmonella allinar~u, prepared by using SDS, contained the mucopeptide layer. However, if the walls were prepared by disruption of cells in the M ckel tissue disintegrator followed by washing, they were found to be deformed indicating damage to the R-layer. They were also extensively disaggregated when treated with SDS. The use of SDS during the preparation of the walls appe,-red to have inactivated the autolytic enzymes. It is possible that such enzymes could have damaged the walls of the marine bacteria during their preparation resulting in their solubilisation in phenol and extensive disaggregation by SDS.

An unusual feature found in the walls of the marine bacteria was the absence of sugars; only glucosm-ine was found to be present.

..








The absence of sugars was also evident from the low reducing values
which were almost the same as the hexosamine values, the former values being a reflection of the hexosamine contents of the cell walls. A similar absence of sugars has been reported for the cell walls of three halophilic bacteria, Vibrio costicolus, Micrococcus halodenitrificans and Pseudomonas salinaria (Gibbons et al,, 1955). However, in the cell walls of another marine bacterium, the only one previously analysed, glucose and a heptose have been reported (Brown, 1960). Evidently, the marine bacteria vary in this regard. The cell walls of P. aeradnosa were found to contain glucose and rhamnose; similar sugars have been found in the cell walls of other Gram-negative bacteria.
The cell walls of the marine bacteria were found to contain a lower amount of hexosamine than that found in the walls of P. aeruxinosa and that reported for the walls of other Gram-negative bacteria. In this respect, the observations were similar to Brown's findings (1960). From the data, some relationship was indicated between the hexosamine contents of the walls and their osmotic fragility, although a perfect correlation was not obtained in the case of all the three marine bacteria. M.B. 98, the most osmotically frnzile of the three, contained the lowest amount (0.9 per cent) of hexosamine in its walls; M.B. 65 and M.B. 29 contained 1.7 and 1.9 per cent respectively. Of these two,, M.B. 65 was the most resistant to osmotic lysis,

..








However, if these data are considered from another angle, a more definite relationship is indicated. As mentioned earlier, the electron micrographs of the cell walls indicated that the cell walls of .B. 65 appeared to be heavier looking, with more body to them, than those of M.B. 29 and M#Bo 98 which appeared to be thin and fragile in nature. It is reasonable to assume that the total amount of hexosamine per cell wall would be greater in M.B. 65 than the other two bacteria and least in M.B. 98. On this basis, the hexosamine content of the cell walls would appear to influence the degree of osmotic fragility.

With our present limited state of knowledge regarding the finer details of the wall structure in bacteria, the exact role of hexosamine in determining the strength of the wall can only be speculated upon. There is little doubt, however, that hexosamine is an important component of the mucopeptide layer. Studies on this layer (R-layer) of E. colt cell walls (Ieidel at al., 1960) have clearly shown that glucosamine, together with muramic acid, is a component of the links vfich join the spheres in the layer. A backbone of amino sugar, with chains of peptide linked through the -COOH group of muramic acid, .has been visualised as the basic structure of the mcopeptide layer. The peptide- chains link the adjacent spheres giving a comb-like layer. By the action of enzymes such as lysozyme and enzyme from bacteriophage T2, the links are broken resulting in the disengagement of the spheres from one another (Weidel et al., 1960). These facts point

..








towards the key role of hexosamine in the structure of the mucopeptide layer. A small amount of this sugar can affect the rigidity since polymers of such compounds are believed to be responsible for the mechanical strength of walls.

The results of this investigation suggested that the weak nature of the cell walls of the marine bacteria was due to a low sugar content. It was considered possible that the amount of sugar in the wall influenced the degree of osmotic fragility exhibited by these organisms. The cell wall appeared to be composed of a soft lipoprotein layer interspersed with a mucopeptide complex, the whole structure being able to confer a characteristic shape to the cells but not osmotic stability.

..












SUMmARY


Marine bacteria are osmotically fragile and their susceptibility to lysis varies from species to species. The cause of this fragility is believed to be the weakness of the cell wall, and the presence of a low amount of amino sugar in it has been suggested as the cause of this weakness. Three marine bacteria, differing in osmotic fragility, were selected and their cell wall compositions were compared. As these bacteria were suspected to be pseudomonads, the cell walls of Pseudoronas aeruginosa were also studied to provide comparison between marine and non-marine species.

The cell walls of the marine bacteria were prepared by shaking the cells, suspended in 0.5 M K-phosphate buffer, with glass beads in a Mickel tissue disintegrator followed by repeated washing with dilute buffer. The cell walls of P. aeruginosa were prepared by a similar method but the cell walls were washed with NaCl and distilled water. The electron micrographs showed differences in the appearances of the walls; the wall of the more osmotically fragile marine bacterium appeared to be thinner than those of less fragile forms.

The cell walls of the marine bacteria were predominantly composed of lipoprotein., No sugars, except glucosamine, were detected, the reducing values being low and comparable to the respective

..









hexosamine values. In the marine bacteria, the wall hexosamine content was lower than that of P. aeruginosa and of other Gram-negative, nonmarine bacteria which have been reported. Some relationship between the hexosamine content of the walls and osmotic fragility was indicated, although a perfect correlation was not obtained.

The walls of the marine bacteria were probably composed of a soft lipoprotein coat; although muramic acid was not detected, the presence of mucopeptide was indicated by the presence of a penicillin-sensitive component and of diaminopimelic acid. The complete solubility of the walls in phenol suggested that the mucopeptide did not form a separate layer but was interspersed throughout the lipoprotein layer. The amount of hexosamine in the walls was related to the osmotic fragility exhibited by these organisms; it is possible that the thinness of the walls also played a roleU in'this property.

..









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Knox, K. W. and Bandesen, J. W. 1962 The isolation of components from cell wall of Lactobacillus casei. Biochem. J., 1v 15-23.

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Mitchell, 'P. and Moyle, J. 1956 Osmotic function and structure in bacteria. In Bacterial Anato Sixth Symposium of the Soo. Gen. lIcrobiol., Cambrido Univ. Press, Groat Britain.

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Park, J. T. and Strominger, J. L. 1957 node of action of penicillin. Science, 2, 99-101.

Partridge, S. Me, 1949 Aniline-hydrogen-phlthalate as a spraying recent for chromatography of sugars. Nature, 164 443.

Partridge, S. M. and iWestall, R. 1948 Filter paper partition chromatography of sugars. Biochem. J., 42, 238-250.

Primsigh, J.3, Pelzer, H., Naass, D., and V'eidel, W. 1961 Chemical characterisation of mucopeptides released from the cell wall of Eo celi by enzymic reaction. Biochim. et Biophys. Acta, .69 68-rT.
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Richrnond, Y1. H. and Perkins, H. R. 1960 Possible precursors for the synthesis of murmic acid by Stanhylococcus aureus. BMoohem, J., L, 4p.
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Salton, M. R. J, 1961a The anatovq of bacterial surface. Bacteriol. Rev,, 2, 77-99.

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Salton, M. R. J. 1961b In Ilicrobial Cell Walls. John Wiley and Co., New York.
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Salton, H. IR, J. and Horne, R. W. 1951a Electron microscopical observations on heated bacteria. Biochim, et Biophys. Acta, z, 19-42.

Salton, M# R. J. and Horne, W. 1951b Methods of preparation and some properties of cell walls. Biochim. et Biophys. Acta, 7, 177-197,

Salton, M. R. J. and Williams, R Co, 1954 Electron microscopy of the ceil walls of Bacillus megaterlum and Rhodospirillum ubrum# Biochim, et Biophys. Acta, 14, 455-458.
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Shockman, G. D., Kolb, J. J., and Toennies, G. 1958 Relation between bacterial ce wall synthesis, growth phase, and autolysis. J. Biol. Chem., Q0, 961-977.
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Snell, 'E. E., Radin, N. S,., and Ikawa, 14. 1955 The nature of D-alanine in lactic acid bacteria. J. Biol. Chem., L11, 803-818.

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BIOGRAPHICAL ITEMS


Inder J. Sud was born in Agra, India, on January 22, 1927. He graduated from St. John High School, Agra, in April, 1943. He attended the Agra University for six years, reoieving the degree of Bachelor of Science in November, 1947, and the degree of Master of Science in November, 1949. He served as a teacher in Agra College until August, 1959, when he joined the University of Florida, He is now a candidate for the degree of Doctor of Philosophy.

Inder J. Sud is married to Asha Sud and has two sons. He is a member of Sigma Xi.

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This dissertation was prepared under the direction of the candidate's supervisory committee and has been approved by all members of that committee. It was submitted to the Dean of the College of Agriculture and to the Graduate Council, and was approved as partial fulfillment of the requirements for the degree of Doctor of Philosophy.


April 20, 1963




Dean, College of Agriculure



Dean, Graduate School Supervisory Committee:



/haiftanan

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Plate 2 Electron micrographs of M.., 29 and M.B. 98 showing
flagellation.

Fig. 4 M.B, 29 (x 17,000).
Fig, 5 M.B. 98 (x 8,000).




Full Text

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MARINE BACTERIA: WALL COMPOSITION AND OSMOTIC FRAGILITY By INDER JIT SUD A DISSERTATION PRES13NTBD TO THE GRADUATE COUNOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA April, 1963

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ACKNOWLEDGMENT The.author wishes to express his appreciation to '. Dr.. Max ETyler and Dr. Darrell B. Pratt or the guidance during this study~ This work was supported p~ by a NSF research grant (no 021471) and by a Public Health Service training grant {no 20-869) from the General Hedical Science Division, Public Health Service .. ii

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TABLE OF CONTENTS Page LIST OF LIST OF PLATES AND FIGURES u .,. ....... ... . v INTRODUCTION ,. 1 LITERATURE REVIEiv. - 4 11ATERIALS MID EXPERIMENTAL RESULTS .35 .. SU?~"l.Y -. ., 80 BIBLIOGRAPHY 82 iii

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LIST OF TABLES TABLE Page 1. CARBOHYDRATE METABOLISM OF THE MARINE 41 2. PHYSIOLOGICAL CHARACTERISTICS OF THE MARINE BACTERIA .. .. . .... u .,. .. .. 4 3 ~ SENSITIVITY OF MARINE BACTERIA TO ANTIBIOTICS . 44 4. LYTIC PROPERTIES OF MARINE 45 5. COMPOSITION OF CELL WALLS OF MARINE BACTERIA AND .f. .AERUGINOSA 64 6 SUBSTANCES IDENTIFIED IN CELL WALL HYDROLYSATES OF MARINE BACTERIA A..'ID J: AERUGINOSA ,. . 66 iv

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LIST OF PLATES AND FIGURES Page PLATE 1. Phase contrast llliorographs of M; B. 29, M. B. 65 and M . B. 98, ............................ ._ 37 PLATE 2. Electron miorographs of M . B. 29 and M .. B. 98 showing flagellation ....... 40 PLATE 3. Electron micrographs of M. B. 29 after lysing and washing with various procedures '49 PLATE 4. Electron micrographsor cells or M. B. 29 after lysia in water and treatment with' various enzymes . .................... ~.. 52 PLATE 5. Electron micrographs of' oell walls-of M. B. 29 prepared by mechanical disintegration and washing with various procedures ......... ... 56 PLATE 6. Electron micrographs or cell walls of M, B. 29 obtained by mechanical disintegration and action or various enzymes .. ,58 PLATE 7. Electron micrographs of cell walls or 1'!. B. ~ M. B .. 6.5, M. B. 98, and ~ aeruwosa .,. ..... 60 FIGURE 2J. Disaggregation of cell walls or M. B, 29 and E. aeru~osa by sodium dodecyl sulf'ate 62 PLATE 8. Chrorrw.togrmns or ninhydrin positive substances in eeil i-rall hydroiysates of maririe'bacterla' and .!: aerug:inosa . .70 V

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INTRODUCTION A major function of the bacterial cell wall is to maintain cellular integrity in adverse external environments. Thus, most bacteria can survive in media of low osmotic pressure, an ability dependent upon the presence of a cell wall rigid enough to prevent osmotic swelling and bursting of the bacterial protoplasm. The cell walls of Gram-positive organisms can withstand high internal osmotic pressures, g.g. 20 to 25 atmospheres in Staphylococcus aureus (Mitchell and Hoyle, 19,56). In contrast, this pressure in Gram-negative organ isms is low, between 2 and 3 atmosnheres as in Escherichia coli ----(Mitchell and Hoyle, 1956), but it has been shovm that these cells can be grovm in salt-rich media and remain intact when placed in distilled water (Doudoroff, 1940). This protective function of the cell wall is apparently absent in the marine bacteria which have been isolated and studied. Their cells lyse when put in distilled water, the cell wall rupturing allow ing the cellular contents to escape (Johnson~&, 1943). The oc currence of lysis can be shovm by a number of determinations such as decrease in turbidity and viability and release of protein and nucleic acids into the suspending medium. A marine bacterium has been estimated to possess an internal osmotic pressure of about 20 atmospheres (Johnson and Harvey, 1937; Johnson, Zworykin, and Warren, 1943) and the lysis 1

PAGE 7

2 was the result of a critical difference in the osmotic pressure inside and outside the cells. The seat of osmotic frarrility has been shmm (Boring, 1961) to be the weakness of the cell wall; whole cells and penicillin-induced spheroplasts of a marine bacterium were found to be nearly alike in osmotic fragility. It was suggested that the penicillin-sensitive component of the cell wall, while conferring a characteristic shape to the organism, was unable to protect it from osmotic lysis. In non-marine bacteria, it is this component which is believed to give the cell wall its rigidity and mechanical strength. ,,, The cause of weakness in the cell walls of marine bacteria is not known but suf_mestions have been made that it is due to thinness, or to difference from ordinary cell walls in chemical composition or physical structure (Salton 1956). A low hexosa:mine content in the cell wall of a marine bacterium has been implicated in osmotic fra gility (Bro1-m, 1960). This compound has been shown to be a component of the nru.copeptide layer of the cell walls of ~. Q!! (Weidel, Frank, and Martin, 1960), a layer responsible for the rigidity of the cell walls. Some strains have been found to be less fragile osmotically than others (Tyler, Bielling, and Pratt, 1960; HacLeod and Hatula, 1962) indicating the existence of a spectru.in of osmotic fragility in these bacteria.. If the weakness of the cell walls were due to a low hexosamine content, then it might be possible to correlate the wall hexosamine content with the degree of os1110tic fragility exhibited by the marine organism,

PAGE 8

3 This investigation was undertaken to establish and compare the cell wall compositions of three marine bacteria selected on the basis of their lytic behaviour in various test media, the extent of lysis being taken as an indication of their.degree of osmotic fra gility. Of the three organisms, H.B. 65 was the least and H.B. 98 the most susceptible to lysis while H.B. 29 was intermediate. The cell walls of a non-marine pseudomonad, Pseudomonas aeruginosa, were also isolated and analysed as earlier studies had indicated that the selected marine bacteria might be pseudo~...onads. After considerable difficulty, a method was devised which gave clean cell walls of the marine bacteria. The fragile nature of the cell walls, especially those of H.B. 98 and H.B. 29, was evi dent from the electron micrographs. The analytical results indicated that the cell walls were predominantly lipoprotein in nature. They were completely soluble.in phenol and were extensively disaggregated by sodium-dodecyl-sulf'ate. No sugars, except glucosamine, were de tected; the hexosamine values were low as compared to those of E neruginosa and other Gram-negative bacteria. The data suggested that the hexosa.mine content of the cell wall influenced the degree of osmotic fragility. From the electron rnicrographs, it was considered possible that thirmess of the wall was partly responsible .r or the weak nature of the cell walls of the marine bacteria.

PAGE 9

LIT&'1ATURE REVIEW The literature, pertinent to tho subject of this thesis, has been revimved under two sections; (a) bacterial cell walls and (b) osmolysis of marine bacteria. Bacterial Cell Walls The external structures of bacterial cells, responsible for the rigidity and integrity of cells, are generally referred to as the "cell walls." In Gram-positive bacteria, the existence of a wall as a separate and distinct entity has been demonstrated. in a variety of ways. This has not been possible in Gram-negative bacteria; whether they possess a wall distinct from the cytoplasmic membrane is uncertain. Apart from their obvious mechanical function, very little is knovm about their biochemical activities. It is generally agreed that the isolated cell walls of Gram-positive bacteria are devoid of any enzymatic activity. The situation with the cell walls of Gram-negative bacteria is less certain; several reports on the en zymatic activities associated with the "envelope" preparations of such bacteria have appeared (Marr, 1960; Hunt, Rodgers, and Hughes, 1959; Salton, 1961a). 4

PAGE 10

5 The removal of the cell wall, partially or completely by the action of agents such as lysozyme and inhibition of its syn thesis by the action of penicillin on whole cellst results in the loss of the protective function and spherical bodies are produced. These are osmotically fragile in contrast to whole cells and have also lost the characteristic shapo if they happened to be derived from rod-shaped cells. The bodies from Gram-positive bacteria, produced by the action of lytic enzymes, have been shown to be free from wall components (Freimer, Krause, and McCarty, 1959; Vennes and Gerhardt, 1959) and are called protoplasts. Gram negative bacteria, on the other hand, give rise to bodies which re tain some of the wall constituents (Shafa and Salton, 1958; Salton, 1958) and their membranes react positively with cell wall anti bodies (Holme et al., 1960). The term spheroplast was suggested to describe such bodies. Both protoplasts and spheroplasts are reasonably stable in media of appropriate osmotic pressure. They resemble whole cells in permeability properties and in their ability to carry out bio synthetic activities such as protein synthesis, indicating that the cell wall is not essential for such activities as long as protection from osmotic effects is provided. 'l'he presence of a cell wall, however, seems to be necessary for multiplication since these bodies have been found to be unable to reproduce. Cell walls may also act as reservoirs for certain metabolites; Gerhardt (1959) and Butler!:_ al. (1958) obtained evidence suggesting that certain amino acids ma.y be stored in the walls.

PAGE 11

6 The rigidity and the mechanical strength of the cell wall is believed to be due to its nmcopeptide component. Salton (1958) demonstrated this ~Tith the isolated cell walls of Rhodospirillum rubrum; treatment of the walls with lysozyme resulted in their be coming spherical in shape and the mucopeptide components were re leased. The formation of spherical bodies, by the action of lyso zyme or penicillin on whole cells, is due to the disorganisation of the mucopeptide of the wall; lysozyme acts by degrading the mucopeptide enzymatically whereas penicillin interferes with its synthesis. In both the cases, the rigid part of the wall is weakened and the wall is unable to perform its mechanical function. Attempts at isolating the cell walls were first made in 1887 when Vincenzi obtained what he believed to be the walls of Bacillus subtilis by extraction with 0,5 per cent NaOH. It is now known that treatment with alkali and acids leads to the degradation of part of the ,rall structure (Abrams, 1958; Baddiley et al 1958). Cell walls can be obtained by mechanical disintegration of cells followed by differential centrifugation (Salton and Horne, 1951a, b). The use of mechanical methods for disruption of bacterial cells is by no means of recent origin; as early as 1901, MacFadyen and Rowland had used agitation with fine sand for disrupting the typhoid bacillus. King and Alexander (1948) refined this method by using small glass beads. Dawson (1949) showed that the cells of Staphylococcus aureus, on vigorous shaking with glass beads, gave a preparation containing

PAGE 12

7 cell walls. He used the Mickel tissue disintegrator (Hickel, 1948) for providing rapid and vigorous shaking of the cell suspension. Salton (1956) and Cummins and Harris (1956a} hav~ clearly shown that the residues from mechanically disrupted cells consist. primarily of cell walls and this method is now vr.i.dely used for obtain ing bacterial cell walls. It is preferred since.it avoids drastic chemical alteration of the walls. The method, as described by Salton and Horne (1951b), involves shaking of heavy cell suspensions ~"1th an equal volu.~e of glass beads (0.15-0.20 mm in diameter) in a Mickel tissue disintegrator. The cell walls are separated from the suspension by differential centrifugation. They are then either washed repeatedly with appropriate solutions or treated with enzymes for the removal of cytoplasmic material from them, Various solutions have been used for the cleaning of cell walls. Washing with 1 H NaCl or with phosphate buffer was found to be more effective than washing with water alone (Salton, 1953). Holdsworth (1952) used washing with sodium acetate solution followed by extrac tion with 90 per cent phenol for cleaning the cell walls of Cor;yne bacterium diphtheriae. The cell walls of Gram-negative bacteria, however, have been found to be less amenable to cleaning by washing. In their studies of' bacterial cell walls, Cummins and Harris (1956b; 1958) employed enzymes for removing cytoplasmic material from cell walls. Since then, the use of proteolytic enzymes, ribonuclease 9 and deoxyribonuclease has become v,D.despread. It is now lmorm that

PAGE 13

8 bacterial cell walls are not altered in physical appearance by these enzymes but it is possible that surface components are ren10Ved. Salton (195:3) reported. that trypsin removed the M protein from. strepto .. cocoal cell walls and that the amino acid composition Va$ simpler after.trypsin treatment, suUur-containing and aromatic amino acids being eliminated. The removal of surface protein antigen by the action or pepsin on Corynebacteriu.11 (Cummins, 19.54} and solubilisa.tion of s.bout 40 per cent of isolnted-eell walls of St~eptococcu~ by the Action of trypsin (Barkulis and Jones, 1957) have been reported. Similarly Kno:x; and Bandesen (1962) found that trypsin released a number of low molecular-weight peptides from isolated cell walls ot Lactobacillus oasei. Salton has reconnnended that the use of enzymes, especially crude enzymes which may contain -wall degrading enzymes as well as other insoluble protein material, should be carefully controlled. other methods for obtaining bacterial cell wll.s a.re also nvaila ble. Those .involving disintegration or cells with sonie and ultra sonic vibrations, decompression rupture, and pressure cell disinte gration suffer i'rom the disadvantage of fragmentation and solubilisation ot the wall (Slade Md Vetter, 1956; Marr and Cota-Robles, 19.57). Foster. C6wan, and Haag (1962) have recently described a device for :rupturing of bacteria., under contx-olled conditions, by explosive decom pression in a closed system. Autolysis .and osmotic lysis ot Whole cells also yield cell walls; Weidel (19.51) used toluene for autolysing the cells of'! Z?!! and a lytie principle, associated with the cultures of Bacillus eereus, was !'o,md to digest cell contents or a number

PAGE 14

9 of Bacillus spp. (Norris, 1957) giving clean cell walls. These methods have not been widely used because of the risk of degrading the wall enzymically. The most widely used criterion for the purity of cell wall pre parations is the absence of cytoplasmic material as determined with the electron microscope. Although it is not a very satisfactory cri terion, wall preparations free from nucleic acids, electron-dense cytoplasmic material, and intracellular pigments can be obtained by careful control of the procedure employed. In certain cases, the purity of a wall preparation can be determined by using any special property of the cell walls under examination. Thus, with Micrococcus lysodeik ticus, Sarcina lutea, or Bacillus megaterium, the purity can be deter mined by dissolving the walls with lysozyme and weighing the lysozyme insoluble residue. Isolated bacterial cell walls have been repeatedly examined by electron microscopy. They generally retain the shape and outline of the organism from which they had been derived. The walls of many Gram-positive bacteria, such as Staphylococcus aureus and Streptococcus faeoalis, have a homogeneous appearance, although the walls of Bacillus megaterium give a vague impression of being fibrous (Salton and Williams, 1954). In the walls of some bacteria, such as Rhodospirillum rubrum and Halobacterium halobium, a spherical macromolecular type of fine structure has been observed (Salton and Williams, 1954; Houwink, 1956). In some bacteria, such as E.coli, the isolated walls appear

PAGE 15

10 homogeneous by the usual method of examination in the electron micro scope, but thin sections of cells have clearly established the multilayered nature of the wall. The walls of E.coli appear to -have three layers visible in the electron microscope (Kellenberger and Ryter, 1958). The existence of these layers has been confirmed by their separation by chemical methods; Weidel .ill: (1960) were able to separate an outer lipoprotein layer soluble in phenol, an inner insoluble and rigid layer containing the mucopeptide consti tuents, and a middle lipopolysaccharide layer. The thich-ness of bacterial walls has been estimated from measurements on thin sections of isolated walls or cells and on the shadows cast by the walls during electron microscopy. It varies from 100 to 200 i, the walls of Gram-positive bacteria being thicker than those of Gram-negative organisms (Birch-Andersen and Ha.aloe, 1953; Kellenberger and Ryter, 1958) . The cell wall accounts for about 20 per cent of the dry weight.of the cell (Mitchell and Moyle, 1951; Cummins, 19.56; Salton, 1956), but this value may v~ depending upon the.phase of gro,rt.h or cultural conditions as in Streptococcus faeoalis (Shockman al., 19.58; Toennies and Shockman, 1959). Chromatography of cell wall digests has proved to be an in valuable tool in studies on the chemical composition of bacterial cell walls. Such studies have revealed. a number of unusual components in the walls and the list is steadily gro~Ti.ng. Host of the information available concerns the Gram-positive bacteria because of the relative

PAGE 16

11 simplicity of their wall composition as well as structure and the ease with which they can be prepared. The cell walls of Gram negative bacteria are more complex in nature. both chemically and structurally. Some excellent reviews on this subject have appeared within the last few years, the more recent being those or Work (1961) and Salton .(1961a, b, 1962}. Bacterial cell walls have been found to contain a variety of chemical components: amino acids, lipids, carbohydrates, and phos phorus, but no nucleic acids, purines, and pyrimidines. A more complete picture of the chemistry of bacterial walls became availa ble with the discovery of diaminopimelic acid (DAP} by Work (19.51} and its detection in the cell walls of various bacteria, the iso lation of muramic acid by Strange and Dark (1956} and its presence in the walls of a.11 bacterial species so far examined' (Salton, 1957; Cummins and Harris, 1956a; Work, 1957}, the detection of D-amino acids (Salton, 1957; Snell, Radin, and Ikawa, 1955; Ikawa and Snell, 19.56; Park, 1958}, and the discovery of teichoic acids (Baddiley !:. !!, 19,58; Abrams, 1958} and teichuronic acids (Janczura !:. &, 1960, 1961). The cell walls of Gra..~-positive bacteria differ noticeably from those of Gram-negative bacteria; the formAr contain a limited variety of a.mi.no acids a small amount of lipid material, and a high amino sugar content, whereas the latter contain proteins with the usual variety of amino acids, a high amount of lipid, and a

PAGE 17

12 small content of amino sugar, The cell walls of a large number of Gram-positive bacteria were examined by CUIJllllins and Harris (1956a, b; 1958). The main components, invariably found, were glucosamine, rnuramic acid, glutamic acid, alanine, lysine, and diaminopimelic acid; in some cases there were also up to five sugars, one or two other amino acids, or galactosamine. A recurring type of "basal unit" was soon recognised (Work, 1957) and the term "mucopeptide" was proposed by Mandelstam and Rogers (1959) to describe this unit. It is a complex of amino acids and a.mi.no sugars and is now recognised as the structural "backbone" common to the cell walls of Gram positive bacteria. In addition to these components, some other polymeric substances have been isolated from walls and partially or fully characterised. These include oligosaccharides, polysaccharides, teichoio aoids, and teichuronic acid. These polymers are less widely distributed and teichu.ronic acid has been reported in walls or Bacillus subt1lis (Janczura et!!!, 1960, 1961) only, The chemical structure of teichoic and teichuroriic acids has been established. The name teichoic acid refers to the polymers of glycerophosphate and ribitol phosphate; the former type of polymer was first detected in the walls of Staphylococcus aureus (Mitchell and Moyle 9 1951) and later ort was found in the cell walls of other bacteria. Ribitol phosphate polymers were detected. in the cell walls of Bacillus subtilis and Lactobacillus arabinosus (Baddiley al 1958). The teichoio acids

PAGE 18

13 of 1?. subtilis (Armstrong 21, al., 1961) and aureus H (Baddiley, Buchanan, Martin, and Rajbhandhary, 1962) contain ribitol units joined by phosphodiester linkages and most of the ribitol units carry ester linked Dalanine (Baddiley, Buchana.11, Rajbhandhary, and Sanderson, 1961) and glucosyl residues. Teichuronic acid, isolated from the walls of g. subtilis, is composed entirely of acetyl-galactosamine and glucuronic acid (Janczura fil &, 1960, 1961). Dianrl.nopimelic acid (DAP) and muramic acid are two important constituents of bacterial cell walls which are generally absent in other types of organisms (Rhuland, 1960). DAP is present in most bacteria with the exception of some Gra.~-positive cocci and lactobacilli (Work, 1951; Hoare and Work, 19.57). Mu.ramie acid, first isolated from a product obtained from the exudates of germinating spores of Bacillus megaterium (Strange and Dark, 1956), is glucosamine carrying ether-linked lactic acid at the 3-position (Strange and Kent, 1959) and probably originates from glucosamine (Zillikin, 1959; Richmond and Perkins, 1960). The key role of this a.'llino acid in the structure of the bacterial wall nmcopeptides has been recognised and a broad outline of the nmcopeptide structure has been established from. the studies of products isolated. from walls and nmcopeptides after digestion with lysozyme and streptomyces amidase (Salton, 1956, 1951; Ghuysen and Salton, 1960; Ghuysen, 1961; Primosigh fil al., 1961),

PAGE 19

14 There is little doubt now that the mucopeptide forms the rigid backbone component composed of covalently bonded amino acids and amino sugars. The relation ofother wall compounds to the muco peptide component is less certain although it seems likely that they are attached to the mucopeptide by weak linkages. Evidence for this is provided by the extraetibility of teichoic acids (Archibald et &, 1961) and teichuronio acid (Janozura et al., 1961) with trichloro acetic acid in cold and the removal of oligosaccharide and poly saccharide residues with both picric acid (Holdsworth, 19.52) and formamide (Krause a11d i-rccarty, 1961). In all the eases the wall polymers have been obtained in solution leaving behind insoluble mucopeptide residues still possessing the structural rigidity and the appearance of the original cell walls as seen in the electron mici,oscope ( Archibald ~. 1961; Krause and McCarty, 1961). 'he walls of Gram-negative bacteria show :more complexity in chemical composition and structure than those of Gram-positive bacteria (Salton, 1961b). The protein, lipid, and polysaccharide complexes form part of the cell wall and, in addition, the specific rnucopeptide constituents are also .present. The existence of a rigid rnucopeptide layer has been clearly demonstrated by the studies of Weidel .ill: (1960) on the cell walls of coli, although the overall concentration of mucopeptide components is lower than in the walls of Gram-positive bacteria. Thus, there is now enough evidence that the walls of some Gram-negative bacteria possess a

PAGE 20

15 r:mcopeptide of similar composition to that found in the cell walls of Gra:n-positive bacteria and it is the mucopeptide component which :represents the 0 basal structure 11 of the walls of most bacteria.. The bacterial cell walls are disaggregated by the action of surface-active agents such as sodium-dodecyl-sulfate (SDS). The hemolytic action of SDS has long been knoim and the mechanism has been explained in terms of the "collapse" of oriented lipid and cholesterol layers in the red cell membrane (Schulman _tl fil:, 19.55). The Grara-negative bacteria are generally resistant to the bacteri cidal action of certain surface-active~ anionic compounds (Baker et al. 1941) but there have been reports of killing by high cone entrations (0.2 per cent) of SDS (Lominsky and Lendrum, 1942). As the walls of these bacteria contain appreciable amounts of lipid, it is likely that the disaggregation of cell walls on exposure to SDS involves a physico-chemical change in the wall structure (Sha.fa and Saltont 1960). Complete disaggregation or isolated bacterial cell walls has been reported and it has been suggested that the nru.copeptide.compl:ex of thewallsrorms a network extending across the mu1 tilayered wall rather than a continuous; separate layer (Sha.fa and Salton, 1960). However, Weidel et& (1960) used SDS (0.4 per cent) during the isolation of the nru.copeptide layer from the cell walls of.. .1!, the detergent removing some of the protein and lipid from the outermost layers, a process which was completed by the action of phenol leaving behind the rigid nru.co peptide layer.

PAGE 21

16 The taxonomic importance of the cell wall composition has been streased by some workers (Cumrains and Harris, 1956a, b; Cummins, 1956, 1962)~ Each bacterial genus and even each species often has a particular pattern of amino acids, amino sugars, and sugars super imposed on the basal nmcopeptide unit. Such patterns may prove useful in bacterial classification, although in some cases con siderable variation in wall composition of a particular species has been reported (Slade and Slamp, 1962). Osmolysis of Harine Bacteria The internal osmotic pressure of one marine bacterium has been estimated to be about 20 atmospheres (Vdtchell and Hoyle, 19.56) which approxi.~ately counterbalances that of the sea water. In media of low osmotic pressure, the cell walls of these bacteria are unable to withstand the internal pressure and the cells undergo lysis. In contrast, the terrestrial bacteria, though living in environments of low osmotic pressure, pos\ess an internal pressure which may be as high as 20 to 25 atmospheres as in the case of Grrun~positive bacteria {Mitchell and Moyle, 19.56). In Gram-negative bacteria this pressure is low, between 2 and 3 atmospheres (Hitchell and Hoyle, 1956) Their cells, after having bee11 grown in media of hieh osraotic pressure, are protected against osmotic shock when transferred to dist:llled wr,;r,or (Doudoroff, 194-0) While growing

PAGE 22

17 in such media, thecells probably develop a high internal osmotic pressure(Christia.n and Ingram, 1959). Thus, the cell walls of terrestrial bacteria are strong enough to resist large differences . in external and internal osmotic pressures. Early studies on the osmolysis of 1narine bacteria were carried out with two luminous species, Photobacterium fisheri and Photo bacterium harveyi. In 1915, Harvey made the original observation of the cytolysis of a marine bacterium while studying the phenome non of biological luminescence. He demonstrated the dependence of luminescence on cellular i.~tegrity by showing that a dense cell suspension gave a bright light in oxygenated sea water, but no light in oxygenated tap water. He suggested that the cells were lysed in tap water, thus disrupting the system responsible for luminescence, The osmolysis off fisheri was investigated by Hill (1929). He estimated lysis by measuring the disappearance of luminescence in diluted sea water and salt solutions. In distilled water con taining 6 per cent sea water and in 0.0312 H NaCl, the luminescence disappeared within a few minutes but appropriate concentration of sucrose was found to protect it. He suggested. that the disappearance of lur.Jinescenco was an osmotic effect. The observation, that micro scopic examination revealed lj_ttle chang~ in the appearance of cells which had censed to give off light in diluted sea water, led him to suggest that t.he cells were sur:r'Qtrnded. by a rigid envelope

PAGE 23

18 which did not swell but ruptured. at a critical difference in osmotic pressure between the cells and the medium. Korr (193.5) pointed out that the disappearance of lumines cence in hypotonic solutions might not indicate complete lysis and loss of viability. He found that some cells remained viable for several hours in distilled. water. A majority of cells, however, undergo lysis in hypotonic media; Johnson and Harvey (1937) found that when dense cell suspensions were diluted with distilled water, the suspensions became clear and foamy, the motility and lumines cence ceased, and the optical density and viability decreased con siderably. They (1938) also made quantitative measurements of viability, respiration, and luminescence during cytolysis off. harveui. Their results, in general, showed that the above three unctions showed a gradual decrease vrith progressive dilution of sea water with distilled. water. Some salts and sucrose, at certain concentrations, showed protective effects. Direct visible evidence of lysis of marine bacteria was fur nished by Johnson~& (194;). Electron micrographs of marine cells 1 which had earlier been placed in distilled water, showed evi dence of lysis; the cell wall was found to be ruptured resulting in exudation of intracellular contents. That the cell walls are involved in osmotic fragility of marlne bacteria is indicated by the recent work of Boring ( 1961). He compa:.:c
PAGE 24

19 bacterium by estimating the degree of lysis in a series of graded concentrations of NaCl. The whole cells were protected .from lysis in 0.06 to 0.08 11 Na.Cl solutions, while the spheroplasts required 0.08 to 0.10 M solutions. These values indicated that as far as the osmotic fragility was concerned, whole cells and spheroplasts were almost equally fragile and the rigid, penicillin-sensitive component of the cell wall did not confer any added protection to the cells against osmotic lysis. Brown (1960), in his studies on a marine bacterium, found that the hexosa.i-nine content of the isolated cell walls was lower than that reported for non-marine, Gram-negative bacteria. He sug gested that the weakness of the walls of marine bacteria was due to the low content of amino sugar. In this connection. it is interest ing to note that the cell walls of a non-marine bacterium, Vibrio metschnikovii, have been found to be low in amino sugar content (Sha.fa and Salton, 1958) and the cells of this organism are subject to osmotic lysis in distilled water. Amino sugars have been shown to be components of the mucopeptide layer of bacterial cell walls (Weidel et al., 1960) and a low amount of such components can re sult in a. weakened mucopeptide layer. In the present study, three marine bacteria were selected to represent a spectrum of susceptibility to osmotic lysis. Such differences in fragility in marine bacteria have been reported (Riley, 1955; MacLeod and :Hatula, 1962). It was hoped that a compru:-ison

PAGE 25

20 of the chemical compositions of the cell walls of selected. marine bacteria might indicate some relationship between osmotic fragility and wall composition.

PAGE 26

}1ATERIALS AND METHODS The methods, used in this study, are presented in the follow ing sections, each section corresponding with a. particular phase of the investigation. General Methods Artificial sea water (ASH) was used in cultivating the three marine organisms used in this study. It was composed as follows: NaCl, 23.5 g; MgS04. 7B20, 6.2 g; HgClz.6Hz0, 5-1 g; KCl, O. 75 g; distilled water, 1,000 ml. One per cent tryptiaase (B.B.L.) in ASW was used as a source of nutrients for the organisms. Stock cultures were maintained on trypticase-ASW slants containing 2 per cent agar. The same amount of agar was added to the basal medium whenever solid medium was used, unless otherwise stated. Nutrient broth was used for the cultivation of Pseudomonas aeruginosa. Two per cent agar was added to nutrient broth for preparing solid medium. All media were sterilised at 15 pounds pressure and 121 C for 1.5 min. All glassware was initially cleaned in chromous acid so lution; subsequent to any use, it was then cleaned wi:t.h Haemo-sol (Meinecke and Company, Ine.) and rinsed several times with tap 21

PAGE 27

22 water and three times with distilled water. For nitrogen esti mation, the glassware was further rinsed with deionised water. All chemicals used were of reagent or chemically pure grade. Organisms The three marine organisms ca'1l.e from a collection of marine baeteria isolated by Bielling (19.58) from coastal Atlantic waters off Florida. The isolation was based on the ability of the isolates to grow in media containing sea water or sea water salts, but not in media lacking in them. The organisms, designated as M.B. 29, H.B. 6.5, and M.B. 98, were selected mainly on the basis of their lytic properties. A loopful from each of the three stock cultures, kept under oil, was inoculated into tryptioase ... ASW broth. The growth was streaked on solid medium in Petri dishes and well-isolated colonies were picked and transferred to slants. The inability of these cultures to develop in trypticase-distilled water medium was checked before starting work. !: aeruginosa 1 used in the comparative study, was isolated by W. s. Silver in the department. Growth Conditions and Harvesting The marine bacteria were routinely cultivated in 1 per cent trypticase-ASti broth. For obtaining small quantities of cells, Erlenmeyer flasks (2.50 ml) containing .50 ml of the liquid medium

PAGE 28

23 were inoculated from starter broth cultures and shaken at 30 C; 4 to 6 hr old cultures were used. For obtaining large crops of cells to be used for cell wall preparations, several Erlenmeyer flasks (1,000 ml) containing 200 ml of the trypticase-ASW medium uere inoculated with starter cultures and the flasks were shaken at 30 C for 12 hr, The amount of in oculum was so adjusted that the culture was ready for harvesting when it had just reached the stationary stage. An essentially similar procedure was used for the cultivation of~. aeruginosa with the exception that nutrient broth'was used in place of trypticase-ASW medium and the temperature of incubation was 37 C instead of 30 C. For centrifuging large volumes of cultures, a continuous flow centrifuge (Servall ss .. 1, type KSA-1) was used. For preparing the eell walls, 6 liters of a culture were handled at a time and the cells were used directly without washing. Hethods for Characterisation of H.B. 29t M.B. 65, and M.B. 98 Mon,holOFQlThe morphology oi' the marine organisms was studied under phase optics and v1et mounts of young broth cultures were examined for motility under the light microscope. For de termining the type of flagellation, the cells were fixed by adding

PAGE 29

24 2 drops of a 1 per cent solution o:f osmic acid to 1 ml of a young ; "'! broth eul.ture .. After standing. for 5 min, the cells wer,e q~ntri.fuged' ana resuspended in a few drops of distilled water.. The 'suspension was sprnyed on copper grids previously coated With' a. ' ' ' collod.ion film; the grids were allowed to dry. and then e?..amined with an electron microsoope. '. . : . "' PhysioloaCertain physiological properties or marine o,rgan1SJ:llS l-f~e e,:~~ed. The m.ethod of Hugh and Leifson {19.53) tias used or determining tht:: t)'Pa of carbohydrate metabolism.. 'l'he :medium was slightly modified to suit the organisms; tvYPt1o~se, ~.2 per cent, .was substit-qted. for peptone and JS:1 for Wacl. Dupli cate tubes or. the. medium were inoculated by stabbing; one. tube wa.s .$ea.led with,~ layer.or pet~latum and designated as the "5Jlosed tube.'' The tubes were incubated at :;o C and exa,:nined periodically ' 1 ' over, a periO?.o:r. one month. T~e aeticm or the, roold,ng antipiotios on these bacteria. was studied~ .chlortetracycline,.ohloramphenicol, erythronzy-cin, ' . penieillln,. dihydrostreptonzy-ein, triple sulfa, ozjrtetracyollne,, ' . . tetracycline,, the. vibrio.static ,agent 0/129 ,(diamino-di ... isopropy1pteridine, supplied by nr. J, M, Shewan),. One drop of a YQ.ung broth culture va.s spread over the surf'aee or solid medium in a "< Petri dish and the in()culum was allowed to dry !'er a short time. Sensitirlty disc:, w.ere then placed on the sm-faee of the agar and the plates were in.eubat$d at :;o C. For testing the vibriostatic

PAGE 30

25 agent 0/129, a saturated solution was put in a small cavity in the agar surface on which the organism had already been spread. These plates were incubated in an upright condition. The various plates were examined after 24 hr and the presence of a zone of inhibition of growth around the disc or the cavity was taken as an indication of sensitivity. The oxidation of tetramethyl-p-phenylene-diarnine (oxidase test) was determined by Kovacs method (1956). Some other physiological activities of these marine bacteria had previously been studied by Tyler et !1 (1960) and some of the data have been included in this thesis to give a more complete picture of the physiology of these bacteria. Lytic susceptibility.The lytic susceptibilities of the marine organisms were examined according to the method of Tyler al. (1960). The test solutions, in which the extent of lysis was determined, were distilled water, ASW, and 0.5 Mand 0.05 M concentrations of KCl, NaCl, MgCl2.6IlzO, and potassium phosphate buffer of pH 7.0. The test solutions were dispensed, in 10 ml amounts, in optically matched test tubes. Cells from a shaken broth culture were collected by centrifugation and were resus pended in sea water. The suspension was adjusted to give an O,D. of 0.80 at 500 mu when diluted 1:100; 0.1 ml was added to each tube. After mixing by inversion, the tubes were incubated at 37 C, and their optical densities were read at SOO mp., after 15 ll'lin, in

PAGE 31

26 a "Spectronic 20" spectrophotometer (Bausch and tomb). The per cent residual turbidities trere calculated by taking the optical density of the control (.Amv suspension) as 100 per cent residual turbidity. Preparation of Cell Walls The method finally adopted for preparing the cell walls of the marine bacteria was as follovro. The cells were suspended in 0.5 M potassium phosphate.buffer, pH 7.0, to give a heavy suspension. Equal volu..111es of the suspension (3 ml) and "Ballotini" glass beads, approximately 0.007 mm in diameter (C. A. Brinkrr.ia:1 Co., New York), were shaken for 20 min at maximu.rn. oscillation in a Hickel tissue disintegrator. The disrupted and viscous cell suspension was trans ferred. to a beaker; the cuvettes wore washed with M/15 phosphate buffer, pH 7.0, and the washings were added to the beaker. The contents of the beaker were thoroughly Illi.xed and then left stand ing, .9.f'ter addition of a drop of DNA-ase solution in b'.lffer (0.05 mg per ml), for 20 min. The now watery suspension was then carefully decanted and the beads were washed 3 times with M/15 buffer, the washings being added to the supernate. The superna.te was centrifuged at 1,100 x g for 10 min to roF.ove unbroken cells and coarse debris. The crude cell wall fraction was removed and centrifuged at high speed and the cell wall residue was then washed 10 times vrlth dilute buffer. After the final washing, the

PAGE 32

27 oell wall suspension was again centrifuged at 1,100 x g for 10 min to remove any remaining cell debris. 'rhe supernate, which contained the cell walls, 1-ras examined by electron microscopy and > ' the preparations, which were sufficiently free of cytoplasmic contamination, were pooled and centrifuged at high speed. The cell wall residues were resuspended in a small amount of suspending medium to give about 20 mg dry weight of cell walls per ml. The cell walls of H.B. 29 were kept suspended in M/15 buffer. Electron microscopy showed evidence of disintegration of the walls when they were washed with distilled water. This was not observed in the case of the cell walls of H.B. 65 and H.B. 98; hence, after the final washing with dilute buffer, the cell walls were washed 4 times with distilled water and finally suspended in distilled water The cell wall preparations were stored in tightly stoppered tubes at O C. The cell walls off aeru.sinosa were prepared as follows. The cells, a.i'ter harvesting, were suspended in distilled water to give a heavy suspension. Equal volumes of the suspension and "Ballotini" glass beads were shaken in a Mickel tissue dis integrator for 40 min. The contents of the cuvettes were trans ferred to a beaker, diluted with distilled water, and stirred. No increase in viscosity due to the release of intracellular DNA was observed. Presumably, the organism produces an extracellular DNA-ase; several strains off. aeru.ginosa have been found to pos sess this property (Streitfeld et al., 1962).

PAGE 33

28 The suspension was decanted from the glass beads and centrifuged at 1,100 x g for 10 min to remove the coarse debris. The supernate was poured off carefully and centrifuged at high speed; the residue was then washed 4 times with 1 1-1 NaCl solution and 4 times with distilled water. The complete removal-of the chloride ions was checked by adding a Ag1m 3 solution to a small quantity of the suspension. The final suspension in distilled water was again centrifuged at 1,100 x g to remove any remaining coarse debris and the supernate was then centrifuged at high speed. The residue was resuspended in distilled water and e..xamined with an electron microscope. It was stored at O C until used. Electron Microscopy of Cell Walls The cell wall preparations were mounted on copper grids previously covered by a collodion film. The preparations were sha.dotred with chromium st ll.n angle of 25 degrees and then e~-camined with o. Phillips EM-100 electron microscope. The electron microscopy was performed by Hr. T. Carlisle and Hr. E. J. Jenltins, Physics Departmont, University of Florida. Disasgregation of Cell Walls With Detergent The disaggregation of the isolated cell walls in sodium dodecyl-sulfate (SDS) was studied by adding 0.1 ml of a cell wall suspension to 10 ml of a 0.1 per cent solution of sns. The contents

PAGE 34

29 were mixed quickly by inverting the tubes and the optical densities were read at 500 in a 11 Spectronic 20 11 spectrophotometer. The tubes were again read after specified intervals upto a period of 30 min. The per cent residual turbidity was calculated for each reading, taking tho optical density in distilled water as 100 per cent residual turbidity. Action of Phenol on isolated. Cell Walls It was shown by Weidel El: (1960) that phenol can solu bilise the lipoprotein and lipopolysaocharide components of the walls of~~, thus exposing the rigid mucopeptide layer of the wall. A similar reaction was tried with the cell walls of H.B. 29. About 200 mg cell walls, dry weight, were transferred to a 2.50 ml Erlenmeyer flask and 10 ml of 95 per cent phenol were added and the flask was shaken to suspend the walls homogeneously. An ad .. ditional 90 ml of the phenol solution were added and the flask was put on a rotary shaker at 37 C. 'he cell walls were completely solubilised giving a clear solution. Water was gradually added to see if there was any precipitation. The mixture became milky and two layers were found to separate after standing while a white precipitate accunmlated at the interface. The top layer was de canted and the remaining liquid was filtered.. The residue was scraped from the filterpaper and washed several times with distilled

PAGE 35

water. After dialysis against water for 24 hr, the material was dried first on filter paper pads and then in a previously weighed dish to a constant weight in an oven at 100 C The weight of the recovered material was determined and weighed amounts 1-rere used for estimating protein, lipid, hexes ... amine, and reducing S'..lbsta.T1ce using the methods described in the fallowing section~ Chemical Analysis of Cell Walls All quantitative analyses were done in duplicates with cell ~ll Sfu'1If)ie~ prepared at different times~ Dry weights... Aliquots of cell wall samples were dried, in weighing dishes,in an oven at 110 C. The dishes were weighed at intervals uritil they reached a constant weight~ Total nitrogen.Nitrogen was estimated colorimetrically by Nessler' s reaction. One to 2 mg of cell walls (dry weight) were digested t-d.th o.4 ml oi' 2,5 per cent (v/v)'lI 2 so4 ih Kjeldahl flasks. Digestion was continued until the mi.."'Cture beca.-ne brm-m. The flasks were then cooled, a drop of 30 per cent H20z was added and the heating was resumed until the solutions became colorless. The flasks wet-e allowed to cool and the :f'ollov-tlng solutions were then added to each flask in the order given: 1 ml of 6 per cent sodiUlll citrate, 3,5 ml of 1 N NaOH, 20 ml of deionised. water, and 1 ml of Messler's reagent. After the last addition, the contents

PAGE 36

31 were mixed quickly and the optical densities were read immediately at .50.5 m in a "Spectronic 20" spectrophotometer. The Nessler's reagent was made from the commercial Folin and Wu reagent according to the directions given. A standard curve was prepared with known amounts of glycine and cell wall nitrogen was estimated by reference to this curve. Two samples, containing known amounts of glycine, were always run with the cell wall batches to serve as a check of the procedure. Total phosphorus.This was estimated by using a colorimetric method described by Fiske and Subbarow ( 1925). Between 2 and 3 mg cell walls (dry weight) were used for digestion with concentrated HzS04. The amount of phosphorus was estimated by reference to a standard curve of KH2P04 solutions. Total lipid.Lipid material was determined as described by Salton (19.53) with a slight modification. The ethereal extract of the cell wall hydrolysates was washed 4 times with distilled water to remove the non-lipid material which was found to have been picked up du.ring ether extraction of the hydrolysa.tes. The lipid material was estimated by evaporating the solvent in a hood at room temperature and weighing, Reducing substance.The cell walls were hydrolysed as de scribed by Salton (19.53) and the hydrolysates were analysed colorimetrically for reducing substance using the anthrone reagent.

PAGE 37

32 Glucose was used to determine a standard curve which served as a reference. The reducing substance wa.s expressed in terms of glucose. Hexosrun:tne.This was estimated by us:i.ng the Elson and Horgan reaction as described by Kabat and Mayer ( 1948) K novm quantities of g1ucosa.nrl.ne hydrochloride were used to prepare . a standard curve and the resu.its were e'.t..-pressed in terms of glucosamine, Protein~ ... Protein was estimated by the method of Lowry et & (1951) using the Folin-Ciocalteu reagent, About 4 mg of cell walls (dry weight) were suspended iri 1 H 1fa0H and the suspension was incubated at 37 c overnightA sta."ldard curve, using crystaii:tne bo,11no albumin; iias prepared and used as a reference. Paper chromatog.caph;v. Detection of a'11ino acids and carbo hydrates w:1s done by p.:1.per chroniatography Whatn.tan no 1 i'ilter paper was used throughout . Glass tm\seu..'111 jars -were u$ed .t' or ascending and a chromatocab (Research Equipment. Corp., Oakland; California.) for descending chror.w.tography .Amino acids and amino sugars.Cell walls (20 mg dry weight) were hydrolysed i.11 6 ml of 6 N HCl :i..n a sealed tube for 16 hr at '120 C, The hydrolysato ,ms de colcirised with activated charcoal, filtered, and then dried on a steam bath. The residue was dissolved in a small a:10unt of distilled water and then rodried in a vacu\U'll desiccator over lfaOH pellets and concentrated 112s0 4 The inal residue was dissolved in 0,.5 ml of distilled water.

PAGE 38

33 A platinum wire, with a small loop (2 mm diameter) at one end, 'rms used for spotting on the chromatogram paper. The amino acids and amino sugars were separated by two dimensional chro matography. The first solvent used was n-butanol:acetic acid:water (60:1,5:2.5, v/v) and the second solvent was phenol:ammonia (1 ml a'11Ulonia added to 200 ml of phenol-water solution; 1 lb phenol+ 113 ml water). After development, the solvents were removed by evaporation at room temperature in a chemical hood. For de scending chromatography, the spotted paper was first equilibrated with the solvent for 10 to 15 hr before development, The amino acids were detected by reaction with 0.2.5 per cent solution of ninhydrL11 in acetone. After sp1'aying the reagent, the paper was allowed to dry and then heated at 100 C for 10 min. The amino acids were identified by position or color and by comparison with chromatograms of k.novm a.mi.no acids. For detecting the hexosamines, the Elson. and Horgan reaction (Partridge and Westall, 1948) was used on the paper chromatograms. Sugars.Cell wall samples were hydrolysed in sealed tubes with 6 ml of 2 N H 2 S04 for 2 hr at 100 c. The hydrolysates were neutralised with a solution of Ba(OH)2 to pH 6.5; the precipitate of BaS04 was .filtered off and the filtrate was evaporated in a. vacuum jar containing Na.OH pellets and concentrated H 2 so4. The residue was dissolved in a f ow drops of distilled. wat~. The chromatogram papers w-ere spotted using the platinum

PAGE 39

wire loop and the solvent used was isopropa.nol:water (160:40). Multiple ascending development was used.for obtaining a greater separation of sugars. The reducing sugars were detected by spraying with aniline hydrogen-phthalate reagent (Partridge, 1949). other reagents such as na.phthoresorcinol and phloroglucinol were used for the detection of ketopentoses (Smith, 1960). The colorimetric reaction of Dische (1953) was employed. to detect any beptoses; the reaction was carried. out with the extracts of cell walls prepared for the sugar chromatography.

PAGE 40

EXPERIMENTAL RESULTS This study involved the preparation and analysis of the cell walls of three .selected. marine bacteria. It was extended to include the isolation and analysis of the cell walls of a non marine pseudomonad as well as the examination of some of the morphological and physiological properties of the marine organisms. The marine bacteria, H.B. 29, M.B. 65, and M.B. 98, de veloped rapidly in shaken cultures at 30 C in tryptioase-.ASW broth and maximum growth could be obtained in 12 hr. The grol'rth in stationary cultures, though less rapid, attained approximately the same level. A heavy pellicle was observed on the surface of the stationary cultures, especially in the case of 11,B. 29 and M.B. _98. kl abundant growth was obtained on tryptioase-ASW slants in 24 hr. Twenty-four hr old colonies of M.B. 29 and M.B. 98 were_about 2 mm in diameter while those of M.B. 65 were smaller. The colonies were smooth, entire, circular, and slightly raised; they were cream colored in the case of' M,B,. 29 and M.B. 98, and golden-yellow in the case of H.B. 65. Shaken broth cultures, 4 to 6 hr old, were examined under phase optics (Plate 1). The marine organisms were pleomorphic in nature, from straight to slightly curved rods.. The cells of M.B. 65 35

PAGE 41

Plate 1 Phase eontrast micrographs of M.B. 29, M.B. 65t and M .. B. 98 (x 2,400). Fig, .1 11.B. 29 Fig. 2 M.B. 65 Fig. 3 M,B. 98

PAGE 42

J7 PLATE 1 I F ig. 1 _ ............___,__ ., Fig. 3 ' Fig 2 ..

PAGE 43

38 were thinner and longer (1.5 to 3.0 microns long) than those of M.B. 29 and M.B. 98 (1,.0 to 2,0 microns long). Some hemispherical and less phase-dense areas were observed in the case of M.B. 29; they appeared to be protruding from the cells. Their significance was not investigated. The marine bacteria were found to be Gram-negative. Wet mounts of broth cultures of H.B. 29 and H.B. 98 showed the cells to be motile and the electron micrographs of their cells, fixed with osmic acid showedthe flagellation to be polar and mono .. trichous,(Plate 2). Broth and slant cultures of M.B. 65 were examined at different stages of growth but no motility was ob served. The Hugh and Leifson technique (1953) was used for de termining the type of carbohydrate metabolisn1 (Table 1). H.B. 29 and H.B. 98 produced acid from some of the carbohydrates in the open tubes; no reaction was observed in any of the closed tubes. All the positive tests were visible within 24 hr and pro .. longed incubation revealed no adaptive response. M.B. 65 did not produce acid from any of the carbohydrates tested, in either the open or the closed tubes. The marine organisms gave a positive oxidase test, i.e. the oxidation of tetra-methyl-p-phenylene diamine vdth the formation of .:i. blue spot on the filter paper. The control tests vdth f. aeruginosa and li were positive

PAGE 44

Plate 2 Electron miorographs of H.:a_ 29 and H.B. 98 showing flagellation. Fig. 4 M.B. 29 (x 17,000) Fig. 5 98 {x 8,000). I.

PAGE 45

40 PLATE 2 _._._,,:. _. s,-:,-,j,' Fig. 4 Fig, .5 ,,.,[ i ;,

PAGE 46

41 TABLE 1 CARBOHYDRATE METABOLISM OF THE MARINE BACTERIA l Carbohydrate Open tube M.B. H.B. 29 65 Xylose 2 Arabinose i:J Glucose Mannose Galaotose A Mannitol A Sorbitol Rhamnose Sucrose A Lactose A Maltose A Rat:f'inose 1 Incubated at 30 C for 72 hr. 2 = alkaline or no reaction. 3 A= acid production. Closed tube M.B. M,B. M.B. H.B. 98 29 65 98 ... A ... A A ... .. A A .. A

PAGE 47

42 and negative respectively. Some other physiological properties or these tnarine organisms had been previously examined by Tyler !t. !1 (1960) Their data, along with the properties exam1ned above, are given in Table 2. The sensitivity or th(tse organisms to antibiotics was studied and the data are given in Table:,, Chlora.nrphenicol and erythrorrzyein inhibited all three organisms, wher~as triple sulfa inhibited M.B. 29 and M.B. 98 but not M.B. 6.5. The latter was inhibited by chlortetraoycline and oxytetracyoline, The Yi.brio ... static compound 0/129, considered. to be a speoil'ic inhibitor of vibrios, inhibited M,B. 6,5 but not M.B. 29 and M,B, 98. ;r...-tt,ic Properties of Cells The lytic properties of the marine organisms in various t.ist solutions were examined by a proeedUl"e similar to that used by Tyler~& (1960).. The extent of lysis in test solutions, a.s measured by the per cent residual optical density, was taken as . an indication of the degrte of' osmotic fragility. The results are presented in Table 4. They indicated that M.B-. 65 was more resistant to J.ysis than M.B. 29 and M.B. 98. The solutions or 0.0.5 M K ... phospha.te buffer were most effective in lysing the cells, the per oent residual turbidities being 9 per cent and 8 per cent for M.B, 29 and M.B. 98 respecti-vely as compared to 65 per cent tor M.B. 6.5. In di$tilled water, M.B. 6.5 underwent Vfft'y little

PAGE 48

43 TABLE 2 PHYSIOLOGICAL CHARACTERISTICS OF THE !1A.rnNE BACTERIA Property M.B. 29 H.B. 65 M.B. 98 goldenPigment cream cream yellow Motility +1 2 + Oxidase test + + + Starch hydrolysis,:,. + + Gelatin liquifaction* + + + Nitrite from nitrate* H S production~' 2 .. Indol production* *data from Tyler~ al. (1960). 1 t J + = posi ive esc. 2 = negative test.

PAGE 49

44 TABLE 3 SENSITIVITY OF MARINE BACTERIA TO ANTIBIOTICS Zone of Inhibition Antibiotic lm'l:Jllnt/disc M.B. 29 H.B. 65 Chlortotracycline 5ug .. ++ Chloramphenicol Sug ++ ++ Erythrornycin 2ug +++ +l+ Penicillin 2 units .. Dihydrostreptomye:in 2ug .. Triple sulpha 5Qug ++ Oxytetracycline 5ug + Tetracycline 5ug Vibriostatio compound o/ 129* ++ +,+i-,+++ 1 = relative degree of inhibition. ... = uninhibited; M.B. 98 ... ++ +++ ... + a saturated solution was put in a small cavity in the agar.

PAGE 50

., 45 TABLE 4 LYTIC PROPERTIES OF MARINE BACTERIA Per cent residual turbidity 1 Test Medium M,B:. 29. M11B. 6.5 Distilled. Water 62 95 0,0.5 M NaCl ?J 95 0.5 M UaCl 98 95 0.0f M KCl 43 90 0,5 M KCl 88 92 0.05 M MgC1 2 112 109 0,5 M MgC1 2 11.5 110 0.05 MK-phosphate bui'er 2 9 65 0. 5 M K, ,phosphate buf'fer 2 91 100 1 Per cent residual turbidity :er G.D. in suspending med.iW4 x 100 O.D. in ASW M.B . 98 36 63 9S :;6 :-77 100 102 82

PAGE 51

46 or no lysis as compared to 62 and':36 per cent residual turbidities of H.B. 29 and M.B. 98 respectively .. In both concentrations of MgCl2, there was no decrease in optical density; in fact, residual optical densities of more than 100 per cent were observed. The results of this experiment-showed that M.B. 65 was the least .sus ceptible to lysis with 90 per oent or more residual turbidities in all test media except 0.05 MK-phosphate buffer. H.B. 98 was found to be the most susceptible of the three organisms with N.B. 29 showing intermediate susceptibility. Preparation of Cell Walls C Considerable difficulty was encountered in obtaining satis factory preparations of the cell walls of the marine bacteria. Various methods for the disintegration of cells and for the re moval of cytoplasmic material from cell walls were tried before a suitable method could be devised. The earlier attempts were con fined to M.B. 29, and once the final method was adopted it was used without change for the preparation of the cell walls of the other two marine bacteria. No difficulty, however, was encountered in obtaining clean cell walls off aeruginosa. The methods tried for the isolation of the cell walls gener. ally consisted of the following steps! (a) the disruption of cells, (b) differential centrifugation to remove the coarse debris, and (c) the treatment of the residue, obtained from the supernate. by

PAGE 52

47 different washing and enzymatic treatments. The cells or M.a. 29 weredis:rupted.by lysis in cold or hot water, 1n 1 p$r cent aqueous n-butanol, in 0.1 per cent sodium-dodecyl ... sula.te, and also by the treatment of cells with S per cent trichloroacetic acid (TCA). A large majority of the cells were disrupted ss observed \dth the electron miorosoope. :rn the case of n-butanol, sodium-dodecyl sulfate, and TCA, there was considerable fragmentation. of the treated cells (Figure 6); henoe these methods weredisoarded. Inother oaaes, partly empty cell walls could be observed with considerable cytoplasmic material rahering to them, Lysis of' cells. with water was carried out by a. 20-fold dilution into distilled. water of a heavy cell suspension in 1\SW. After removal of the coarse debris by centrifugntion at 1,100 x g or 10 min, the supernate 1-ras examinErl by an elect~on microscope (Figure ?) Almost all the eens appeared to have lysed and in most cases the cell wall was clearly seen to have broken allowing the oellular contents to escape. The cell walls were, however, heavily contami. ' . nated with cytoplasmic material. These cell .walls were then subjected to various treatments. Further washing with I M NaCl and/or dis tied. water did not have eny efteet; after washing 4 times with Na.Cl, there was some evidence of fragmentation into smaller, irregu ... larly-shaped fragments which were flat in appearance and free or cytoplasmic material (Figure 8). or particular interest was the presence or small circular discs which 'Were, later on, observedin

PAGE 53

Plate 3 Electron micrographs or M. B.~>29 after lysing and washing with various procedure~. Fig. 6' .An ASW suspen~ion of cells was diluted 20-fold.with 0.1 per cent SDS; the suspension was centri.fuged at 1,100 x g for 10 min.. The supernate was centrifuged at high speed and residue was washed 4 ~imes with water(~ 9,000). Fig, 7 An ASW suspension ot cells was diluted 20-fold with distilled vra.ter and the coarse debris was removed as above; the supernate was examined (x 9.000). Fig. 8 Cells were lysed and the coarse debris iras removed as above, The residue from the supernate was 11ashod 4 times with 1 11 Na.Cl and 4 times with distilled water (x 8,000).

PAGE 54

49 PLATE 3 F ig. 6 F ig, 7 ~1 8

PAGE 55

50 almost every preparation. These discs were of rather uniform size and flat in nature. Such discs have been observed in the cell wall preparations of Salmonella typhimurium (Herzberg,.personal communi cation) obtained in this department. Their nature remained doubtful. The ad.11ering cytoplasmic material appeared to be protein in nature ... Ultraviolet absorption spectra of TCA extracts did not show a.11y appreciable absorption at the 260 mu wavelength at which nucleic acid shows absorption. In an effort to remove adherent matter, various enzymes (RHA-ase, trypsin, ficin, pepsin, bromelin), singly and in combination, were tried. All enzymatic treatments were ca.."Tied out at room temperature with the material suspended in appropriate potassiu.~ phosphate buffer solutions of M/15 concen tration. The time of incubation was usually 2 hr. When the material was to be treated with a second enzyn1e, the hydrogen-ion concen ... tration of tho suspension was adjusted and the second enzyme added. The suspensions were then centrifuged, washed 3 times with H/15 buffer; pH 7.0, and the.final residues, still suspendod'in buffer, were examined by electron microscope. The results did not show any detectable decrease in the amount or cytoplasmic material. With trypsin alone extensive fragmentation of the cell walls was observed (Figura 9). Most of the fragments were free from cytoplasmic conta11lination. With trypsin and RNA.;.nse together, the effect of trypsin,was even less than when it was used alone; the cytoplasmic material waseven more pronounced (Figure 10).

PAGE 56

, Plate 4 Electron mierographs of cells 0 M.B .. 29 after lysis in water and treatment with -various enzymes. Fig,. 9 Cells were lysed by dilution with water and coarse debris was removed, by centrifugation at 1. 100 x g for 10 min; the residue from supernate was suspended in H/15 buffer, pH a.o, and trypsin (2mg per ml of suspension) was added. Mixture was incubated for 2 hr a.t room temperature; suspension was centrifuged and residue washed J times nth M/15 buffer, pH 7.0 (x 6,.500). Fig. 10 The procedure used was same as above, except that RNA-ase (0.0.5 mg per ml of suspension) was added with'trypsin (x 13.500). Fig. 11 The proeed.ur~ used was same as above, except that only ficin (2 mg per ml of suspension} was used .. (x 9,000).

PAGE 57

52 PLATE 4 Fig 9 F ig. 10 Fig 11 _j

PAGE 58

53 The 'Satne, kind of results was obtained with .f'ioin, alone or in' combination with RNA-ase (Figure 11); and other enzymes~ .~ an a~tempt .to improve the clennliness 9f the wall preparations, the oonventiOnal method of breaking the cells by vigoroua shaking with. glass beads it;1 a Mickel. tissue disintegrator .. was employed~ Hel'e also a variety Of. conditions t-rere ,tried. For $Uspending the cells during disintegration, .3 media were tried. ,i ,' These were ASW, l 1 NaCl, and 0,5 M K-phe>sphate buffer, pH 7,0. Washi11g procedures tried involved l M NaCl, M/15 K-phosphate buffer, pH 7 .o, and distilled water. Along with these, various enzymes t were tried Wlder the conditions described earlier~ Equal volumes of a heavy cell suspension and 0 Ballotini 0 glas~ beads (0.007 m diameter) wore shaken in the Mickel tissue dis~tegrator ror 20 min. The length of time used was arbitrary but later results shmred. it to be satisfactory.. The incranoed viscosity was reduced by the addition of a trace of DHA-ase and, after removsl of' tl+ebeads 1 .the 8Uspension was centrifured $t 1,100 x g for 10 111in. The supex:nate was then centrifuged a:t high Sl'f:!led and the residue :was subjected to various treatments. Dis int,egration. in sea water euspension f'ollowed by 4 washings With l M ,~aCl ,solution gave a heavily contaminated preparation. (Figure 12). Similar results were obtained when l M NaCl or 0.5 M buffer was used as the suspending mediui and 1. M NaCl solution was used for washing (Figure 13). However, batter results :were. obtained.

PAGE 59

when the suspending medium was 0.5 M buffer and the cell walls were washed with M/15 buffer (Figure 14). The cell walls were stll contaminated but there was practically no fragmentation. The cell walls, obtained after this treatment, were used for further trials. Washing with water (Figure 1.5) and the use of various enz,ymes did not remove the cytoplasnrl.c materiD.l. With RNA-ase and trypoin, there was considerable fragmentatj_on of the cell walls as was ob served earlier (Figi.tre 16). Ficin, alone or in conbination with RNA-ase, gave similar results (Figure 17), as did pepsin (Fizure 18). Since the results obtained by disintecration in the presence of 0 .5 1-1 buffer followed by washing with :.1/15 buffer were the best so far, further washing of the cell walls with the dilute buffer was tried. A gradual decrease L'1. tho amount of cytoplasmic con tainination was observed and, after 10 washings, the suspension was found to contain cell walls uhich were practically free from cytoplasmic naterial (Figure 19). The preparation was considered to be satisfactory for chemical analysis, despite some fragmen tation of the cell walls. 'I:he same procedure was then used for preparing the cell walls of H.B. 65 and H.B. 9/3 and the appearance of the preparations obtained is shovm in Figures 20 and 21 respectively. The cell walls of .f aeruginosa were prepared as described in Materials and Hethodo. Clean cell walls were obtained tn the first trial (Figure 22).

PAGE 60

Plate 5 Electron .micrographs o.r eell walls of M.B. 29 pre ... pared by mechanical disintegration and washing with various procedures. Fig. 12 Cells suspended in ASW and disrupted in Mickel tissue disintegrator; coarse debris was removed by centrifugation:at 1,100 x g for 10 nrl.n. Cell walls were washed 4 times with 1 M NaCl and 3 times 'tttith distilled water (x 7,500). Fig. 13 Procedure used was the same as used above, except that cells weresuspended in 0.5 Mbu:f'f'er, pH 7.0, during disruption (x 7,500). Fig. 14 Procedure used was aame as for Fig. 1'.3, except that H/15 buffer, pH 7.0, 't>tas used for washing in place of !faCl solution and dist,illed water (x 4,500). Fig. 15 Procedure used was.same as for Fig, 14, except that distilled water was used after washing with M/15 bufi'er (x 14,500}.

PAGE 61

1-~ I .56 PLATE .5 Fig 12 F ig 13 Fig 14 Fig 15

PAGE 62

Plate 6 Electron mi.orographs 0 cell walls of M.B. 29 ob tained by mechanical disintegration arid action of vm-ious enzymes. Fig. 16 Cells were suspended in 0.5 M buffer pH 7.0, and disrupted in t-!ickel tissue disintegrator. Coarse debris was removed by centrifugation at 1,100 x t ror 10 min; residue from supernate was suspend
PAGE 63

5 8 PLATE 6 F ig 16 Fig 17 Fig 18

PAGE 64

Pl.ate 7 Eleetron micrographs of.cell ua.lls of M.B. 29, M.B, 6,5, H.B. 98, and f.aeruginosa. Fig. 19 Cell walls of M.B. 29 (x 4,500). Fig., 20 Cell mills of H.B. 65 {x 5,500). Fig. 21 Cell walls of 11.B. 93,(x 4,500). Fig. 22 .Cell walls off. ~~rug!nos~ (x 11,500}.

PAGE 65

60 PLATE 7 F ig. 1 9 Fig 20 F ig. 21 F ig. 22

PAGE 66

61 It ~.ight be emphasized here that the method finally adopted for preparing the cell walls of the marine bacteria and f aeruginosa did not involve the use of any enzyme, except DNA-ase which was used to reduce the viscosity produced during the breakage or the cells of the marine bacteria. From the electron microgl"aphs, the walls of .H.B. 29 and H.B. 98 appeared to be thin and fragile in nature as compared to those or M.B. 65 and .E aeruginosa. The cell walls of H.B. 6,5 appeared to be thicker than those of the othertwo marine bacteria but thinner than those of .E aerugino~~ Disaggregation of Isolated Cell Walls.by Sodiu.m-dodecyl-sulfate The extentof disaggregation of the isolated cell walls of the marine bacteria and f. aeruginosa was examined in 0.1 per cent solution of the aniontc detergent. The marine cell walls were dis aggregated to a greater extent than the cell. walls of the nonmarine pseudornonad. Thus, the per cent residual turbidity for H.B. 29 was 15 as compared to 55 per cent for f. aeruginosa (Figure 23). The values for H.B. 65 and H.B. 98 were 10 and 14 per cent respective ly, The greater sensitivity of the cell walls of the marine bacteria to SDS was also evident during the attempts to prepare the cell walls of H.B. 29, when extensive frai,nentation of the walls was ob served by treating the cells with the detergent (Figure 6).

PAGE 67

62 DISAGGREGATION OF CELL WALLS OF M.B. 29 AND P. AERUGINOSA BY SODIUM DODECYL SULFATE 10 0 ., 10 Time (minutes) Figure 23 .E aeruginosa M.B. 29 20

PAGE 68

Action of Phenol on Isolated Cell Walls of M.B. 29 The isolated cell walls of M.B. 29 were shaken with phenol in an attempt to remove the lipoprotein component of the cell walls. It was hoped that an analysis of the insoluble material left behind might indicate the presence of the components detected in the rigid layer of the cell walls of~ ..Q11. On shaking with phenol, the cell walls of M.B. 29 were completely dissolved by the solvent. The material, precipitated after the addition of water, was ana lysed. The data obtained were: protein, 75 per cent, lipid, 18.,5 per cent, hexosamine (as glucosn..une); 2.01 per cent, reducing value (as glucose), 0.97 per cent. This composition of the pre cipitate was found to be very similar to that of the cell walls of M.B. 29. The cell walls of H,B. 65 and M.B. 98 were also dissolved when shaken with phenol. However, the material precipitated after the addition of water was not enough for quantitative studies. Chemical .Analysis of Cell Walls Quantitative data.The values for the quantitative ana lytical data have been expressed. as per cent dry 'lreight of the cell walls. The results are shovn1 in Table 5, The nitrogen contents of the cell walls of the marine bac ... teria ranged from 12.2 to 12.8 per cent as compared to 8.4 per cent

PAGE 69

64 TABLES COMPOSITION OF CELL WALLS OF MARINE BACTERIA AND PSEUDOMONAS AERUGINOSA Chemical Per cent dry weight cell wall constituents M.B. M.B. M.B. P. aeru29 65 98 ginoo'a Nitrogen 12 .5 12.2 12.s 8.4 Phosphorus 0.9 1.2 1.1 1.2 ,Protein 76.0 74.8 76.2 6.5.3 Lipid 19.4 18 .5 18.2 19.3 R$ducing substance 1.8 1.5 1.0 10.0 Hexoarunine** 1.9 1.7 0.9 2.6 ex:pressed as glucose ** ' expressed as glucosamine

PAGE 70

65 for the cell walls of.!! aeruginosa. The protein contents of the marine cell walls were also higher, ranging from 74.8 to 76.2 per cont, in contrast to 65,3 per cent for the walls of 1: aeruginosa. The: lipid contents of the walls varied from 18.2 to 19,1-J. per cent which were about the same found in the walls off aeruginosa. However, in the mnrine bacteria the lipid and protein together comprised. 93 to 96 per cent of the total dr-
PAGE 71

66 TABLE 6 SUBSTMJCES IDENTIFIED IN CELL WALL HYDROLYSATES OF MARINE BACTERIA .ti.ND PSEUD01IDNAS AERUGINOSA Substance M.B. M.B. H,B. P. aeru ... 29 65 98 i1nosa Sugars Glucose + Rhn.mose .. + Glucosamine + + + + Hu.ramie acid* ... + .Amino acids Aspartic acid + + + + Arginine + }~ + + Diaminopimelic acid + + + + Gl.utamic acid + + + + Glycine + + :1+ Serine + + "+ + Proline + + +. Hydro::cyproli.ne + + Leucine -~ + + + Isoleucine + + + + PhenylaJ.anine + + + + Alanine + .+ + + Threonine + + + + Tyrosine + + + .i.. ,, Va.lino !-,+ + + Cysteic a.~id ... + Methionine + + + + . . Identification based on R~ value. ~,

PAGE 72

the size of the spots and the depth of color with ninhydrin gave an approximate idea a.bout the relative concentrations of someor these constituents. On paper chromatograms, only hexosa."lline was detected in the walls of the marine bacteria. It gave a positive test with the Elson and Morgan spray reagent and was identified as glucosamine from its Rg* value(Smith, 1960). Glucose and rhrunnose were found in the walls of 1: aeruginosa. The spot of rhamnose was quite weak as compared to that of glucose. The presence of two hexosamines was also detected; their spots were close to each other and the one with the Rg value of 64.2 was identified as glucosamine, The lower spot, with the Rg value of 61, did not correspond with any of the known amino sugars that were used as references. From its Rg value, it was believed to be muramic acid (Smith, 1960). This substance was not available for direct comparison. The hydrolysis of the walls appeared to be complete since discrete spots were obtained by spraying with ninhydrin. However, poor separation of methionine and valine, and of leucine, iso leucinet and phenylalanine wasobtained. The extent of migration of these two groups of amino acids is nearly the same in various *n = distance substance travels from origin x 100 g distance glucose travels from origin

PAGE 73

68 solvents. The spots of these amino acids, obtained with the un .. knowns, were roughly comparable in size and shape to those found on the chromatograms of known amino acids. Most of the amino acids, generally found in the hydrolysates of proteins, were detected in the cell walls of the organisms studied (Plate 8). Of the sulfur-containing amino acids, cysteic acid and methionine were found in .E aeruginosa; methionine was also detected in the walls of the marine bacteria. Proline and hydroxyproline were both present in H.B. 98; the former was. absent in M.B. 29 and the latter in H.B. 65 and f aeruginosa. Diamino pimelic acid was detected in the walls of all the organisms studied. Spots identified as due to glucosamine were detected in the case of M.B. 65, H.B. 98, and!: aeruginosa but not in M.B. 29, al though it was found in the latter during sugar chromatography. From the aiz~ of the spots and the depth of color with ninhydrin, arginine, lysine, alanine, aspartic acid, and gluta.mic acid appeared to be present in greater concentrations than the rest of the amino acids; this was found to be true in the case of all the cell wall hyd.rolysates examined.

PAGE 74

Plate 8 Chromatograms or ninhydrin positive substances in cell wall hydrolysates or marine bacteria and f, ae:ru.ginosa. *dimninopim.elic acid was identified by one di mensional chromatography in each solvent system.

PAGE 75

70 LATE 8 ("l.1 5 9 16 s o 00 0 111 s O :, 0 D~ .. (],' M,B, 29 lOD t7c:::) 18 150 19 0 16 M B, 98 10 0 17 c;;:::>1 9 q ~ known ino acids ~ 0 ll 10 0 ~ 18 1 (..--/ o. ,o Q t .a. 6.5 10 0 -c;:) 6 q a1, l = alanine 2 upart.ic acid :, = cysteic acid 11 = diaminop lie acid 5 = glucos 6 = lutaaic c 1 7 glycine aerin 9 hydroxyproUn 10 prol1n 11 ar 1n1ne 12 lyain 1:, t.hr o in 14 t.yroain 15 t.hio in 16 valine 17 ph n7lalanine 1 l cine 19 aoleuc:ine I UT OL t .ACETIC ACID t AT

PAGE 76

DISCUSSION The marine bacteria examined in this study can be tenta tively placed in the family Pseud.omonadaceae on the basis of the properties studied. These organisms occur both as straight and curved rods but, as noted by Hayes and Burkholder (Bergey's }Ia.nual, 1957, P 90), the borderline between the straight rods found in Pseudornonas and curved. rods found in Vibrio is not sharp. According to Shewan, Hodgkiss, and Liston (1954), a sharper differ entiation between pseudomonads and vibrios can be made by the use of a vibriostatic agent reported to be a specific inhibitor of vibrios. Since H.B. 29 and H.B. 98 were insensitive to this agent as well as penicillin, were polarly flagellated, and metabolised carbohydrates oxidatively, they couid be placed in the genus Pseudomona.s. If the salt requirements of the genus Halobacterium were less rigidly defined, H.B. 65 could be placed in this genus on the basis of production of a golden-yellow pigment (presumably carotenoid), its inability to produce acid from carbohydrates, and its pleomorphic nature. This genus includes species re quiring at least 12 per cent salt for growth, a property not possessed by M.B. 6.5. The genus Pseudornonas has been placed in 71

PAGE 77

72 the family Pseudomonadaoeae together with bacteria which "attack glucose and other sugars either oxidatively or ferrnentatively. 11 However, the Hanual includes in this genus species (Pseudomonas gelatica, Pseudomonas nigrifaciens) which are without apparent action on sugars. With this precedent, and the fact that this genus includes some non-motile species, I1.B. 65 could also be placed in this genus. These facts exemplify difficulties caused by delineating genera on the basis of a single physico-chemical property. In some marine bacteria, the presence of a cell wall does not offer protection against osmotic lysis (Boring, 1961). If the osmotic fragility of these bacteria is due to the chemical composition of the walls (Brown, 1960), then it was considered possible that some relationship between the degree of osmotic fra gility and wall composition might exist. The marine bacteria, selected for this study, differed in lytic susceptibility; such differences have been reported during studies on these bacteria ('I'yler et al., 1960; Macleod and Matula, 1962). The difficulties encountered during the attempts to prepare clean cell walls of the marine bacteria were two-fold; the fragmen tation of the cell walls which was observed during the various washing and enzymatic treatments, and the failure to remove the cytoplasmic material from the walls. The cell walls of Gram negative bacteria are generally less amenable to various washing

PAGE 78

73 methods which usually succeed in the case of Gram-positive bacteria. In the present study, methods which have been used for the prepa ration of cell walls of other Gram-negative bacteria were found to be unsuitable. Washing with NaCl and the use of various enzymes resulted in the fragmentation of the walls without the removal of the cytoplasmic material. The fragile nature of the cell walls was evident from these observations. The'proteolytic enzymes, such as trypsin, generally do not affect the integrity of isolated cell walls although they may re move some surface components such as the M protein in streptococcal cell walls. The fragmentation of the cell walls of M.B. 29 by the action of trypsin and other enzymes may be indicative of a nature different from that of other bacteria. It is possible that the predominantly lipoprotein nature of the cell walls of the marine bacteria, together with other features such as the absence of sugars, renders them susceptible to degradation by such enzymes. The failure of enzymes to remove the adhering cytoplasmic material from the walls could not be explained unless it was as sumed that either the material was somehow inaccessible to the enzymes, or it was of a nature not susceptible to such agents. From the electron micrographs, it was difficult to determine whether the material was trapped inside the walls or attached on the outside. The method finally adopted was the simplest of the various methods tried. It did not involve the use or any enzyme except

PAGE 79

74 DNA-ase which was used to reduce the viscosity produced during the breakage of cells. The method was found to be satisfactory :r or the other two marine bacteria, especially in the case of M,B. 65. Some differences in the appearance of the cell walls of the org~isms were noticed in the electron ndcrographs. The walls of H.B. 65 appeared to be thicker than those of 11.B. 29 and H.B. 98 but thinner than those of E. aeruginosa. The cell walls of the latter two marine organisms were thin and fragile looking. Since H.B. 6.5 was found to be the least susceptible to osmotic lysis, it appeared possible that the thickness of the walls was partly responsible for resistance to lysis. From ultra thin sections of cell walls and whole cells, it has been possible to measure the thickness of the walls of some bacteria, Similar studies on marine bacteria might give some useful information in this regard. rhe analytical data revealed a close resemblance in the gross chemical compositions of the walls of the marine bacteria. Their nitrogen contents were considerably higher than those found in the cell walls off aerur,inosa and other Gram-negative bacteria. However, the cell walls of certain halophilio bacteria have a high nitrogen content (Gibbons et al., 1955), in which respect they resemble the marine bacteria. The lipid and phos phorus contents of the walls of the marine bacteria and .E aeruginosa were comparable to those reported for other Gramnegative bacteria (Salton, 1953). Lipid and protein together comprised

PAGE 80

75 -about 94 to 96 per cent of the dry weights of the walls of the marine bacteria. Similar data on protein contents of walls of other Gram-negative bacteria are not available but, from their reported nitrogen contents, the amount of lipoprotein in their walls should be much like that off aeruginosa (85 per cent). Some minor differences in the amino acid complement of the walls were observed but, these may not be significant. Although no attempt was made to qua:ntitate the amino acids, some of them ap peared to be present in greater amounts than others; these were arginine, lysine, alanine, aspartic acid, and glutamic acid. :fu.ramic acid, a characteristic component of the wall mucopeptidet was not detected in the walls of the marine bacteria though it was found in those of!: ~erup.nosa, It is possible that it was present in amounts too small to be detected by the methods used in this study. The formation of spheroplasts from marine bacteria (Boring, 1961) by the action of penicillin on whole cells points tonards the. existence of a penicillin-sensitive component in the cell walls. The effect of penicillin, in the case of the cells of~ <214., is believed. to involve an inhibition of the synthesis of the muco peptide and its incorporation into the walls of the bacterium (Park and Strominger, 1957; Weidel &, 1960). By this analogy, presence of a roucocornple..~ in the walls of the marine bacteria is indicated.. Horeover, the detection of' diaminopimelic acid (DAP)

PAGE 81

76 in the marine cell walls indicated the presence of such a complex since this amino acid can be considered as an indicator for the presence or absence of the R-layer links (Weidel et a.1~, 1960). -The cell walls of the marine bacteria were .found to be solu ble in phenol and they were extensively disacgregated on treatment with sodium-dodecyl-sulfate (SDS). Both these chemicals were used by Weidel .fil. l!l (1960) for the isolation of the R-layer of the cell walls off coli. In a paper published after the conclusion of this study, Weidel, Frank, and Leutgeb (1963) have pointed out that autolytic enzymes can damage the mucopeptide layer (R-layer) if suitable precautions against their action are not taken during the preparation of cell walls. They showed that the cell walls of Salmonella gallinanu.~, prepared by using SDS, contained. the mucopeptide layer. However, if the walls were prepared by disruption of cells in the Hickel tissue disintegra.tor followci by washing they were found to be defonr1ed. indicating d,am.age to the R-l2yer .. They were also e:>..-tensively disaggregated -:when treated with SDS. The use of SDS during the preparation of the walls appeared to have inactivated the autolytic enzymes. It is possible that such enzymes could have damaged the walls of the marine bacteria during their preparation resulting in their solubilisation in phenol and ex tensive disaegrega.tion by SDS .An unusual feature found in the walls of the marine bacteria was the absence of sugars; only glucosmnine was found to be present.

PAGE 82

77 The absence of sugars was also evident from the low reducing values which were almost the same as the hexosamine values, the former values being a reflection of the hexosamine contents of the cell walls. A similar absence of sugars has been reported or the cell walls of three halophilic bacteria, Vibrio costicolus, Miorococous halodenitrificans and Pseudomnas salinaria (Gibbons ,!!:. !!, 19.5.5). However, in the cell walls of another marine bacterium., the only one previously analysed, glucose and a heptose have been reported (Brown, 1960). Evidently, the marine bacteria var:, in this regard. The cell walls off aaruginosa were found to contain glucose and rhamnose; similar sugars have been found in the cell walls or other Gram-negative bacteria, The cell walls or the marine bacteria were found to contain a lower amount of hexosand.ne than that found in the walls off aeruginosa and that reported or the walls of other Gram-negative bacteria. In this respect, the observations were similar to Browns findings ( 1960) From the data, some relationship was indicated between the hexosamine contents of the walls and their osmotic fragility, although a perfect correlation was not obtained in the case df all the three marine bacteria. M.B. 98, the most osmotically fr'):1,le of the three, contained the lowest amount (0.9 per cent) of hexosamine in its walls; M~B. 65 and M.B. 29 contained 1.7 and 1.9 per cent re ... spectively. or these two, M.B. 6,5 was the most resistant to osmotic lysith

PAGE 83

78 However, if these data are considered from another angle, a more definite relationship is indicated. As mentioned earlier, the electron micrographs 0 the cell walls indicated that the cell walls or M.B. 6.5 appeared to be heavier looking, with more body to them, than those or. M.B. 29 and 11.B. 98 which appeared to be thin and fragile in nature, It is reasonable to aS'sume that the total amount of nexosamine per cell wall would be greater in M.B. 6.5 than the other tw bacteria and least in M.B. 98, On this ba.sis 1 the hexosamine content of the cell walls would appear to influence the degree of OS!llOtic fragility. With our present limited et ate of lmowledge regarding the finer details of the wall structure in bacteria, the exact role of, hexosamine in determining the strength of the wall can only be spe
PAGE 84

79 towards the key role of hexosa.mine in the structure or the mucopeptide layel" A small amount of this sugar can at.feet the rigidity since polymers of .such compounds are believed to be responsible for the mechanical strength of walls, The results of this investigation suggested that the weak nature of tho cell walls of the marine. bacteria 'W'as due to a low sugar content. It was considered possible that the a.mount of sugar 1n the wall influenced the degree of osmotic fragility exhibited by these organisms. The eell wall appeared to be composed ot a soft lipoprotein lay-er interspersed With a mucopeptideeontplex, the -whole structure being able to confer a characteristic shape to the cells but not osntotio stability.

PAGE 85

SUMMARY Marine bacteria are osmotically fragile and their suscepti bility to lysis varies from species to species. The cause of this fragility is believed to be the weakness of the cell wall, and the presence of a low amount of a.."'llino sugar in it has been suggested as the cause of this weakness. Three marine bacteria, differing in osmotic fragility, were selected and their cell wall compositions were compared. As these bacteria were suspected to be pseudomonads, the cell walls of Pseudomonas aeruginosa were also studied to provide comparison between marine and non-marine species. The cell walls of the marine bacteria were prepared by shaking the cells, suspended in 0,5 MK-phosphate buffer, with glass beads in a Mickel tissue disintegrator followed by repeated washing with dilute buffer. The cell walls of~aeruginosa were prepared by a similar method but the cell walls were washed with NaCl and distilled water. The electron micrographs showed differences in the appearances of the walls; the wall of the more osmotically fragile marine bacterium appeared to be thinner than those of less fragile forms. The cell walls of the marine bacteria were predominantly com posed of lipoprotein. No sugars, except glucosamine, were detected, the reducing values being low and comparable to the respective 80

PAGE 86

81 hexosamine values. In the marine bacteria, the wall hexosamine content was lower than that of .E aeruginosa and of other Gram-negative, non marine bacteria which have been reported. Some relationship between the hexosamine content of the walls and osmotic fragility was indi cated, although a perfect correlation tvas not obtained. The walls of the marine bacteria were probably composed of soft lipoprotein coat; although muramic acid was not detected, the presence of mucopeptide was indicated by the presence of a penicillin-sensitive component and of diaminopimelic acid. The comp~ete solubility of the walls in phenol suggested that the muco peptide did not form a separate layer but was interspersed throughout the lipoprotein layer. The amount of hexosamine in the walls was related to the osmotic fragility exhibited by these organisms; it is possible that the thinness of the walls also played a role in : this property.

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BIBLIOGRAPHY Abra.ms, A. 1958 0-aoetyl groups in the cell walls of St~epto~ coccus fa.eoalis. J. Biol. Chem., 2,Jq, 949 .. 9.59. Archibald, A. R., Armstrong, J,. J., Baddiley, J., and Hay, J.B. 1961 Teiohoic acids and the stX"llCture of bacterial walls. Nature, 191, 570 ... 572. Armstrong, J. J., Baddiley, J,., and Buchanan, J. a. 1961 Further studies on the teichoic aoid from Bacillus eereus. Biochem. J., ~. 2,54-261. Baddiley, J., Buchanan, J. o., and Carss, B, 1958 The presence or ribitol phosphate in bacterial cell walls. Biochim, et Biophys. Acta, gz, 220. Baddiley, s. Buchanan, J. a. Rajbha.ndhary, U, L., and Sand.er$on, A. R. 19~:h Teichoic acid from the cell walls or Staph:lloeocous aureus H. Biochem. J,, 8S, 4;9-4.51. Baddiley-, '1, Buchanan, J. a., Martin, R. o., and Rajbhandhary, u. L. 1962 Teichoic acid .from the walls of Sta12hy:loeoccus aureus H. Biochem. J , ..2,; 49-55 Baker, z., Harrison, R. w., and Benzamint F . M. 1941 Action 0 synthetio detergents on metaboliSDt of bacteria.. J. Exp, Med, 2J, 249-257 Barkulls, s. s. and Jones, M. F. 19.57 Studies of streptqeocoal walls~ J. Baeteriol., 22, 207-216. Bet'ge;z s Manual g! Determinative BacteriolOQ: 19.57 7th Ed. Fd. by R. s. Breed, E. o. n. Murray, and N. R. Smith. The Williams and Wilkins Co., Baltimore. Bielling, M, c. 1958 Marine bacteria: cla.ssirication of species obtained. from Florida. coastal waters. Thesis, Univ. of Florida, Gainesville, Florida. Birch-Andersen, A. and Ma.aloe, o. 19.5:3 High,resolution micro.. gra~hs of sections of!~. Bioehim. et Biophys. Acta.~. 395 .. 400. Boring, J. R. III, 1961 The osmotic fragility of cells and sphe:roplasts of a marine vibrio+ Thesis, Univ. of Florida, Gainesville, Florida. 82

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8.3 Brown, A. D. 1960 Some properties of a Gram-negative, hetero trophio marine bacterium. J. Gen. Microbiol~, gJ, 471-48,5. Butler, J. A. vH era.thorn, A. R., and Hunter, a. n. 19.58 The site or protein synthesis in Bacillus megaterium. Bioohem. J 22, 544-.5.SJ. Christian, J. H.B. and Ingram, M. 19.59 The freezing points of baQterial cells in relation to halophilism. J. Gen, Miorobiol., ~, 27 ... 31. Cumiil111a 11 C/ s. 19,54 Some observations on the natur1:, of antigens 1n the <)ell wall of Cofrebacterium diphtheria.a.' Bi-it. J. Exptl. Pathol,, 12, 1g -180. . Cummins, :a. s. 19$6 The chemical composition of the baeterlal cell wall. Intern. Rev. Cytol., y, 2.5.50. CUnnnins, c. s. 1962 Chemical composition and antigenic structure or cell :walls of Conffiebacteritll11 I1Y;cobaeterium, Noeardia, Aatino&oes, and Arthrobacter . J. Gen. MiorobioL., &, ;5-50. Cunmiins, c. s. and Harris~ H. ;6a The'chemioal composition of' the cell wall in some Gram-positive bacteria and itspossible value as a taxonomic character. J Oen~ Microbiol., !!!:, .583-600. 'Cummins,' c. s. and Harris, H. 19,56b The relationship between oertain members or the-staphylococous .. microeoacua'group as shown by their cell wall composition. Intern.. Bull .. baot. Nomen. tmd Taxon., ,, 111 ... 123. : Cu.mmins, c. s. and Harris, H. 1958 Studies on the cell'wall co~sition and taxonomy of Aatinomyoetales and. related groups. J. Gen. }ficrobiol.; is. 173.188. ...... ... Dawson, I. M. 1949 In The. Nature 0 the Bacterial. Surface. First Symposium, So
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84 Fiske, c. and Subbarow, Y. 192.5 The colorimetric determination of phosphorus. J. Biol. Chem., &s, :,75 ... 400. Foster, J. w., Cowan, R. :M., and Maag, T. A. 1962 Rupture of bacteria by explosive deooupression. J. Bacteriol., J, :;.30 ... :3,z.. Freim.er, E. H., Krause, n. M., and McCarty, M, 19.59 Studies or t forms and protopla$tS or group A streptococci. 1. Isolation, gl:'Owth, and bacteriological characteristics, J Exp. Med., !!Q., a.5:,-874. Gerhardt., P. 19.59 The protoplast membrane or bacteria . Univ. Mi.ch~, Med"' Bull,, &.2, 148-1.59 . : . Ohuy:aenl J. M. 1961 Precisions sur la. structure des complexes disac~haride-:peptide libres des paroia,de Microcoecus ,l:ys~ deikticus sana 1 action des beta~(1-4)-N .. aoetylhexo~idase. Bioohim. et Biophys. Acta, !!;2, 561 .. .568. . Qhuysen, J. M. and Salton, M. n." J,, 1960 ketyihexosandne compound enzymically released trom Mi.crocoecua l;y!eikticus cell walls . 1, Isolation and. composition of a.oetylhexo~amine ... peptide .complexes. Biochitn. et Biophys. Acta, !!Q., 462-472. Gibboni:1, N. E., Smithies, w. n., and Bayley, s. T. 19.5.5 Chemical composition or cells and cell walls or some halophllic bacteria. Can,. J, Microbiol., !, 605.613. Harvey, E. N. 1915 Studies on light prod.uoticm by l'UI!linous bacteria. Am. J. Physiol., JZ, 2.:30 .. 239. Hill,. s. E . 1929 The penetration of li.Llllinous baoter:l.a by ammonium salts of lower ratty aoids . J, Gen, I->hysiol.~ 12, 86,3-872 _., ',' ,. '~' Hoare, D. S~ and Work, E. 1957' The stereoisomers of dia.minopimelic aeid, 2. . Their distribution .in the bacterial. ordev Aotino!4.'leetales and in certain Eubaeteriales. Biochem. J. 22 441-447. ' Holdsworth, E. s. 1952 The nature 0 the cell wall.ot Co!"J'lle baaterium di;ehtheria~. Isolation of en ollgosaeeharide. Biochim. et Biopbys. Acta, 2, 19-28. Holme,. T., Malro.borg A. s., and Cota-Robles, E. 1960 Antigens of spheroplast membrmie propax-a.tions from~st
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85 Houwink, A. L. 19.56 Flagella, gas vacuoles, and cell wall. atrueture in Halobaotsrium. halobium; an electron microscope study, J. Gen, Miorobiol,, lli 14.5 ... 1.50, Hugh* R. and Leifson, E, 1953 The taxonomic significance 0 ternientative ve:rsus oxidative metaboli~ qarbohydrates by various Gl'am-negative bacteria. J Baoteriol,, ,22, 24-26, Hunt; A, L.,Rodgers, A., and Hughes, D, E, 19.59 Subcellular p~ioles and the nicotinic a.oid hydroxylase system in extracts ot Paeti.donlOnas i'luoresoens KB 1. Bioahim, et Biophy~, Acta, l!;, 3.54-;72. . Iko.wa, i,I. and Snell, E,. E. 1956 D .. glutrunic acid and amino sugal"S as cell wall constituents in lactic acid bacteria, Bioqhm. et Biophys. Acta, !2, 576-578, Janc~a, E., Perlo..ns, H. n.,. and Rodgers, H, J~ 1960 Isolation 0 a mucopolysaooharide t'ron1 cell wa.11 prepal"ations of Bacillus subtills. Bioohom. J., .z!!:, 7P Janozura. E., Perkins, H. R.; and Rodgers, H, J, 1961 'l'eichu ... ronic.aoid: a mucopolysaecharide present in wall prep~ations from ve.getativecells of Bacillus subti.lis. Biochem. J,, ~,.82"!'93,. ,\ ,1 Johnson, F" H. and Harvey, E. N. 1937 The osmotic prope:rt~e5iof marine luminous bacteria. J. Cellular Comp. Physiol,, .2., Jo:3-379~ I > ? ,t Jolmson, .F, H. and. Harve-.r, E11 N.: 1938 Baoterial lwni.neacenee, respiration, and viabUity in :relation t:o oamotic pressure Md $pec1f1e salts of sea water. J, CeUulDJ:' Comp~ Physiol,., .:.12, 213-2:;1. Johnt1011, F. H.' Zworykin, N.... and ,Warren, (},. 194,3 A study or lwnino'U.$ bacterial eellsand cytolysates with the electron micro. scope. J,. Bacteriol., ~' 167-185.. Kabat, E. A. and Mayer, M,. .M. 1948 Experimental Imnn.inochemist;ry, Springfield., Illinois: Charles C:. Thomas. Kellenberger, E. and Ryter, 19.58 Cell.wall and cytoplasmic membrane of ! coli. Ji. Biophya~ Biochem~ Cyi;,Ql, .!, 32:3,.:326,. l King, R. K. and Alexander, H. 1948 The mechanical de$.truction of bne:t~ria. J. Gan. Mierobiol .. ;';15-324.

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86 Knox, K. w. and Bandesen, J. w. 1962 The isolation of components from cell.wall of' La.etobacillus casei. Biochem. J ., 1~-23. Korr, I. M. 193.5 The relation between cell integrity and bacterial. luminescence. Biol. Bull., ~. 374-354. Kovac, M. 1956 The id.entif'ication of Pseudomona.s J?Z09W1ea by the oxidase reaction. Nature, 118; 70J. Krause, R. H. and HcCar,ty, M. 1961 Studies on the oha"l'lical structure of the streptococcal cell wall, 1. The identli'ieation 0 a muoopeptide 1n the cell walls of group A and A-variant streptococci. J Exp-. Med,., ill, 1.2?-140. wminaky, I. and Lendrum, A. c. 1942 The ef'!eet ot :aur.face ... active agents on~ proteus. J~ Pathol. Baoteriol., ,!!:, 421-4,o. lowry, O. H., Rosebrough, N. R,., Fan-, A t., and Randall, n. J, 19.51 Protein measurement with the Folin phenol reagent. J. Biol. ~hem., .!2J, 265-27.5 MaoFa.dyen, A. and Rowland s, 1901 Upon the intracellular constituents of the typhoid bacillus. Zbl. Bakt. Abt,, l, _JQ, 48. M.acteod, R. A. and 1-Jatula., T, I. 1962 Nutrition nnd metabolism. or marine bacter10.. XI. Some characteristics of the lytic phenomenon.. Can. J. Microbiol., .t 883 .. 896. . Mandelstam, J . and. Rodgex-s, H. J. 1959 The incorporation ot amino acids.into cell wru.1 mucopoptide or staphylococci and the effect or antibiotics on the process. Biochem. J., zg, 654.;,.662. Ms:rr, A. o. 1960 .Localisation of enzymes in bacteria. In The Bacteria, vol~ I, PP 443-468 . Ed.' I. C. Gunsa.lus and R. Y. stonier. New York: Academic Press Inc. Marr, A. G,. and Cota.-1\obles, E. H. 19.57 Sonic disruption of Azotobaeter vinelandii~ J. Bacteriol. 2!, 79 .. 86. Mickel, m . 1948. Tissue disintegration~ J. Roy* Microaeop. Soo., 68, 10. ...... ~ Mitchell, P. and J1oyle, J . 19.51 The glycerophosphoprotein compl~ envelop ()fMicroeopcus pyogenes. J. Gen. Microbiol 2t 981 .. 992.

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87 Mitchell, p. a.nd Moyle, J. 1956 Osmotic function and structure in bacteria. In Bacterial klatomy, Suth Symposium of the Soo .. Oen. Mierobiol., Cambri.dgc Univ. Press, Great Britain. Norris, J., R. 195? A bacteriol.ytio principle associated W'ith cultures ot Bacillus eereua. J. Gen. Mio:robiol,, !Q., 1 ... 8. Park, J .. T. 19,58 In The Strategy of Chemotperapy, PP 49. Cambridge Univ. Press, London and Ne'ir York. Park, J. T. and Strominger, J. t. 19.57 Mode oi' action of penicillin. Science, 12,2, 99-101. Partridge, s. M. 1949 Aniline-bydrogen ... phthalate as -a spraying reagent fo~ ehroI11.atography of sugars. Nature. ~. 44). Part.ridge, s. M. and Westall, R. 1948 Filter paper ~ition chromatography of sugars. Biochem. J., fr&., 238-250. Primosigh, J., Pelzer, H., Maass, D., and Weidel, w. 1961 Chemical characterisation or muaopeptides released from-. the cell wall of .E. coli by enzymie reaction. Biochim. et Biophys~ Acta., !I:., 68-81. . Rhuland, L. E. 1960 Diaminopimclic a.cid: its distribution, synthe sis, and .metabolism. Nature, ,185, 224-225. Richmond, M. H. and Perkins, H .n. 1960 Possible precursors f'or the synthesis of mu.ramie acid by St~:i:ph:yloaoc~ au.reus . Bioehem. J . 1.., 1p. R1.ley, w . u. .1955 .A study ot factors causing lytd.s -or a mnrine bacterium . Thesis, Univ. or Florida, G:,.inesville., Florida. SaJ.tont 1,1. R. J. 1953 Cell structure and enzymic lysis of bacteria. J" Gen. m.crobiol.; 2, ,512-.52J. Salton, !1. R. J,. 19.56 In Bacterial Anatomy, Smh symposium .of the Soc. Gen. f'.d.cl'Qbiol., PP 81..;~10, Cambridge Univ. Press, tor.ldon .a:nd New York. Salton, u. R. J, 1957 Cell wall ruiu.no aoids and amino $ugars. Na't;,ure, 1.Q., ,>3 .. :339. Salton~ M. R. J. 19.58 The lysis of microorga.rusms b-.r lysozyrae and related enzYI!les. J. Gen. Hicrobiol., 1., 481-490. Salton; M. R. J . 1961a The anatomy of' bacterial surface. Baoteriol. Rev., ~, 77 .. 99.

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88 Salton, M. R. J. 1961b In Microbial Q.fil Walls. John Wiley and Co., New York. Salton. : M. R. J. 1962 Cell, wall structure and synthesis. J. Gen. M:1.crobiol., 29, 1; .. 2:3, ' Salton, M . R. J,. and Horne, R. W, 1951a Electron microscopical observations on heated bacteria. Bioehim at Biophys, Acta~ Z, 19-42. Salton, M., R. J. and Horne, R. w . 1951b Methods or preparation and some properties or cell Wa.Uih Bioehini. et Biophys Acta, 2, 177-197 Salton, M,. ~ ,J. and WU~ams, R. c. 19.54 Electron microscopy or the .cell 'walls of Bacillus megaterium and Rhodospirillum rubrum, Bioehim, et Biophys. Acta, 1-!f, 455 ... 458. Schulman, J . H., Pethica, B. A., .Few, A. V , and Salton, M. R. J. 1955 In Progress Biophysics. Fd. J. A. V. Butler and J. R.' Randall. Pergamon Press, wndon. Shara, F, and Salton, M. R. J. 19.58 Some change, in the surf aoe structure of'Grom-negative bacteria induced by penicillin action. Nature, }.81,. 1321-1:,zlh Sha.fa,, F~ and Salton, M. R. J. 1960 Disaggregation or bacterial cell. walls by anionic detergents. J. Gen. Microbiol., ,l, 1:37""141. Shew-an, J. M., Hodgk1$e, w. and Liston, J. 19.54 A method for the rapid di:t"f'erentiation ot certain non-pathogenic, a.sporogenous bacilli . Nature, lZ,l, 208.:.209. , . Shoakman, G. D'., Kolb, a. J., and Toennies, a. 19,58 Relation between'.ba.cterial ce wall synthesis, growth ph~e, and,a.utolysis. J, Biol. Chem., 230, 961-977. '' Slade, H, D. and Vetter, J. K.' i956 : Stud.i9S on Stre;gtoooccus pyogenes. J . Bacterial Z!, 2J~r24J. . Slade, H, .D . and Sl~, w., c. 1962 Cell wall compositi~n W'ld the grouping of antigens of. streptococci. .J. Be.cteriol., .!!., ;45-3.51. ' '' Smith, I .. '1960, C~omatogrtip~ic and. Electrophoritic Techniques.' Ed. Ivor Smith, vol. 1, pi: 24,. Snell, ,E. E., Radin, u. s., and llcawa, M. 19.55 The nature or D-a.1anine in laotic acid bacteria. J. Biol. Chem., 217, 803-818.

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89 Strange, R. E. and Dark, F. A . 19.56 An unidentified amino sugar present 1h cell walls and apor8~ oi' various bacteria. Nature m. 1a6..;1as. Strange, R . E~ and Kent, L. H. 19.59 The isolation, characterisation, l:lbd chemical synthesis or murano.c acid. Biochem. J., ,Z!, 333 .. 339. '' ' . Streitfela, M, M., Hoffman, E~.M~,and.JanklOl-t, H . M. 1962 Evaluation orextracellul~ deoxyribonuolease activity in Psetidomonas, J. Bacteriol., 77 .. 79.,, ~oerlnies, o. and Shockman, o. n. 1959 Growth ohemS.stry of' Streptococc~s. i'aeoalis. 4th, In~ern. Congr. BiochEllm,, ,llll, jt;.5~393. Tyler,M.E.~ Bielling, M., and Pratt, n. B. 1960. Mineral re quirements.and other characters or selected marine bacteria. J. Gen~ Microbio1., ~, 15;-i61. Vennes, J., ~1. and Gerhardt, p . '19.59 .Antigenic analysis .or cell struotures isolated from Bacillus megaterium. J, Baoteriol. ZZ.r S~1-~92! , Weidel, w. 19.51 Uber die Zellmembran von E. coli B. z. Naturi"orsch., '.2Ja, 251.2.59~ . . Weidel, W., }frank, H., and ?:tart.in, H,. H. 1960 The rigid layer of .the'oeU,uallof E! coli. J! Gen. Miel"Obiol.,g&, 158~166. Weidel~ w., Fr~k, H., and, teuigeb, w. 1963 Autolytie em~ymes as _l:\source or error .in the preparation and study of Gram--negative oell tfttlls~ J~ oen. M1ci'obiol., JQ, 127-1JO. . Work, 'E. '19.51 .The isolation of diaminopi.Iri.elic acid from Co;r:,me ba.cterium ~iphtheriae and ~lvcobacterium tuberculosis. Biochem. J., !, ~7-2J. Work, E. 19.57 Biochemistry or the cell wall. Nature; .!12, 841-847. Work, E. 1961 The mucopeptide of bacterial cell. walls. .J. Gen. Microbi:ol., i, 167-189,' . Zillikin, F. 19.59 Chemistry of bacterial cell walls. Fed. Proc., 18, 966 .. 973.

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BIOGRAPHICAL ITEMS Inder J. Sud was born in Agra. India, on January 22. 1927 He graduated from st. John High School, Agra, 1n April, 1943, He attended the Agra University tor six years, reo1ev1ng the degree of Bachelor of Science 1n November, 1947, and the degree or Master or Science in November, 1949, He served as a teacher in Agra College until August, 1959, when he joined the University or Florida. He is now a candidate or the degree of Doctor of Philosophy. Inder J. Sud is married to Asha Sud and has two sons. He is a member of Sigma Xi.

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This disse%'tat1on vas prepared under the direction or the candidate's supervisory committee and bas been approved by all members of that committee. It was submitted to the Dean of the College or Agriculture and to the Graduate Council, and was approved a.s partial i'ulf'illm.ent of the requirements or the degree of Doctor of Philosophy. April 20, 1963 Dean, College of Agricul,;ure Dean, Graduate School