• TABLE OF CONTENTS
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 Title Page
 Acknowledgement
 Table of Contents
 List of Tables
 List of Figures
 Introduction
 Literature review
 Materials and methods
 Experimental results
 Discussion
 Summary
 Bibliography
 Biographical sketch
 Copyright














Title: Marine bacteria: wall composition and osmotic fragility
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Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
    List of Tables
        Page iv
    List of Figures
        Page v
    Introduction
        Page 1
        Page 2
        Page 3
    Literature review
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
    Materials and methods
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
        Page 33
        Page 34
    Experimental results
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
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        Page 49
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        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
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        Page 60
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        Page 62
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
        Page 69
        Page 70
    Discussion
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
    Summary
        Page 80
        Page 81
    Bibliography
        Page 82
        Page 83
        Page 84
        Page 85
        Page 86
        Page 87
        Page 88
        Page 89
    Biographical sketch
        Page 90
        Page 91
    Copyright
        Copyright
Full Text











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




















ACKIO;'LEDGrIENT


The author wishes to express his appreciation to
Drv Max E. Tyler and Dr, Darrell B. Pratt for the guidance
during this study. This work 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 Medical Science
Division, Public Health Service*















TABLE OF CONTENTS


Page
LIST OF TABLES......................,a................iv

LIST OF PLATES AND FIGURES...... .................... v

IITTRODUCTIOiJ...........,i**....(...................,..., 1

LITERATURE REVIEW.*..............,.................. 4

MATERIALS AND METHODS*........... ..............,.........21

EXPERIMENTAL RESULTSe..................., ........... 35

DISUCUSSION* *..*.....**....*.*....... .................,..71

SU-MARtY. ......... ...... ......... ........ ....... .....80

BIBLIOGRAPHY.. *....,................... ............,82


iii













LIST OF TABLES


TABLE Page

1. CARBOHYDRATE METABOLISM OF THE I-IWAIIIE BACTERIA ....... 41

2. PHYSIOLOGICAL CHARACTERISTICS OF
THE MARINE BACTERIA 4,.............,..* ,..... 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













LIST OF PLATES AND FIGURES


Page
PLATE 1. Phase contrast micrographs of M: B. 29,
M. B. 65, and M4.B. 98,..........,,.,.,,,,,, 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
losing 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 M. B. 29,
M. B. 65, M. B. 98, and P. aerurinosa .......,. 60

FIGURE 23. Disaggregation of cell walls of M. B, 29
and P. aerudinosa by sodium dodecyl sulfate .*.. 62

PLATE 8. Chromaatograns of ninhydrin positive substances
in cell wall hydrolysates of marine bacteria
and P. aeruginosa ......,.....*........,..... 70














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.g. 20 to 25 atmospheres in Staphylococcus aureus (Mitchell

and Moyle, 1956). In contrast, this pressure in Gram-negative organ-

isms is low, between 2 and 3 atmospheres as in Escherichia coli

(Mitchell and Moyle, 1956), but it has been shown that these cells can

be grown 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 et al., 1943). The oc-

currence 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 Harvey, 1937; Johnson, Zworykin, and Warren, 1943) and the lysis











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 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 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 fra-

gility (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 Hartin, 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.











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, M.B. 65 was the least and M.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 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-sulfate. No sugars, except glucosamine, were de-

tected; the hexosamine values were low as compared to those of P.

aeruginosa and other Gram-negative bacteria.

The data suggested that the hexosamine content of the cell

wall influenced the degree of osmotic fragility. From the electron

micrographs, it was considered possible that thinness of the wall

was partly responsible for the weak nature of the cell walls of

the marine bacteria.














LITERATURE REVIEW


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 en-

zymatic activities associated with the "envelope" preparations of

such bacteria have appeared (4Marr, 1960; Hunt, Rodgers, and Hughes,

1959; Salton, 1961a).











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 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. 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. 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.










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 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 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, MacFadyen and Rowland had

used agitation with fine sand for disrupting the typhoid bacillus.

::ing and Ale:xnder (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










cell walls. He used the Mickel tissue disintegrator (Mickel, 1948)

for providing rapid and vigorous shaking of the cell suspension.

Salton (1956) and Cummins 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 obtain-

ing 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 Class 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 M NaC1 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 Coryne-

bacterium diphtheria. 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 widespread. It is now known that










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 strepto-

coccal 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 Corynebacteriuin (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 nurnbr of low

molecular-weight peptides from isolated cell walls of Lactobacillus
case. Salton has reconiended that the use of enzymes, especially

crude enzymes which may contain wall degrading enzymes as Iall as

other insoluble protein material, should be carefully controlled.

Other methods for obtaining bacterial cell walls are also availa-

ble. Those involving disintegration of cells with sonic and ultra-
sonic vibrations, decompression rupture, and pressure cell disinte-

gration suffer from the disadvantage of fragmentation and solubilisation

of the wall (Slade and Vetter, 1956; Marr and Cota-Robles, 1957).

Foster, Cowan, and Naag (1962) have recently described a device for

rupturing of bacteria, under controlled conditions, by explosive decom-

pression in a closed system. Autolysis and osmotic lysis of whole cells

also yield cell walls; Ueidel (1951) used toluene for autolysine 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











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

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










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 et al. (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 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 Maaloo, 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 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 growing. Most of the information

available concerns the Gram-positive bacteria because of the relative










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 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 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 (1951)

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 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











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 recognized (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 recognized

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 characterized.

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 teichoic acids










of B. subtilis (Armstrong et.al., 1961) and S. aureus 1 (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 N-

acetyl-galactosamine and glucuronic acid (Janczura et al., 1960,

1961).

Diaminopimelic 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 Gram-positive cocci and

lactobacilli (Work, 1951; Hoare and Work, 1957). Huramic 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, 19591,

Richmond and Perkins, 1960). The key role of this amino acid in

the structure of the bacterial wall mucopeptides has been recognized

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; Ghuysen, 1961; Primosigh et

al., 1961).











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 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 extractibility of teichoic acids (Archibald et al.,

1961) and teichuronic acid (Janczura et al., 1961) with trichloro-

acetic acid in cold and the removal of oligosaccharide and poly-

saccharide residues with both picric acid (HIoldsworth, 1952) and

formamide (Krause and McCarty, 1961). In all the cases the wall

polymers have been obtained in solution leaving behind insoluble

rmcopeptide 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 'cCarty, 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

cormplexes 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 E. 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










mucopeptide of similar composition to that found in the cell walls

of i". .r:1-i:ositive bacteria and it is the mucopcptide componeut 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 bacteri-

cidal action of certain surface-active, anionic compounds (Baker

et al., 1941) but there have been reports of killing by high concen-

trations (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 walls forms a network extending across

the multilayered wall rather than a continuous, separate layer

(Shafa and Salton, 1960). 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 muco-

peptide layer.











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 super-

imposed on the basal mucopeptide 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 Marine Bacteria


The internal osmotic pressure of one marine bacterium has

been estimated to be about 20 atmospheres (Mitchell 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 (Mitchell and :loyle, 1956). In Gram-negative bacteria

this pressure is low, between 2 and 3 atriospherc3 (Mitchell and

Moyle, 1956). Their cells, after having been grown in media of

high osmotic pressure, are protected against osmotic shock when

transferred to distilled wator (Doudoroff, 1940). While groirin-











in such media, the cells probably develop a high internal osmotic

pressure (Christian and Ingram, 1959). Thus, the cell walls of

terrestrial bacteria are strong enough to resist large differences

in external and internal osmotic pressures.

earlyy studies on the osmolysis of marine bacteria were carried

out with two luminous species, Photobacterium fisher 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 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. fisher 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 II IaCl, 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 micro-

scopic examination revealed little change in the appearance of

cells which had ceased to give off light in diluted sea water, led

him to sugiest that the cells were surrounded by a rigid envelope










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 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 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 fur-

nished by Johnson et al. (1943). Electron micrographs of marine

cells, 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 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










bacterium by estimating the degree of lysis in a series of graded

concentrations of NaC1. The whole cells were protected from lysis

in 0.06 to 0.08 M iaCI 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 hexosamine 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

(Shafa 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 Matula, 1962). It was hoped that a comparison







20


of the chemical compositions of the cell walls of selected marine

bacteria might indicate some relationship between osmotic fragility

and wall composition.














MATERIALS 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 (ASJ) was used in cultivating the three

marine organisms used in this study. It was composed as follows:

NaCl, 23.5 g; sT 04.7iH20, 6.2 g; MgC1z.6HO1, 5.1 g; KC1, 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. "utriont 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 chro;nous acid so-

lution; subsequent to any use, it was then cleaned with Haemo-sol

(i;einecl:e and Company, Inc.) and rinsed several times with tap










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 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

1MB. 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-AS"W 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 trypticaso-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-ASW broth* For obtaining small quantities of cells,

Erlenmeyer flasks (250 ml) containing 50 ml of the liquid medium











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 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 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

!orpholor.,.- The morphology of the marine organisms was

studied under phase optics and wet mounts of younr broth cultures

were examined for motility under the light microscope. For de-

termining the type of flagellation, the cells were fixed by adding











2 drops of a 1 per cent solution of osmic acid to i ml of a young

broth culture. After standing for 5 min, the cells were cen-

trifuged and resuspnhded 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.

PhysioloMrj. Certain physiological properties of the marine

organisms were c::a-ined. The method of Hugh and Leifson (1953)

was used for determining the type of carbohydrate metabolism. The

medium was slightly modified to suit the organisms; trypticase,

0.2 per cent, was substituted for peptone and ASW for IlaC1. Dupli-

cate 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--rcin,

penicillin, dihydrostreptonycin, triple sulfa, oxytetracycline,

tetracycline, and the vibriostatic agent 0/129 (diamino-di-isopropyl-

pteridine, supplied by Dr. J. TM. 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 C. 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 oxidasee

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 susceptibility,- 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, AS'!, and 0.5 M and 0.05 M

concentrations of KC1, NaCl, IgCC12.6I20, 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 500 mu, 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 (ASW 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. Brinkrman Co., New York),

were shaken for 20 min at maximum oscillation in a Nickel tissue

disintegrator. The disrupted and viscous cell suspension was trans-

ferred to a beaker; the cuvettes were washed with M/15 phosphate

buffer, p:T 7.0, and the washings were added to the beaker. The

contents of the beaker were thoroughly mixed and then left stand-

ing, Lfter addition of a drop of DNA-ase solution in buffer (0.05

mg per ml), for 20 mrin The now watery suspension was then

carefully decanted and the beads were washed 3 times with M/15

buffer, the Tashings 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

contrifugoc at high speel 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 con-

tamination, 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 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 M.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 IUckel 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 of P. aeruginosa have been found to pos-

sess this property (Streitfold et al., 1962).










The suspension was decanted from the glass beads and cen-

trifuged 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 M NaCl solution

and 4 times with distilled water. The complete removal of the

chloride ions was checked by adding a A-10, 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 Microscopy of Cell Walls


The cell wall preparations were mounted on copper grids

previously covered by a collodion film. The preparations were

shadowed with chromium at an angle of 25 degrees and then e::amined

with a Phillips EM-100 electron microscope. The electron microscopy

was performed by Mr. T. Carlisle and Mr. E. J. Jenkins, Physics

Department, University of Florida,


Disaggregation 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 SDS. The contents











were mixed quickly by inverting the tubes and the optical densities

were read at 500 mU, 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 solu-

bilise 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 ad-

ditional 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 de-

canted 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 C.

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 Analysis 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 0.4 ml of 25 per cent (v/v) HI2SO in Kjeldahl

flasks. Digestion was continued until the mixture became brown.

The flasks were then cooled, a drop of 30 per cent H202 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: 1 ml of 6 per cent

sodium citrate, 3.5 ml of i N NaOH, 20 ml of deionised water, and

1 ml of Nessler's reagent. After the last addition, the contents











were mixed quickly and the optical densities were read immediately

at 505 mi 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

H2SO4. The amount of phosphorus was estimated by reference to a

standard curve of 1KH2PO4 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 de-

scribed 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'

HIexosamine*- This was estimated by using the Elson and

N1rgan reaction as described by Kabat and Mayer (1948), Known

quantities of glucosamine hydrochloride were used to prepare a standard

curve and the results were expressed in terms of glucosamine<

Protein, Protein was estimated by the method of Lowry et

ali (1951) using the Folin-Ciocalteu reagent* About 4 mg of cell

walls (dry weight) were suspended in 1 I NaOH and the suspension

was incubated at 37 C overnight A standard curve, using crystalline

bovine albumin, was prepared and used as a reference.

Paper chromatography.- Detection of amino acids and carbo-

hydrates was done by paper chroriatography. .Piatrian no 1 filter

paper was used throughout Glass imisezLu jars were used for ascending

and a chromatocab (Research Equtipcnt Corpo, Oakland, California.)

for descending chromatography.

Amino acids and amino sugars*- Cell walls (20 mg dry weight)

were hydrolysed in 6 ml of 6 N HC.1 in a sealed tube for 16 hr at

120 Ci The hydrolysato ras 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 IHSO4. 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 chro-

matography. The first solvent used was n-butanol:acetic acid:water

(60:15:25, v/v) and the second solvent was phenol:ammonia (1 ml

ammonia 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 developnont.

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

hexosamines, the Elson and Morgan 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 H2SO4 for 2 hr at 100 C. The hydrolysates were

neutralized with a solution of B3.(0O1)2 to pTI 6.5; the precipitate

of 3D304 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 (160:40).

Multiple ascending development was used for obtaining a Creator

separation of sugars.

The reducing sugars were detected by spraying with aniline-

hydrogen-phthalate reagent (Partridge, 1949). Other reagents

such as naphthoresorcinol and phloroclucinol were used for the

detection of kotopentoses (Smith, 1960). The colorinstric 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.














EXPERI I TAL 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, M.B. 29, M.B. 65, and M.B. 98, de-

veloped 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 M.B, 29 and

M.B. 98. An abundant growth was obtained on trypticase-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 1I.8. 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 1 Phase contrast micrographs of M.D. 29, T1.S. 65, and
M.B. 98 (x 2,400).

Fig. 1 I.B. 29
Fig. 2 1.B. 65
Fig. 3 M.B. 98














PLATE 1


Figs 1 Fig, 2


Fig. 3











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 hcrispherical

and less phase-dense areas were observed in the case of 1T.D. 29;

they appeared to be protruding from the cells. Their significance

was not investigated.

The marine bacteria were found to be Gran-negative. Wet

mounts of broth cultures of M.B. 29 and M.B. 98 showed the cells

to be motile and the electron micrographs of their cells, fixed

with osmic acid, showed the flagellation to be polar and mono-

trichous.(Plate 2). Broth and slant cultures of M:.D. 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 metabolism (Table 1), M.B.

29 and M.B. 93 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 with the formation of a blue spot on the filter paper.

The control tests with P. aeruginosa and E. coli were positive
























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).









40a








PLATE 2








i ,.. *
9r .
U.. %r' ~

r, ' '*-





Fig 4











Fig 5












TABLE 1


CARBOHYDRATE METABOLISM OF THE MARINE BACTERIA1


Carbohydrate Open tube Closed tube
Carbohydrate ....... .... .. .. . . ... ....
M.B. M.B, M.B. MB3. M.B. M.B.
29 65 98 29 65 98


Xylose 2 -
Arabinose -
Glucose A A
Mannose -
Galactose A A -
Mannitol A A -
Sorbitol -
Rhamnose -
Sucrose A A -
Lactose A A -
Maltose A A -
Raffinose -




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










and negative respectively. Some other physiological properties

of these marine organisms had been previously examined by Tyler

et al* (1960), Their data, along with the properties examined

above, are given in Table 2.

The sensitivity of those organisms to antibiotics was

studied and the data are given in Table 3. Chloramphenicol and

erythrormycin inhibited all three organisms, whereas triple sulfa

inhibited M.B. 29 and M*B. 98 but not L.B. 65. The latter was

inhibited by chlortetracycline and oxyftetracycline. The vibrio-

static compound 0/129, considered to be a specific inhibitor of

vibrios, inhibited M.B. 65 but not hI.B. 29 and IB. 98


Lytic Properties of Cells


The lytic properties of the marine organisms in various
test solutions were examined by a procedure similar to that used

by Tyler e~ al. (1960). 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 II.B. 65 was more

resistant to lysis than M.B. 29 and M.B. 98. The solutions of

0.05 M K-phosphate buffer were most effective in lysing the cells,

the per cent residual turbidities being 9 per cent and 8 per cent

for H.B. 29 and 1M.B 98 respectively as compared to 65 per cent

for 1*B* 65. In distilled water, I.B, 65 underwent very little







43


TABLE 2


PHYSIOLOGICAL CHARACTERISTICS OF THE :mIRIUE BACTERIA


Property M.B. 29 M.B. 65 M.B. 98

golden-
Pigment cream cream yellow

utility + 2 +

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 MARINE BACTERIA TO AiTIBIOTICS


Zone of Inhibition
Antibiotic

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


Chlortotra-
cycline 5ug ++

Chloramphe-
nicol 5ug ++ ++ ++

Erythro~ycin 2ug --+ +++ +++

Penicillin 2 units -

Dihydrostrep-
tomycin 2ug -

Triple sulpha 50ug ++ +

Oxytetracycline 5ug + -

Tetracycline 5ug -

Vibriostatic
compound 0/129* ++ -


+,++,+++, = relative degree of

= uninhibited.

a saturated solution
in the agar.


inhibition.



was put in a small cavity










TABLE 4

LYTIC PROPERTIES OF MARINE BACTERIA


Per cent residual turbidityt
Test Medium

M.B, 29. MB. 65 M.B. 98

Distilled Water 62 95 36

0.05 1M aCl 73 95 63
0,5 M Nacl 98 95 95
0.05 M KC1 43 90 36
0.5 1 KCI 88 92 77
0.05 1M MIcCl2 112 109 100

0.5 M MgCl2 115 110 102
0.05 M K-phosphate
buffer2 9 65 8
0.5 M K .phosphate
bufr2 91 100 82


IPer cent residual turbidity
'D. in suspending medium i00
0.D. in ASW
pH 7.0











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 sus-

ceptible 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 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 of P. 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











different washing and enzymatic treatments. The cells of M.B. 29

were disrupted by lysis in cold or hot water, in 1 per cent aqueous

n-butanol, in 0.1 per cent sodium-dodecyl-sulfate, and also by the

treatment of cells with $ 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, -odium-dodecyl-

sulfate, 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 ASW. 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 contami-

nated with cytoplasric material. These cell walls were then subjected

to various treatments. Further washing with I M NaCI and/or dis-

tilled water did not have any effect; after washing 4 times with

NaCI, there was some evidence of fragmentation into smaller, irregu-

larly-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. B.o,29 after lysing and washing
with various procedures.

Fig. 6 An AMSW suspension of cells was diluted 20-fold. with 0.1
per cent SDS; the suspension was ccntrifuged 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 AS'.- 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 M NaC1 and 4 times with distilled water
(x 8,000).













PLATE 3


Fig. 6 Fig. 7


Fig. 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 typhimuriur (Herzberg, personal conmiuni-

caLion) 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 (RUIA-ase, trypsin, ficin, pepsin, bromelin),

singly and in combination, were tried, All enzymatic treatments

were carried out at room temperature with the material suspended

in appropriate potassium phosphate buffer solutions of M/15 concen-

tration. The time of incubation was usually 2 hr. :When the natorial

was to be treated with a second enzyme, the hydrogen-ion concen-

tration of the suspension was adjusted and the second enzyme added.

'he suspenzicns were then centrifuged, washed 3 times with M/15

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. 'Tith trypsin alone ex-tensive fragientation

of the cell walls was observed (Figure 9). Most of the fragments

were free from cytoplasmic 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 microLTaphs of cells of M.B. 29 after lysis
in water and treatrmsnt 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 1/15 buffer, pH 8.0, and trypsin (2ng per ml
of suspension) was added. U,-ture was incubated
for 2 hr at room temperature; suspension was
centrifuged and residue washed 3 times with M/15
buffer, pH 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


'5

'4


Fig. 9


Fig. 10


Fig. 11











The sano kind of results was obtained with ficin, alone or in
combination with RIIA-ase (Figure 11), and other enzymes.
In an attempt to improve the cleanliness of the wall propa-
rations, the conventional method of brclking the cells by vigorous

shaking with glass beads in a Iicl:el tissue disintegrator was

employed. Herealso a variety of.conditions rcre tried. For

suspending the cells during disintegration, 3 media'were tried.

These were ASIJ, I M NaCi, and 0,5 M K-pihophate buffer, pH 7,0.

Washing procedures tried involved 1 M NaCi, I:/15 1--phosphate buffer,

pH 7.0, and distilled water. Alon writh these, various enzymes

were tried under the conditions described earlier,

Equal volumes of a heavy cell suspension and "Ballotini"
Glass beads (0*007 rma diaamter) were shaken in the .ickel tissue
disintegrator for 20 min. The length of time used was arbitrary

but later results showed it to be satisfactory. The incr'-.r:c.l

viscosity was reduced by the addition of a trace of DI:A-~,s and,

after removal of the beads, the suspension was centrifuged at
1,100 x g for 10 tin. .The supernate was then contrifuged at high
speed and the residue was subjected to various treatments. Dia-

integration in sea water suspension followed by 4 washings with

1 M ~IaC solution gave a heavily contaminated preparation (Figure

12). Similar results were obtained when 1 M NaCI or 0.5 M buffer

was used as the suspending medium and 1 : iaC1i 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 still con-

taminated 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 RNA-ase and trypsin,

there was considerable fra[.':ent-tion of the cell walls as was ob-

served earlier (Figure 16). Ficin, alone or in combination with

RiIA-as.e, gave similar results (Figure 17), as did pepsin (Figure

18).

Since the results obtained by disintegration in the presence

of 0.5 M buffer followed by washing with 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 con-

ta.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 fragmen-

tation of the cell walls.

The same procedure was then used for preparing the cell

walls of I.B. 65 and M.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.1 in I;ntcrials and

Ueathods. Clean cell walls were obtained in the first trial

(Figure 22).




















Plate 5 Electron micrographs of cell 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 min* Cell
walls were washed 4 times with 1 '' :!aCl 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 NaCI solution and distilled water
(x 4,500).
Fig. 15 Frocedure used was, same as for Fig, 14, except
that distilled water was used after washing with
M/15 buffer (x 14,500).



















PLATE 5


Fig. 12 Fig. 13


Fig. 14


'
, .
1


Fig. 15






















Plate 6 Electron micrographs of cell walls of M.B. 29 ob-
tained by mechanical disintegration and action of
various cnzemecz.

rig. 16 Cells were suspended in 0.5 M buffer, pH 7.0,
and disrupted in Mickel tissue disintegrator.
Coarse debris was re-roved by centrifugation at
1,100 x j'or 10 min; residue from supernate was
suspenddt'in 1M/15 buffer, pH 7.0, and PJUA-aso
(0.05 mg per ml of suspension) and trypsin (2 mg
per ml of suspension) were added; midxure was
incubated for 2 hr at room temperature. 1li:ture
S 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 suspension) 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).














PLATE 6


Fig. 16 Fig. 17


Fig, 18


























Plate 7 Electron micrographs of cell walls of i. 3. 29, U.B. 65,
M.B. 98, and P. aeruginpsa,

Fig. 19 Cell walls of M.B. 29 (x 4,500).
Fig. 20 Cell walls of M.B. 65 (x 5,500).
Fig. 21 Cell walls of M.B, 93, (x 4,500).
Fig. 22 Cell walls of P. aerauinosa (x 11,500).








60





PLATE 7


Fig. 19 Fig. 20


Fig. 21


Fig. 22










It might be emphasized 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 appeared

to be thicker than those of the other two marine bacteria but thinner

than those of P. aeruginosa.


Disaggregation of Isolated Cell Walla.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 maFiAa cell walls were dis-

aggregated to a greater extent than the cell walls of the non-

marine pseudomonad. Thus, the per cent residual turbidity for 1M.B.

29 was 15 as compared to 55 per cent for P. aeruginosa (Figure 23).

The values for M.B. 65 and M.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 IM.B. 29, when extensive fragmentation of the walls was ob-

served by treating the cells with the detergent (Figure 6).














DISAGGREGATION


OF CELL WALLS OF M.B. 29 AND P. AERUGINOSA
BY SODIUM 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 ana-

lysed. The data obtained were: protein, 75 per cent, lipid, 18.5

per cent, hexosamine (as glucosamine), 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 :,.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 ana-

lytical data have been expressed as per cent dry weight of the cell

walls. The results are shown 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











TABLE 5

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. aeru-
29 65 98 ainosa


Nitrogen 12.5 12*2 12.8 8.4
Phosphorus 0.9 1.2 1.1 1.2
Protein 76.0 74,8 76.2 65.3
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 frorn 74.8 to 76*2 per

cent, in contrast to 65.3 per cent for the walls of P. aeruginosa.

Th: 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.

Hort-:'ver, in the marine bacteria the lipid and protein together

co'mrised 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 hexosanine 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. neru1inosn. The reducing values for the walls of

the marine bacteria were close to the respective hexosamine values,

Since no sugars were detected in their cell walls, 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 rha.nose in the cell walls.

Chromatographic analysis.- The presence of amino acids,

amino sugars, and sugars in the cell walls was detected by paper

chromatograph:- of the wall hydrolysates. The various substances

identified are given in Table 6. They were identified by compari-

son with chromatograms of known compounds and from their Rf values.

No attempt was made to quantitate any of the contitl-unts; however,







66



TABLE 6


SUBSTANCES IDENTIFIED IN CELL WALL HYDROLYSATES OF MARINE
BACTERIA AND PSEUDOMONAS AERUGINOSA




Substance
M.Bu MbB. ..B P. aeru-
29 65 98 pinosa


Sugars
Glucose +
Rharmose +
Glucosamine + + + +
MIranic acid* +

Amino acids*
Aspartic acid + 4 + +
Arginine + + + +
Diaminopimelic acid + + + +
Glutamic acid + + + +
Glycine + + + +
Serine + + +
Proline + + +
Hydroxyproline + +
Leucine + + + +
Isoleucine + '+ + +
Phenylaanine + + + +
Alanine *+ + +
Threonine + + + +
Tyrosine + + + +
Valino + .+ + +
Cysteic acid +
Ilethionine + + + +



Identification based on Rg value.
0











the size of the spots and the depth of color with ninhydrin gave an

approx:diate 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 !organ spray reagent and was identified as glucosamine

from its Rg* value (mith, 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 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-

leucine, 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 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 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. 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, I'.B. 98, and P. aeruginosa but not in M.B. 29, al-

though 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 ChromatoCrams of ninhydrin positive substances in
cell wall hydrolysates of marine bacteria and P.
Ceraginoon.

diaminopimelic acid was identified by one di-
mensional chromatography in each solvent system.







70





PLATE 8



W-'l 1o
9
5, 0 oi
50 0( 00 O0"
0 ,



-. f
' M.. 29 0 6 N.. 65


10 10
L^17I8 107 CD is
YO 19
91

90 O op SSK




B r. ,M



w5 ~- .. ...
6 gamaein ld
8 ..rin..
11 WSgl asse
*B12 iala
13 til i e
16 vdkai
; Illu~17
;~c~ ul~air~ei~i~ ~ ; i I u~to













DISCUSSION


The marine bacteria examined in this study can be tenta-

tively 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 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 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 re-

quiring 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 annual l 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, M.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

(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 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











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 NaC1 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 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 M.B. 65 appeared to be thicker than those of i~.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 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.

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 phos-

phorus contents of the walls of the marine bacteria and P.

aeruginosa were comparable to those reported for other Gram-

negative 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 ap-

peared to be present in greater amounts than others; these were

arginine, lysine, alanine, aspartic acid, and glutamic acid, :1uranic

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. aeruinosa, 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 muco-

peptide and its incorporation into the walls of the bacterium (Park

and Strominger, 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 al., 1960).

The cell walls of the marine bacteria were found to be solu-

ble in phenol and they were ex-tensively disaggregated on treatment

with sodium-dodecyl-sulfate (SDS). Both these chemicals were used

by Weidel et al. (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, Weidel, Frank, and Leutucb (1963) have pointed out

that autolytic enzymes can damage the mucopeptide layer (R-layer)

if suitable precautions against their action are not taken durin-:

the preparation of cell walls. They showed that the cell walls

of Salmonella gallinarum, prepared by using SDS, contained the

mucopeptide layer. However, if the walls were ri-roprcd by disruption

of cells in the Mickel tissue disintegrator followed by washing,

they were found to be deformed indicating d.'nte to the R-layer.

They were also extensively 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 disaggregation by SDS.

An unusual feature found in the walls of the marine bacteria

was the absence of sugars; only glucosrince was founi 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, Iicrococcus

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. aeruginosa 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.

aeruginosa 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 fr-nile

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. Of these two, MB. 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 M.B. 65 appeared to be heavier looking, with more body to them,

than those of M.B. 29 and M.B. 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. coli cell walls (Weidel et al., 1960) have clearly shown that

glucosamine, together with muramic acid, is a component of the links

uilch join the spheres in the layer. A backbone of amino sugar, with

chains of peptide linked through the -COOH group of Tnranic acid, has

been visualised as the basic structure of the mecopeptide layer. The

peptide- chains link the adjacent spheres giving a comb-like layer.

By the action of enzymes such as lysozyme and enzyme from bacterio-

phage T2, the links are broken resulting in the disengagement of the

spheres from one another (Weidel et ali, 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 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 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 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 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 com-

posed 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, 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 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 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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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, relieving 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.










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 Agriculture



Dean, Graduate School
Supervisory Committee:



Chaii ipan

/ ,J I

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CT-a/i (^L /-'/'L-^ ^










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