Life cycles in the methanogenic archaebacterium Methanosarcina mazei

MISSING IMAGE

Material Information

Title:
Life cycles in the methanogenic archaebacterium Methanosarcina mazei
Uncontrolled:
Methanosarcina mazei, Life cycles in the methanogenic archaebacterium
Physical Description:
vii, 147 leaves : ill. ; 28 cm.
Language:
English
Creator:
Robinson, Ralph Wendell, 1953-
Publication Date:

Subjects

Subjects / Keywords:
Methanobacteriaceae -- Growth   ( lcsh )
Genre:
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1985.
Bibliography:
Includes bibliographical references (leaves 136-146).
Statement of Responsibility:
by Ralph Wendell Robinson.
General Note:
Typescript.
General Note:
Vita.

Record Information

Source Institution:
University of Florida
Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000504179
notis - ACS4260
oclc - 22777150
System ID:
AA00003818:00001

Full Text














LIFE CYCLES IN THE METHANOGENIC ARCHAEBACTERIUM
METHANOSARCINA MAZEI









By

RALPH WENDELL ROBINSON


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


UNIVERSITY OF FLORIDA


1985















ACKNOWLEDGMENTS


This dissertation is dedicated to my parents, for
without their help and encouragement someone else's name
would be on the cover.

I would like to thank my fellow graduate students
Debbie Akin and Donna Williams for their help on this
project. Also, special thanks to my committee and to
Drs. Greg Erdos and Howard Berg.


An unsigned pastel drawing of a thin section of
Methanosarcina mazei by the late Doris Murray 1982
(conpa' Lt'iLh ?ij. '1I).


















TABLE OF CONTENTS


ACKNOWLEDGEMENTS.................................... ii


ABSTRACT........................................... iii


CHAPTER
1. INTRODUCTION .....................................1

2. LITERATURE REVIEW.................................5
Early Descriptions and Taxonomy...................5
Isolated Strains and Ultrastructure............... 6

3. MATERIALS AND METHODS ..........................13

Cultures and Growth Conditions...................13
Light Microscopy.. ..............................15
Sample Preparation for Electron Microscopy.......15
Method 1. For General Morphology using TEM......15
Method 2. For Gold Labelling
and X-ray Microanaysis..................16
Method 3. For Silver Staining and UV
Fluorescence Analysis...................17
Method 4. For Scanning Electron Microscopy......17
Method 5. For Freeze-Fracture..................18
Method 6. For Negative Staining................18
Changes in Cytoplasmic Fluorescence............ 19
Cytochemical Analysis of the Cell Surface........ 19
Silver Stain................................. 19
Fluorescein-Labelled Lectins.................20
Gold-Labelled Lectins........................21
Enzyme Extraction ............................22
Cytochemical Analysis of the Granules
and Polyphosphate Bodies.............23
Elemental Analysis...........................23
Enzyme Extraction ............................23

4. RESULTS.......................................... 25

Morphology of the Complex Life Cycle............25
Cocci ....................................... 25
Matrix Development and Division Patterns.....26
Sarcinal Colonies............................28
Colony Disaggregation.......................30
V












Morphology of the Limited Life Cycle............58
Methanosarcina mazei Strain S6...............58
Resting Forms................................70
Methanosarcina barker Strain 227 ...........73
Disaggregation in Methanosarcina
strain LYC................. 78
Freeze-Fracture analysis .....................81
Cytochemical Analysis of the Cell Surface....87
Silver Stain............................... 87
Fluorescein-Labelled Lectins............... 90
Gold-Labelled Lectins...................... 93
Enzyme Extractioi. ..........................93
Cytochemical Analysis of the Granules
and Polyphosphate Bodies ..........101
Elemental Analysis........................101
Silver Stain.............................. 104
Enzyme Extraction......................... 104

4. DISCUSSION..................,,............... 110

LITERATURE CITED ................................ 136

BIOGRAPHICAL SKETCH............................. 147
















Abstract of Dissertation Presented to the Graduate
School of the University of Florida in Partial
Fulfillment of the Requirements for the Degree of Doctor
of Philosophy

LIFE CYCLES IN THE METHANOGENIC ARCHAEBACTERIUM
METHANOSARCINA MAZEI
by

RALPH WENDELL ROBINSON

May, 1985

Chairman: Henry C. Aldrich
Major Department: Microbiology and Cell Science


Methanosarcina mazei strains S6 and LYC along with

Methanosarcina barker strain 227 were utilized to study

the structure and differentiation of the aggregating

methanogens. Cultures harvested under varying

conditions are described at the ultrastructural level

and qualitatively examined using cytochemical

techniques. Cells are irregular and 1-2 pm in size.

Cells of Ms. mazei strain S6 are enclosed by a protein

layer 12 nm thick in contact with the plasma membrane.

In sarcinal colonies, cells are held in close

association by a fibrous matrix up to 60 nm thick.

Colony maturation was examined in Ms. mazei strain

S6 over a period of one year. Changes occur in the

shape and staining of individual cells. Also, various

inclusion bodies were observed that either persist










throughout colony maturation or are only found at

certain growth stages. The inclusion bodies, cell

surface, and matrix were analyzed with silver methen-

amine, fluorescein- and gold-labelled lectins, X-ray

microanalysis, freeze-fracture, and various enzymatic

treatments.

Based on electron microscopic examinations, two

life cycles are described for Methanosarcina. A complex

life cycle is described with two variations for Ms.

mazei strains S6 and LYC. In addition, a limited cycle

is described that contains the only morphotypes observed

in Ms. barker strain 227. This limited cycle can also

be observed in the two strains of Ms. mazei studied.

The complex life cycle of strain S6 begins with

single cells that deposit matrix polysaccharide at the

cell surface; this prevents complete daughter cell

separation. These in turn divide forming sarcinae and,

as more matrix material is deposited, become distorted

into compact sarcinal colonies. The colonies dissociate

by degradation of the matrix. When released cells of

strain S6 are placed in fresh media, they can repeat the

cycle.

Ms. barker is restricted to a limited cycle which

also occurs in the Ms. mazei strains while the matrix

remains intact. These colonies mature from 3-5 microns

into larger masses (>3mm) that multiply by fragment-

ation. When Ms. mazei strain S6 remains in the limited










cycle and does not disaggregate in stationary phase,

several types of possible resting forms are found.


















CHAPTER 1

INTRODUCTION



Methanogenic bacteria are strict anaerobes and

generally obtain their energy from the oxidation of H2 to

CH4 by the reduction of CO2. A few genera can also utilize

methanol, format, methylamines, and acetate, forming

methane as a reduced product (Mah et al. 1977, Mah and

Smith, 1981). In general, methanogens can be isolated

from environments that provide H2 and CO2 and exclude 02.

These include the rumen of cattle, the intestines of

other animals and man, decaying tree trunks, stagnant

water, ocean bottoms, thermal hot springs, and sewage

treatment plants. Some sewage plants obtain much of

their operating energy from collected methane.

The morphology of the methanogens is as diverse as

their habitats. Cells are found as regular or irregular

cocci, as rods, spirals, or as sarcinae in aggregates of

large colony clusters. Such diversity in morphology

contrasted with a relatively uniform and unique

metabolic pathway led to the belief that methanogenesis

is a very ancient process, pre-dating the morphological

changes that occurred during evolution (Woese 1982).

1











Recently, an entire group of bacteria, including

the methanogens, has been recognized as being

phylogenetically separate from other prokaryotes (Balch

1982, Fox et al. 1980, Woese & Fox 1977). This group,

called the archaebacteria, is believed to comprise a

third kingdom, with the eubacteriaa" (other prokaryotes)

and the eukaryotes making up the other two kingdoms.

Archaebacteria are not simply ancestors of the

eubacteria but are a separate and independent group

related to eukaryotes as much as they are to the

eubacteria (Woese et al. 1982). Six characteristics

shared by archaebacteria examined thus far are 1) the

lack of peptidoglycan in the cell wall (Kandler & Koenig

1977, 1978, Kandler 1982, Koenig et al. 1982); 2) the

presence of unusual ether-linked poly-isoprenoid

glycerol lipids in the membrane instead of ester linked

alkane lipids (Tornabene et al. 1978, Langworthy et al.

1982); 3) unique tRNAs and rRNAs (Woese 1982); 4) their

existence in unusual, harsh environments (Woese 1977);

5) a eukaryotic-like DNA-dependent RNA polymerase

structure (Stetter & Zillig, 1980); and 6) unique

cofactors involved in metabolism (Cheeseman et al.

1972). The methanogenic bacteria were the first members

recognized as being uniquely separate from other members

of the Prokaryoteae and they still comprise the largest

and most diverse group in the Archaebacteriaceae (Wolfe











1971, Fox et al. 1977, Mah et al. 1977, Zeikus 1977).

Presently, other members of the archaebacteria include

the aerobic extreme halophiles (e.g. Halobacterium), the

thermoacidophiles (e.g. Sulfolobus and Thermoplasma),

and the thermophilic acidophiles (e.g. Thermoproteus).

Members of the group usually have a low percentage of

rRNA homology with other bacteria and a high percentage

of homology among themselves.

The sarcina-forming methanogens are currently

placed in the genus Methanosarcina. They display a

cellular arrangement that is unique, and several members

exhibit complex life cyles which are rare among isolated

archaebacteria. A life cycle has been partially

described at the light microscope level for Ms. mazei

where sedentary colonies break up into single cells

which can grow into new colonies in fresh media (Mah,

1980). The ability of a colony-forming bacterium to

dissociate into single cells in response to unfavorable

environmental changes provides a unique means for

dispersal. When cells regain a favorable habitat, they

form colonies which attach via sedimentation and

entanglement and are less likely to wash away. Indeed,

colony-forming methanogens are major inhabitants of

fixed bed reactors where the fluid components are

periodically flushed from the system (Zinder and Mah

1979, Robinson et al. 1984).













This group is of prime importance in the final

steps of biomass conversion to methane in mesophilic

environments, with up to 70% of the methane formed

derived from the acetate utilizing methanogens (Mah and

Smith 1981). These two qualities, of displaying a

complex life cycle and being of prime importance in

bioconversion, warrant an in-depth examination of this

group. This dissertation characterizes the structure

and development of these aggregating methanogens at the

ultrastructural level. The general objectives are 1) to

describe the morphology using light and UV microscopy,

scanning electron microscopy, and transmission electron

microscopy; 2) to determine and describe the life cycles

in Methanosarcina mazei; and 3) to obtain qualitative

data on cellular composition using cytochemical

techniques.

















CHAPTER 2

LITERATURE REVIEW





Early Descriptions and Taxonomy



The genus Methanosarcina contains the most

metabolically diverse methanogens isolated thus far. It

was described in 1903 by Maze (cited in Maze 1915 and in

Barker 1936) in mixed cultures as a "pseudo-sarcine"

because of its ability to grow either as large sarcinal

groups or as single cells. Soehngen (1906) later

described a methanogenic sarcina that was more or less

cubical and was not observed as single cells. Smit

(1933) placed both of these bacteria into the genus

Zymosarcina along with other anaerobic sarcinae, with

the type species Z. methanica. In 1936 Kluyver and van

Niel created two genera: Methanosarcina for the methane

producing sarcina described by Soehngen with Ms.

methanica as the type species, and the genus

Methanococcus for the "pseudosarcina" described by Maze

with Mc. mazei as the type species (Barker 1936).











Since Ms. methanica and Mc. mazei had not been

isolated or maintained in pure culture, they were

omitted from the approved list of bacterial names (Ad

hoc committee 1976). Balch et al. (1979) designated

Methanosarcina barker and Methanococcus vannielii as

neotypes. Ms. barker matches the descriptions of Ms.

methanica, but Mc. vannielii is an irregular, highly

motile, marine coccus that does not aggregate and has

little resemblance to the early descriptions for Mc.

mazei made by Maze, Soehngen, or Barker (Jones et al.,

1977). Then, in 1980, Mah reported the first successful

isolation of a pure culture that exactly matched the

"pseudosarcina" descriptions. Thus, the name

Methanococcus mazei was temporarily revived. Recently

though, there has been a re-evaluation of the taxonomic

position of Mc. mazei and its relationship to other

methanococci and methanosarcinae. Consequently,

Methanococcus mazei has been moved into the genus

Methanosarcina as Ms. mazei, with Ms. barkeri designated

the type species (Mah & Kuhn 1984a and b).



Isolated Strains and Ultrastructure



Terminology. Since all methanogenic genera start

with the letter "M," the abbreviations recommended by

Daniels et al. (1984) will be used. These are as











follows: Mbr., Methanobrevibacter; Mb., Methano-

bacterium; Msp., Methanospirillum; Mc., Methanococcus;

and Ms., Methanosarcina.

There is some confusion over the descriptive terms

used for the various morphotypes of Methanosarcina,

especially Ms. mazei. Mah (1980) designates types I,

II, and III (Fig. 1). Zhilina (1976) had earlier

designated different types I, II, and III (Fig. 2). To

further complicate matters, Zhilina and Zavarzin (1979c)

later described their type III as exhibiting a variety

of morphotypes. Except when specifically describing

others' work, this paper will use the 3 forms shown in

figure 3 and refer to them as cocci, sarcinal colonies,

or resting forms. These three morphotypes can be stable

and long lasting in culture. The cocci correspond to

type III described by Mah and by Zhilina. The sarcinal

colony corresponds to type I described by Mah and type II

described by Zhilina.

Mah's isolate of Ms. mazei, designated S6, has been

described only at the light microscope level (Mah 1980).

It exhibits three morphotypes at different growth stages

(Fig. 1). When cultures are young, cells are found in

small sarcinal aggregates (type I) that measure up to 20

pm in size and may form larger aggregations with each

other. As the cultures mature and enter stationary

phase, sarcinal aggregates go through a transitory stage













1000
coo
C0cc
Ov ei2


Type II


Type I
Mah, 1980


Type III


Type I Type II
Zhilino.1976


Sarcinal Colony


FIG. 1


00
00%
0


Type III


FIG. 2


FIG. 3


0
120


0 -;
GCO
)to


Cocci


Resting Forms











to form coccal cysts (type II). These are up to 100 pm

in diameter and ma- form larger aggregates up to several

millimeters in size. Mah reported that when the coccal

cysts are physically disrupted, a cyst "wall" breaks,

releasing individual cocci (type III) that are 1-3 pm in

size. Each of these can grow into sarcinal forms when

placed in fresh medium.

Several other strains of Ms. mazei have been

reported: "MC3" (Touzel and Albagnac 1983) exhibits a life

cycle that is apparently identical to S6; "Juelich"

(Scherer and Bochem 1983a) for which a life cycle is not

described; and "LYC" (Liu et al. 1985). Strain LYC

exhibits only two morphotypes with a unique mechanism of

disaggregation. A sarcinal colony forms when the cells

are grown at pH 6.0 but when the pH is raised to 7.0 the

colony disaggregates into single cells. Apparently, an

enzyme is activated that hydrolyzes the matrix holding

the colony together. Unfortunately, the single cells

cannot presently be induced to form aggregates so a

complete life cycle cannot be delineated. Ms. mazei

strains have not previously been described at the

ultrastructural level.

The ultrastructure of Methanosarcina spp. from

enrichment cultures were examined by Zhilina (1971,

1976), Zhilina and Zavarzin (1979a) and Bochem et al.

(1982). Pure cultures of Methanosarcina spp. not











designated Ms. mazei were examined by Zeikus and Bowen

(1974), Zhilina and Zavarzin (1979b,c), Pangborn (In Mah

1981), Archer and King (1983, 1984), Scherer and Bochem

(1983a,b), Ollivier et al. (1984), and Sowers et al.

(1984a).

Zhilina (1971) first looked at sarcinal aggregates

found in digester enrichments. They are composed of

irregular cells measuring 1-3 pm with unequal division

zones. Colonies are enclosed by a variably thick

lamellar wall. Numerous gas vacuoles were described

that are 70-80 nm wide and 200-400 nm long with a 2.5 nm

membrane. Also present are electron dense granules

measuring up to 100 nm and polyphosphate-like inclusion

bodies of unreported size. Due to the ability to form

gas vacuoles, Zhilina and Zavarzin (1979b) proposed a

new species, Ms. vacuolata, but this epithet has not

been officially recognized.

Zhilina (1976) also reported on three morphotypes

of Methanosarcina in an enrichment culture (Fig. 2).

Morphotype I corresponds to a large sarcinal aggregate

that possesses a central cavity with a single pore.

Morphotype II is a smaller aggregation of irregular

cells tightly enclosed by matrix material. This type is

a sarcinal colony and corresponds to Mah's type I (Fig.

1). Zhilina's type III is a single cell and corresponds

to Mah's type III. Zhilina's morphotype III was later











reported to occur as single cells, in groups forming

loose aggregates, or in tightly packed cysts (Zhilina

and Zavarzin, 1979c). However, these authors do not

indicate any developmental changes that occur between

the various forms. This biotype may be a strain of Ms.

mazei but this has not been confirmed.

Another isolate, designated Methanosarcina strain

MP, which may be a strain of Ms. mazei was recently

described by Ollivier et al. (1984). They describe

sarcinal colonies that can break up into coccoid

elements in old cultures. However, they only examined

the ultrastructure of the sarcinal colonies.

Sowers et al. (1984a) recently reported on the

isolation and ultrastructure of a third species of

Methanosarcina, Ms. acetivorans, that grows in marine

environments as irregular single cells that are motile.

The cytoplasm contains electron dense granules and is

enclosed by a plasma membrane and a closely associated

protein layer. When cultures enter stationary phase the

single cells begin to aggregate into loose associations

that are eventually enclosed by a thin proteinacous cyst

wall. A matrix is not present and when fresh medium is

added the aggregates break up into single cells once

again.

The electron dense granules and polyphosphate-like

inclusion bodies have recently been the focus of several











papers and presentations. Scherer and Bochem (1983 a

and b) used X-ray microanalysis (see Moreton 1981) on

ultrathin sections of 12 strains of Methanosarcina to

determine the partial elemental composition.

Polyphosphate-like globular inclusion bodies 150-250 nm

in diameter contain Ca, P, and Fe. The electron dense

granules have the same elemental composition but in

lower amounts. Murray et al. (1984) examined crude cell

extracts and reported that a carbohydrate is present

when cells are grown under nitrogen limiting conditions

and that the granules may be carbohydrate storage sites.

However, there is little evidence presented and the

granules have not been purified and analyzed.


















CHAPTER 3

MATERIALS AND METHODS



Cultures and Growth Conditions



Methanosarcina mazei strains S6 and LYC and the type

species Ms. barker strain 227 were examined. Colony

development was examined only in Ms. mazei strain S6. Two

different cultures of the same strain S6 were utilized.

One exhibited a complex life cycle while the other grew

only in sarcinal colonies in a limited cycle and is a

variant of 56. The variant strain was obtained when

cultures were repeatedly transferred in the laboratory.

Ms. mazei strain S6 and Ms. barker were grown at pH 6.8

in Bl medium (Balch et al. 1979) modified according to Mah

(1980) with H2/CO2, acetate, trimethylamine, or methanol used

as energy sources. Ms. mazei strain LYC was grown at pH

6.0 in a medium described by Liu et al. (1985) with H2/CO2 or

methanol used as energy sources.

Cultures were usually grown in serum tubes (Bellco)

containing 9 ml of medium and sealed with butyl rubber

stoppers. Inoculations consisted of 1 ml of a 10-14 day











culture passed through the stoppers using 20 or 25 gauge

needles. All cultures were incubated at 350 C without

shaking.

Sarcina development from single cells and matrix

deposition was examined in a series of tubes inoculated

with cultures that had been filtered through a 3 pm

filter to remove any remaining sarcinal forms. Tubes

were incubated from 1-3 days, then prepared for

transmission electron microscopy (TEM) using method 1

below.

Sarcinal colony maturation in the variant strain of

S6 grown on acetate was monitored by inoculating a

series of tubes through a 5 pm filter to select for the

smallest sarcinal forms. No growth occurred when

filters smaller than 5 pm were used (single cells are

not released by this variant). Cells were collected at

5, 10, 12, 15, 21, 30, 90, 105, and 360 days and

prepared for EM using method 1 below.

Colony disaggregation was monitored using strain S6

that spontaneously breaks apart into single cells.

When a 200 ml culture grown in a 600 ml serum bottle

showed the first signs of turbidity, the colonies were

examined using light microscopy and the culture was

prepared for EM using method 1 below.

Colony disaggregation in Ms. mazei strain LYC was

monitored by growing the cultures at pH 6.0 for 5-7 days











and then raising the pH to 7.0 by adding Na2HCO3 (10% in

reduced medium). Cultures were collected at 0, 12, 24,

36, and 48 hrs and prepared for EM using method 1 below.



Light Microscopy



Two light microscopes were used to examine culture

changes and to record micrographs. A Zeiss WL research

microscope equipped with phase and Nomarski optics was

used to make light micrographs on Kodak Plus-X 35 mm

film. A Zeiss photostrobe was used when photographing

single cells. Alternatively, a Nikon Labophot

microscope equipped with UV epifluorescence was used for

general culture monitoring and the recording of UV

micrographs on Kodak Tri-X film.



Sample Preparation for Electron Microscopy



Method 1. For General Morphology



Samples were fixed in a modified (2.0% form-

aldehyde and 2.5% glutaraldehyde in 0.1M Na-cacodylate

buffer) Karnovsky's fixative (Karnovsky 1965) for 30 min

at pH 7.2 on ice. After washing in buffer for 1 hr,

samples were fixed in 0.1M cacodylate buffered 1% OsO4 at

pH 7.2 for 1 hr on ice. Cells were washed once and then











encapsulated in 1.5% agar (Difco). Samples were then

dehydrated through a graded ethanol/acetone series,

embedded through a resin/acetone series into 100%

Spurr's resin (Spurr 1969), and polymerized in a 600 C

oven for 24 hrs. Thin sections were cut on a LKB

Ultrotome III ultramicrotome using a diamond knife and

collected on copper grids. Sections were poststained

with 1% uranyl acetate for 20 min followed by lead

citrate (Reynolds 1963) for 5 min. Micrographs were

taken on a Hitachi HU-11E or a JEOL JEM 100-CX

transmission electron microscope operated at 75 and 60

KV respectively. For stereo micrographs, semi-thick

sections (gold) were observed at a tilt angle of -5 and

+50 using a goniometer stage (Rieder et al. 1985). The

accelerating voltage was increased to 100 KV.



Method 2. For Gold Labelling and X-ray Microanalysis



Cells were fixed for 15 min in 2% formaldehyde and

0.2% glutaraldehyde in 0.1M cacodylate buffer at pH 7.2

on ice. After washing in buffer, cells were

encapsulated in 1.5% agar then dehydrated in an ethanol

series. Samples were further processed using one of two

methods. In one, they were embedded through a series of

ethanol/Lowicryl K4M plastic (Polaron Equipment Ltd.) to

100% plastic and polymerized by UV radiation overnight.











During this treatment, cells were kept on ice up to the

75% ethanol step and then placed at -400C for subsequent

steps until polymerization was complete. Alternatively,

samples were placed directly in 100% LR White plastic

(Polaron) from the 95% ethanol step and then, after 4

changes of plastic, were polymerized at 60C overnight.

Sections were cut and examined as in method 1 except

they were collected on nickel grids and were not

poststained.



Method 3. For Silver Staining and UV Fluorescence
Analysis


This procedure was essentially the same as method 1

except samples were not exposed to osmium, and

epon/araldite plastic (Mollenhauer 1964) was used for

embedding. Sections were collected on nickel grids and

were not poststained.



Method 4. For Scanning Electron Microscopy



Cells were harvested on a 0.2 pm syringe type

filter and fixed with 2% glutaradehyde in 0.1M

cacodylate buffer at pH 7.2 for 20 min. The cells were

washed, treated with 1% buffered OsO4 for 15 min, then

washed again. The filter and cells were dehydrated in

an ethanol series and dried by the critical point method











using CO2. The cells were then sputter coated with gold

and viewed in a Hitachi S-450 scanning electron

microscope.



Method 5. For Freeze-Fracture



Cells were collected on a 2.3 mm diameter 400 mesh

gold grid, sandwiched between two 3 mm gold discs and

immediately frozen in a propane jet freezing apparatus

(Mueller et al. 1980). The frozen discs were stored

in liquid nitrogen until being separated in a Balzers

BA-360M freeze-fracture machine. The fracture faces

were angle shadowed with platinum-carbon and replicated

with carbon using electron guns. Replicas were cleaned

in 50% aqueous chromic acid for 2 hrs, rinsed, then

followed with sodium hypochlorite (50% household bleach)

overnight before being collected on carbon and Formvar

coated copper grids for viewing using TEM.



Method 6. For Negative Staining



Drops of cultures were placed on Formvar coated

copper grids for 1-5 min depending on culture density.

The medium was blotted away and the adsorbed cells

stained with aqueous 2% phosphotungstic acid or aqueous

1% uranyl acetate for 5 min.













Changes in Cytoplasmic Fluorescence



Twelve-day old colonies of strain S6 grown on

acetate and prepared for TEM using method 3 above were

sectioned using a glass knife to obtain a single

silver-gold thin section. This was poststained and

observed using TEM. When an adequate thin section

through a sarcinal colony was found, a 1 pm thick

section was cut as the very next section. This was

placed on a glass slide and observed with UV microscopy.

A comparison was made between the morphology of cells

observed in the thin section using TEM and the

fluorescent properties of the same cells in thick

section.



Cytochemical Analysis of the Cell Surface



All cytochemical analyses were performed on Ms.

mazei strain S6. Ms. mazei strain LYC and Ms. barker

were not examined.



Silver Stain



Sections prepared by method 3 were etched at room

temperature by floating on 1% periodic acid from 1-15


I











min (Erlandsen et al. 1979). After rinsing, the

sections were stained for 30-45 min at 600C with silver

methenamine (de Martino and Zamboni 1967, modified by

Picket-Heaps 1967). The grids were rinsed and then

floated on Ektaflo fixer (Kodak, diluted 1:100) to

remove non-specific silver deposits. Controls were run

with sections receiving 1) no treatment; 2) periodic

acid only; 3) silver methenamine only; and 4) periodic

acid and silver stain only (no fixer).



Fluorescein-Labelled Lectins



Whole colonies of strain S6 grown on acetate were

treated with 0.1M cacodylate buffered 1% OsO4 at pH 7.2

for 1 min to quench autofluorescence and then rinsed

with 0.1M PBS buffer at pH 7.2 supplemented with 0.1 mM

CaCl2, MnC12, and MgC12. The autofluorescence of single

cells lasts only a few seconds, so for single cells this

step was omitted. The samples were then treated for one

hour with one of the following fluorescein-labelled

lectins (Vector Labs, Burlingame CA): concanavalin A

(Con A); wheat germ agglutinin (WGA); soybean agglutinin

(SBA); Dolichos bifloris agglutinin (DBA); Ulex

europaeus agglutinin (UEA-I); peanut agglutinin (PNA);

or Ricinus communis agglutinin I (RCA120). Controls were

run with 0.1 M sugar (D-galactosamine, alpha-methyl-











mannose, alpha-methyl-glucose, or L-fucose) present in

the incubation mixture. Samples were washed in the

supplemented buffer and fixed in buffered 3%

glutaraldehyde for 20 min. After a final wash, samples

were observed with UV microscopy.



Gold-Labelled Lectins



Colloidal gold approximately 15.0 nm in size was

prepared by reducing chlorauric acid with sodium citrate

according to the method of Frens (1973). A 4% gold

chloride solution was prepared and 0.5 ml added to 200

ml of double distilled water. Four milliliters 1 of 1%

sodium citrate was added and the solution was refluxed

for 15 min After cooling to room temperature, the pH

was adjusted to 7.0 with 0.1M K2CO 3 The minimum amount of

lectin required for stabilization of the gold was

determined by the technique of Horisberger and Bauer

(1976) as modified by Roth and Binder (1978). A 0.1 ml

aliquot of serially diluted lectin was mixed with 0.5 ml

of colloidal gold. After 15 min, 0.5 ml of 10% NaCl was

added and flocculation due to insufficient stabilization

of the gold was determined by a color change from pink

to blue. A 20% excess of lectin needed for stabil-

ization was added to 30 ml of colloidal gold. A 10%

solution of PEG-20 was added for a final concentration











of 0.5 mg/ml and the solution was centrifuged at 28,000

X g for 60 min. The clear supernatant containing any

unbound lectin was discarded. The gold pellet was

washed once by centrifugation in 20 mM NaHPO4-KH2PO4 at

pH 7.2 containing 0.5 mg/ml PEG-20 and 0.02% sodium

azide and then resuspended in the same buffer to 1/10

the original volume.

Sections of strain S6 prepared using method 2 were

floated for 20 min on drops of the lectin-stabilized

gold which had been serially diluted with PBS. Sections

were washed three times with PBS and then once with

distilled water before viewing. Controls were run with

sections receiving 1) colloidal gold only; and 2)

D-galactosamine (0.1 M) in the reaction mixture.



Enzyme Extraction



Sarcinal colonies of strain S6 were exposed to 0.5%

protease (type XIV, Sigma) in 0.1 M cacodylate buffer at

pH 7.0 supplemented with 20% sucrose for 5, 10, and 30

minutes at 370C. These were then fixed and embedded

using method 1 above. Controls were run with colonies

treated only with buffer. Alternatively, cultures

containing single cells were fixed and embedded as in

method 1 above except they were treated with 1.0%

protease for 30 min after fixation. Also, thin sections











of cells prepared according to methods 2 and 3 above

were treated by protease digestion as described below.




Cytochemical Analysis of the Granules and Polyphosphate
Bodies


Elemental Analysis



Thin sections of cells embedded by methods 2 or 3

were examined by energy- dispersive X-ray microanalysis

(Kevex) in a JEOL JEM 100-CX TEM. Sections were held in

a carbon specimen holder tilted 45-50. The TEM mode

was used at 60 KV and the condenser 1 spot size reduced

to 2 or 3 to restrict the diameter of the area being

counted.



Enzyme Extraction



Sections prepared by methods 2 or 3 using 12-day

cells of strain S6 were collected on nickel grids and

floated on 1% periodate for 15 min and washed. The

sections were then exposed to one of the following

solutions at 370 C: aqueous 0.5% protease at pH 7.0

(Anderson and Andre 1968); phosphate buffered 1% amylase

at pH 7.0 for alpha amylase and at pH 5.8 for beta

amylase activity (Palevitz and Newcomb 1970); phosphate

buffered 0.5% pullulanase at pH 5.8 (Palevitz and











Newcomb 1970); bicarbonate buffered alkaline phosphatase

(type 1-S, Sigma) at pH 10.4; or phosphate buffered 1%

cellulase (type 2, Sigma) at pH 4.5. Sections were

frequently monitored by TEM during treatment which

lasted up to 24 hrs. Sections were also exposed to

sequential treatments with pullulanase for 6 hrs

followed by alpha and beta amylase each for 24 hrs.

Controls were included with sections floated on their

respective buffers. Positive controls of Dictyostelium

myxamoebae were included to test for glycogen extraction

by amylase. Embedded filter paper was included to test

for cellulase extraction. Sections of Sporosarcina

ureae which has a protein layer outside the gram-

positive cell wall and protein layers on endospres were

included to test for protease activity.


















RESULTS


Morphology of the Complex Life Cycle



Cocci



When viewed with Nomarski optics, free cells of Ms.

mazei strain S6 are approximately 1-2 pm in size (Fig.

4). The cytoplasm often contains 1 or 2 refractile

granules up to 0.5 pm in size (not shown). Under UV

microscopy, cells display moderate fluorescence which

quenches within a few seconds. Scanning electron

microscopy (SEM) shows cocci 1-2 pm in size and roughly

spherical (Fig. 5). Negative staining (Fig. 6) reveals

an uneven surface, and polyphosphate inclusion bodies

can be seen which are up to 0.8 pm in size. Flagella

or pili were not present.

Thin sections show irregular and regular cells up

to 2.0 pm in size (Fig. 7 and 8) that differ in their

degree of cytoplasmic staining. The nuclear region is

not readily apparent. Electron dense granules are

present in the cytoplasm that range from 40-100 nm in

size. These are spheroid with very rough margins and

25

























Figures 4-7
strain S6.


SSingle cells of Methanosarcina mazei


Fig. 4. Cells viewed with Nomarski optics.

Fig. 5. Cells viewed with scanning electron
microscopy (SEM).

Fig. 6. Transmission electron micrograph of
negatively stained whole cells. One to two
polyphosphate inclusion bodies can be seen
in some cells.

Fig. 7. Thin section of cells showing a variety of
shapes.


P= polyphosphate











CD -^
1 *
4 4 3



t 4

.,



Sq%
:. S .'
at 1


wp


1 pm


0*


AL1


t


0~


I.)


I pm1n


r






















Figures 8-11. Transmission electron micrographs of
single cells of Ms. mazei strain S6.

Fig. 8. Cells containing cytoplasmic granules and
polyphposphate inclusion bodies.

Fig. 9. Smooth surface with protein layer in close
contact with the plasma membrane
(arrows). Material with a membranous
appearance may be shedding from the cell
surface (arrowheads).

Fig. 10. A cell that has lost most of the protein
layer.

Fig. 11. Higher magnification of the cell in figure
10 shows the remaining protein layer as
clumps on the cell surface.


g= granules

P= polyphosphate

L= protein layer


pm= plasma membrane










4k
)I<
Pf


L)c


- .wA

L


Ijim


0.2pm


to O.iu -










are characteristically surrounded by an electron

transparent halo. In single cells, the granules are not

evenly distributed but tend to group into clusters at

the periphery of the cytoplasm. Larger inclusion bodies

similar to polyphosphate are common. The cytoplasm is

bound by a unit membrane that is not very prominent and

an additional closely associated protein (see below)

layer 12 nm thick (Fig. 9). Frequently, material is

observed that may have sloughed off the surface in

strips. These sometimes have a membranous appearance

(arrowheads Fig. 9). Occasionally cells are seen that

have lost most of this layer (Figs. 10 and 11). The

material on these cells is present as clumps

approximately 14 nm in size just outside the plasma

membrane.



Matrix Development and Division Patterns



A matrix material coats the outside of the cell

surface and is probably responsible for cell-cell

adhesion during colony formation. Matrix development

begins as a fibrous layer close to the protein layer on

the cell surface. During cell division and subsequent

development into sarcinae, the matrix becomes thicker,

denser, and more regular, somewhat resembling a gram-

positive wall. As sarcinal colonies mature however, the

matrix becomes very irregular as described below.



























Figures 12-15. Various aspects of single cell division
of Ms. mazei strain S6.


Fig. 12.


Fig. 13.

Fig. 14.


Fig. 15.


Different stages of Cell division viewed
under Nomarski optics.

SEM of dividing cells.

Thin section of a cell initiating septum
formation.

Thin section showing completed septum.
Matrix material is incorporated in the
septum.


M= matrix

g= granules

S= septum















































forr

4b~~~J






-yppl









With Nomarski optics, cells in two-day cultures

appear like diplococci in various stages of division

(Fig. 12). The first division is by symmetrical binary

fission with the two daughter cells remaining in

contact. In SEM (Fig. 13), the dividing cells appear

similar to those observed in light microscopy. Single

cells initiate division with the plasma membrane,

protein layer, and matrix invaginating as an annulus

across the equator of the cell (Fig. 14). The matrix is

60 nm thick and has a fibrous structure.

After the septum has bisected the cell, the cross

matrix thickens from the periphery inward (Fig. 15)

until it is as thick as the outer matrix. The cells do

not separate, presumably because of the tight

association of the cross matrix. The second divisions

that give rise to four daughter cells are initiated in

the same plane with each other but perpendicular to the

first cross septum (not shown). Sarcinae that develop

from single cells have a regular shape when viewed with

light microscopy and fluoresce brightly under UV

microscopy (Fig. 16). In thin sections of three day

cultures, the sarcinae show a fairly regular shape but

the division septa are askew (Fig. 17). Unlike the

first division where the completed septum is almost in

the same plane, the second divisions are completed in an

asymmetrical manner and two perpendicular septa are

rarely seen.


























Figures 16-19. Various aspects of Ms. mazei strain S6
sarcina structure.


Fig. 16.


Fig. 17.



Fig. 18.


A sarcina-containing culture viewed under
UV microscopy.

Thin section of sarcina showing the
involvement of the matrix in the cross
septa.

A culture of cells developing beyond the
sarcina stage.


Fig. 19. Higher magnification of sarcina developing
into colonies. The division planes are
very irregular.


M= matrix

g= granules


s= septa






















1 pm


M
hanFt


a


ha-'r


q %Y-


;IIC


wv










Overall, the sarcinae are approximately 3.5 pm in

size and the matrix is more condensed. Cytoplasmic

granules are more numerous and tend to group towards the

center of the cells. Further growth results in

irregular masses at four days (Figs. 18 and 19). Each

sarcina is derived from a single cell and is capable of

eventually forming a large sarcinal colony. There is no

evidence that sarcinae coalesce to form colonies. A

large inoculum of single cells results in the growth of

numerous small colonies while a small inoculum (i.e. a

few cells) will give rise to just a few large colonies.



Sarcinal Colonies



Sarcinal colonies of strain S6 can reach up to 5 mm

(Fig. 20). Light microscopy using Nomarski optics shows

smaller colonies that are approximately 100 pm in size

with an uneven surface (Fig. 21). Under phase and UV

microscopy at lower magnifications, larger sarcinal

colonies up to several hundred pm in size (Fig. 22)

display a strong fluorescence which does not quench

after several minutes (Fig. 23). The larger colonies

are globular and are composed of smaller sized groupings

which may fragment when disturbed (see limited cycle

below).

SEM of broth cultures reveals a configuration

somewhat like cauliflower (Fig. 24). Cells are either





























Figures 20-23. Sarcinal colonies of Ms. mazei strain
S6.

Fig. 20. Broth-grown culture poured into a staining
dish. The colonies can reach up to 5 mm
in size.

Fig. 21. Individual sarcinal colony viewed with
Nomarski optics.

Fig. 22. Aggregates of colonies viewed with light
microscopy.

Fig. 23. The same group of cells seen in figure 22
viewed with UV radiation. The cytoplasm
fluoresces with a bright blue-green color.































pm --


i :Iv


i.


0.2mm


'tw=


*11(11
































Figures 24 and 25. Scanning electron micrographs of
sarcinal colonies of Ms. mazei strain S6.

Fig. 24. Large colony with an appearance somewhat
like cauliflower.

Fig. 25. Enlargement of boxed area in figure 24
showing the close association of the cells
covered with fibrous matrix material.






40



















255









4d





b.b





Fm






imp
7r t A,

,










closely packed in the colony interior or are arranged in

lobes at the surface. Individual cells can be seen at

higher magnifications but these are difficult to discern

due to the thick matrix material (Fig 25).

In thin sections, colonies are composed of a

heterogeneously staining population of cells that are

more irregular than the earlier sarcinal forms (Fig.

26). The matrix is approximately 40 nm thick and has a

fibrous appearance. The granules are either dispersed

throughout the cytoplasm or are concentrated at the cell

center. The granules possibly come together and fuse to

form the polyphosphate bodies since very compact groups

of granules are sometimes observed which lack the

characteristic electron transparent margins (Fig. 27).

Clear areas are found where polyphoshate bodies have

been removed during processing (Fig. 28). These are up

to 0.8 pm in size and usually possess some residue

around the margins. Occasionally, polyphosphate bodies

are observed where some of the material is present in a

loose reticular network (Fig. 29) or in bodies that are

more condensed (Fig. 30).

A large number of curved tubules 14 nm thick that

appear to contain membrane are trapped between the

protein layer and the matrix (Figs. 27-37). These are

sometimes in close association with the cell surface

(arrows Figs. 31 and 32). When there is a high

concentration of the tubules, they can intertwine with



















Figures 26-30. Thin sections of sarcinal colonies of
Ms. mazei strain S6. Numerous inclusion bodies are
shown along with tubules associated with the cell
surface.


Fig. 26.




Fig. 27.


Low magnification showing the irregular
arrangement of the cells. They contain
numerous inclusion bodies and stain
heterogeneously.

Thin section showing the cytoplasmic
granules that tend to group toward the
center of the cells in thin sections and
may fuse to form the polyphosphate bodies.


Fig. 28. Section through a polyphosphate body where
most of the material has been removed
during processing.

Fig. 29. A cell with two polyphosphate bodies that
contain dark staining material in a
characteristic reticular pattern.

Fig. 30. A polyphosphate body containing a dense
arrangement of the dark staining material.


M= matrix

g= granules

P= polyphosphate


t= tubules



























Figures 31-34. Thin sections of Ms. mazei strain S6
showing the cell surface and the close relationship of
the tubules. Bar = 1 pm


Fig. 31.




Fig. 32.


Micrograph of tubules found between two
cells. The tubules are found in groups on
the cell surface and appear to be attached
in certain areas (arrows).

Micrograph of the tubules and matrix.
Frequently, the tubules are mixed with
fibers of the matrix material. The
tubules appear attached to the cell
surface (arrows).


Figs. 33 and 34. Stereo pairs of micrographs that
can be observed using a stereo viewer to obtain a three
dimensional image. A tubular structure is clearly
demonstrated.


M= matrix

g= granules


t= tubules






45




31 32






tM














Ati


33 34



I9
..
f~W*


.I~ :B
9)e












a~n t
f i.










fibers of the matrix (Fig. 32). Stereo pairs of

micrographs show the irregular arrangement and the

mixing with fibers of the matrix (Figs. 33 and 34). In

cross section (Fig. 35) they are circular and

approximately 15 nm in diameter. They are usually

distributed in clusters at areas on the cell surface;

however, they can be found in grazing sections as

threads stacked roughly parallel to each other and

approximately 20 nm apart (Figs. 36 and 37).

After cells mature into sarcinal colonies during

the complex life cycle, they possess most of the

ultrastructural characteristics of colonies that are

restricted to the limited cycle described below.



Colony Disaggregation



After an unspecified amount of time in stationary

phase but usually not before two weeks, the colony

matrix begins shedding from the cells. In dark field

light microscopy at low magnification, single cells are

seen shedding from sarcinal colonies (Fig. 38). Under

phase contrast at higher magnifications, numerous cocci

are present with sarcinal colonies (Fig. 39). It is

common to find intermediate forms where both sarcinae

and cocci are present. Under UV microscopy (Fig. 40),

the colonies appear as coccal colonies held together by

what remains of the matrix. These coccal colonies



























Figures 35-37. Thin section micrographs of Ms. mazei
strain S6 showing tubules associated with the cell
surface.

Fig. 35. High magnification micrograph of a group
of tubules cut in cross section showing a
hollow tubule structure.

Fig. 36. A cell showing four groups of tubules at
the cell surface. The one at the upper
right is cut in a grazing section of the
surface.

Fiq. 37. High magnification of the grazing section
of the cell surface seen in figure 36.
The tubules are in a parallel arrangement.


M= matrix


T= tubules
































Figures 38-40. Microscopic examination of gross colony
morphology of Ms. mazei strain S6 during disaggreg-
ation.


Fig. 38.



Fig. 39.


Dark field light micrograph of two
disaggregating colonies releasing single
cells.

Disaggregating culture observed with phase
contrast microscopy shows intact colonies
together with free single cells.


Fig. 40. Disaggregating colonies observed with UV
microscopy. Single cells that fluoresce
brightly are held together by the
remaining matrix material.





lbr











joj










usually break up spontaneously or can persist until

physically disrupted.

In SEM, cells in a cluster of colonies are not

arranged in a tight fashion (Fig. 41) and less matrix

material is present between cells. Individual cells are

easily observed at high magnifications (Fig. 42) and

measure 1.3 to 2.2 pm. Lobes and crevices are not as

apparent as in whole colonies (compare with Figs. 24 and

25).

When observed in thin sections, the matrix has a

loose fibrous nature when the colony degrades into a

coccal colony (Fig. 43). The fibrous matrix has a

laminated appearance at higher magnifications and

involves uncompleted division septa (Fig. 44). The

membrane tubules between the matrix and the protein

layer are scattered throughout this region. The matrix

layers may number 20-30 per cell and shed or peel from

the surface (Fig. 45). At very high magnifications of a

cell nearly devoid of matrix, fibers 4-8 nm thick are

shedding from the surface (Fig. 46). Colony

disaggregation is usually completed 48 hrs after

initiation. However, coccal colonies are sometimes not

completely degraded, with the cells held together by the

remaining fragments of matrix (Figs. 47 and 48). Slight

physical pressure can break these colonies apart.

Single cells within these colonies still possess a

remnant of the matrix (Fig. 49). This layer is dense,

































Figures 41 and 42. Scanning electron micrographs of
disaggregating colonies of Ms. mazei strain S6.


Fig. 41.


A disaggregating colony with little matrix
material holding the cells together.


Fig. 42. Higher magnification of colony with
sarcinae on the surface.







I. -y


c*1 r
'9P" rV


;rNwm-ijjj


r,rr~c=r!ra
Jtl~I


6,
~4j


r~





















Figures 43-46. Thin sections of Ms. mazei strain S6
showing the matrix material during colony disaggreg-
ation.

Fig. 43. Disaggregating colony held together by a
loose fibrous matrix.

Fig. 44. Individual cell arrested during division.
The matrix in the septum has the same
fibrous appearance as the matrix on the
cell surface.

Fig. 45. Micrograph showing the matrix peeling or
shedding from the cell surface in layers.

Fig. 46. High magnification of the matrix on a cell
that has almost lost all of the matrix.
The matrix material is present as thin
fibers and tubules are still associated
with the cell surface.


M= matrix

g= granules

pm= plasma membrane

t= tubules


S= septum








































-1 pm


* S1


'!q$4. pr.i I'

p.4



Objn .-h O.lpm
NGP.r


4i I "


S V *


4M


"A


*1$~W


1p~m -
'


'A'It ir


*- P '


1"


s~ ~II
c~ir h


I ,
























Figures 47-49. Thin sections of Ms. mazei colonies in
the later stages of colony disaggregation.


Fig. 47.


Fig. 48.





Fig. 49.


Low magnification of a loose colony held
together by remnants of the matrix.

Higher magnification of a partially
disaggregated colony. A thin layer of
matrix material remains around each cell
in the colony while the associated free
cells lack this material.

A single cell that is surrounded by
remaining matrix. Tubules are present
between the matrix and the cell surface.


M= matrix

L= protein layer

g= granules

pm= plasma membrane


t= tubules






4-

9 ,


/


S*~


20 pm


p1
I
I. I










uneven, approximately 50 nm thick, and is not laminated.

The layer is located several hundred nanometers from the

cell surface. Such cells are less frequent as the

cultures age.



Morphology of the Limited Life Cycle



Methanosarcina mazei Strain S6



A limited cycle occurs in the wild type Ms. mazei

when the colonies are still actively growing before

matrix degradation and colony dissagregation begins.

When the wild type Ms. mazei was repeatedly transferred

in the laboratory, it gradually lost the ability to

undergo the complex life cycle. It failed to

disaggregate into single cells and the matrix remained

intact. This variant strain can only disperse by

fragmentation of the sarcinal colony. Ms. barkeri and

the variant of M. mazei strain S6 are limited to this

cycle and do not break up into individual cells. When

these cultures are passed through 5 pm filters into

fresh media, colonies have the same general morphology

as mature colonies found in the complex life cycle. If

these colonies are disturbed sufficiently by methane gas

evolution or by shaking, they will fragment into smaller

masses. The size depends on the severity of the

shaking, and if very hard (vortexing for 1 min), then










individual units approximately 3.0 pm in size are

released (see boxed area in Fig. 24). These will again

grow into larger masses when placed in fresh media. The

developmental changes of maturing sarcinal colonies of

Ms. mazei S6 were examined in the variant strain over a

period of 360 days. Cultures of Ms. barker were

examined after 14 and 90 days of growth.

In thin sections, the cytoplasm of the cells of

5-day colonies grown on acetate stains rather evenly

(Fig. 50). At this stage the colonies are found singly

in the culture medium and are uniform with a diameter of

3-5 pm. The individual cells themselves are 0.6-1.0 pm

in diameter and are surrounded by a 30-60 nm matrix.

The electron dense granules are scattered throughout the

cytoplasm and polyphosphate bodies are rarely seen.

Membrane tubules between the protein layer and the

matrix are not commonly observed in cultures restricted

to the limited cycle.

Within 10 days, the colonies enlarge to 8-10 pm and

the cells to 1.5 pm (Fig. 51). Cells of this age show a

variability of cytoplasmic staining correlated with

three morphological types. These cell types are

arbitrarily defined and intermediate forms can be found.

The first type (A) has a dense rather dark staining

cytoplasm, while that of the second type (B) appears

less dense and stains lightly. The third cell type (C)

appears highly degenerated and is usually found near the



























Figures 50 and 51. Young colonies of the variant
strain of Ms. mazei strain S6 growing in the limited
cycle.


Fig. 50.


Fig. 51.


Five-day old colony with fairly uniform
staining cells.

Ten-day old colony showing a variability
in the cytoplasmic staining of cells.
Cell type "A" stains rather dark while
cell type "B" stains lighter. Cell type
"C" is highly degenerated with numerous
inclusion vescicles. A granule-containing
membrane bound core is present
(arrowheads).


M= matrix

g= granules































A


bib.
3..'
50 ) <* -i-












*. -----
^te ---^-- ---- --^^^--^^ --- ^


A4


,r
~

~
F3

"~


C










periphery of the colony. This type has numerous

inclusions and membranous structures and does not show

the cytoplasmic staining homogeneity seen in

younger-looking cells.

Two kinds of membrane cores are found in the

cytoplasm of cells restricted to the limited cycle. One

kind is found in cell types A and B (Figs. 52-55 and

arrowheads Fig. 51). They are 90 nm in diameter and

appear to have a double membrane surface 25 nm thick.

The membranes are straight and have a rigid appearance.

A trilamellar unit structure is only rarely demonstrated

(Fig. 54). There can be up to three cores of this kind

per cell and when more than one is present they are

arranged parallel to each other (Fig. 53). Inside the

cylindrical cores are electron dense granules arranged

in single file. These granules are of the same

appearance as those found in the cytoplasm but are much

more uniform in size at 40 nm. These cores are observed

in less than 1% of the cells in thin sections but almost

traverse the cytoplasm and are closely associated with

the plasma membrane. A connection with the plasma

membrane has not been observed and the ends appear to

open into the cytoplasm (Fig. 55).

The second kind of core is associated with the

plasma membrane in cell type C (Figs. 56 and 57). These

are 100 nm in diameter and are shorter than the cores

seen in younger cells (above). They are more numerous
















Figures 52-57. Thin sections of membrane cores found
in Ms. mazei strain S6. Figs. 52-55 are of the
granule-containing cores found in cell types "A" and
"B". Figs. 56 and 57 are of plasma membrane-associated
cores found in cell type "C".


Fig 52.


A high magnification of the core present in
figure 51 (arrowheads). The core nearly
bisects the cell and contains a single
file of cytoplasmic granules.


Fig. 53. A cell with two of the granule-containing
cores arranged parallel to each other
(arrowheads).

Fig. 54. A granule-containing core at high
magnification showing the double membrane
structure.

Fig. 55. A granule-containing core with one end
opening into the cytoplasm.


Fig. 56.



Fig. 57.


A grazing section of a plasma
membrane-associated core. A regular
pattern is observed in the core surface.

A cell containing several randomly
arranged cores, one of which shows a
connection with the plasma membrane
(arrows).


M= matrix

g= granules

im= inner core membrane

om= outer core membrane






























41k4



~q
C. OL


541

,rip



Iku : 16










however, and are not in an ordered arrangement. These

cores do not possess electron dense granules and appear

empty. The inner membrane is continuous with the plasma

membrane (arrows Fig. 57) and in grazing sections, a

regular helical pattern is observed (Figs. 56 and 57).

This regular pattern is probably due to the protein

layer in close contact with the inner membrane. The

ends of the outer membrane have not been resolved.

Twelve-day sarcinal colonies of strain S6 fixed

only with glutaraldehyde and embedded in epon/araldite

still fluoresce when viewed under UV irradiation. A

comparison of cells observed in TEM (Fig. 58) and the

same cells under UV epifluorescence (Fig. 59) reveals a

correlation between the intensity of fluorescence and

the type of cell observed within the colonies (see

above). The dark staining type A cells observed in thin

sections fluoresce brightly while the lighter staining

type B cells and degenerated type C cells fluoresce very

weakly or not at all.

At 12 days the three cell types within a colony

are present in approximately equal numbers but by day 15

the second lighter staining cell type predominates (Fig.

60) and in cultures older than 21 days the degenerative

cell type is most common (Fig. 61). By 90 days only a

few of the younger cell types can be found (Fig. 62),

and at 105 and 360 days (Fig. 63), which appear the

same, only a few single cells look viable. The matrix































Figures 58 and 59. Sections showing the correlation
between cell type and autofluorescence.

Fig. 58. Thin section of colonies showing the
various cell types.

Fig. 59. Thick section of the same cells in figure
58 but viewed with fluorescence
microscopy. The dark staining cells in
thin sections (type "A") are the same
cells that fluoresce brightly in thick
sections.











Wl'


I


4 ='
S


.. v4


1pm -




























Figures 60-63. Thin sections of older cultures of the
variant strain of Ms. mazei S6 grown in the limited
cycle.


Fig. 60.


Fig. 61.


Fig. 62.


Fifteen-day old colony showing the three
cell types (see text).

Twenty-one-day colony with cell type "C"
predominating.

Ninety-day colony where most of the cells
are dead.


Fig. 63. One-hunded-twenty-day colony that appears
the same as one-year-old colonies (not
shown). Only a few viable looking cells
are present.













between degenerated cells is poorly defined and the

granules and polyphophate bodies persist. Possible

resting forms (see below) can be found in these older

cultures that have thick specialized walls.





Possible Resting Forms



Cultures of the variant Ms. mazei strain S6 older

than three months are largely composed of dead cells.

However, several types of structures which may be

resting forms are found (Figs. 64-66). Islands of

viable looking cells can be found toward the center of

the colonies (Fig. 64). In thin sections these are

usually composed of 10-15 cells each surrounded by a

dense layer of matrix material 100-200 nm thick.

Frequently, individual cells can be found with a

specialized uniform layer 40 nm thick that is apparently

derived from the matrix but appears more dense (Fig.

65). Numerous membrane tuhiles may be found outside the

protein layer and division septa were never observed in

these cells. The single-cell forms are usually

spherical but they are sometimes rod shaped. They may

or may not contain cytoplasmic granules or polyphosphate

bodies. A specialized layer sometimes develop around

cells in younger cultures when the other cells still

look viable (Fig. 66).




























Figures 64-66. Thin sections of resting forms found in
old colonies of the variant strain of Ms. mazei growing
in the limited cycle.

Fig. 64. One type of resting form is composed of
many cells enclosed by a thick specialized
matrix.

Fig. 65. Single-cell resting forms surrounded by a
thick specialized layer.

Fig. 66. A group of viable looking cells with a
specialized layer (arrowheads) around one
of them.

R= resting forms

t= tubules

















Methanosarcina barkeri Strain 227



Young colonies of Ms. barkeri strain 227 (Fig. 67)

appear similar to the 5-day cultures of Ms. mazei

(compare with Fig. 50) and exhibit morphotypes similar

to those in the limited cycle. The nuclear regions are

prominent and granules are present in the cytoplasm.

The matrix is up to 150 nm thick, approximately twice

the thickness of the matrix in Ms. mazei, and is closely

packed between the cells. An additional layer

approximately 10 nm thick could be observed between the

plasma membrane and the matrix but this is usually

difficult to discern due to the close association of the

matrix (Fig. 68). Colonies at 90 days (Fig. 69) are

highly degenerated, much more so than older cultures of

Ms. mazei (compare with Figs. 62 and 63). A few viable

looking cells are present but these do not have

specialized layers and do not appear to be resting

forms. The degenerated cells contain laminated or

concentric stacks of membranes that form a variety of

structures (Fig. 70). The rest of the cytoplasm is

filled with membrane particles and electron dense

granules (Fig. 70). Occasionally, membrane bound areas

are found with dark staining spherical bodies (Fig. 71).

These are up to 80 nm in diameter and are made of


























Figures 67 and 68. Thin sections of 14-day colonies of
Methanosarcina barker strain 227.


Fig. 67.


Cells within a colony are enclosed by a
closely associated matrix. The cells
stain rather homogenously.


Fig. 68. High magnification of the cell surface
showing a layer in contact with the plasma
membrane.


M= matrix

g= granule

pm= plasma membrane

Nu= nuclear region

L= probable protein layer


























































M W
A1A4

























69-71. Thin sections of 90-day colonies of Ms.
strain 227.


Fig. 69.




Fig. 70.


Fig. 71.


Degenerative cells in these old cultures
do not show any regular structure.
Membrane vescicles are present along with
the electron dense granules.

A higher magnification showing the
vescicles (arrowheads) and granules.

A membrane bound vescicle containing
several very dark staining structures.
These are apparently made of coils of
membrane (inset).


M= matrix

g= granules

pm= plasma membrane

m= vescicle membrane


Figures
barker










p-*' ~-.


I ,v
.4 "
1I


"' 'LI,


f


0 pm -
** -\


I' 1
\ I


*
b


i i-

_. I


CI)


" -r


I


'7A *1
. c


1


t


>a-
At*










closely packed concentric rings 5.0 nm thick (insert).

The nature of these structures is unknown.





Disaggregation in Methanosarcina Strain LYC



Ms. mazei strain LYC when grown at pH 6.0, has a

morphology and life cycle similar to Ms. barker. When

colonies are viewed in SEM, cells are 1.0-2.5 pm in size

and are held in lobes that make up the sarcinal colony

(Fig. 72). The matrix is not as thick on the surface as

in strain S6 and individual cells can easily be seen.

If cultures are maintained at pH 6, the larger colonies

will fragment into smaller colonies when agitated. When

the pH of a culture is changed to 7.0 however, single

cells begin shedding from the colonies.

When the disaggregating colonies are observed in

SEM, cells on the colony surface are not tightly

associated (Fig. 73). These are similar to cells in

disaggregating colonies of strain S6 (compare with Figs.

41 and 42). Thin sections, however, show that cells are

being released from the matrix at the colony surface and

only the outermost cell layers are affected (Fig. 74).

The cells in the colony interior have a homogeneous

staining cytoplasm which contains only a few electron

dense granules and polyphosphate-like bodies. The

matrix is dark staining and is approximately 50 nm


























Figures 72-75. Disaggregation in Methanosarcina mazei
strain LYC.


Fig. 72.


Fig. 73.



Fig. 74.



Fig. 75.


SEM of a sarcinal colony growing at pH
6.0.

SEM of a colony after changing the pH of
the medium to 7.0. Free single cells are
apparent on the colony surface.

Thin section of disaggregating colony.
The matrix is only being degraded at the
colony surface.

High magnification of the colony surface
showing a single cell breaking through the
remaining matrix. The single cells are
osmotically sensitive and the cell surface
is not well preserved. However, dark
areas are present (arrowheads) that may
correspond to a protein layer.


M= matrix


L







80





72 ~ ~ i



























L ~Li
















I
pI ,II .~ ,


$ ~ -


1pm _


O.SSpm -






81


thick. It is fibrous and generally not in close

association with the plasma membrane. The matrix has a

loose appearance in the colony periphery and is

approximately 150 nm thick. At the very outer edge, the

matrix thins (Fig. 150) until the underlying cell breaks

through and migrates away from the colony. The cells

become contorted in this process and retain an irregular

appearance. A protein layer was not observed outside

the plasma membrane, which is poorly preserved. The

cytoplasm stains lighter than that of cells within the

colony and there are regions along the cell surface that

stain darker than the cytoplasm but lighter than the

matrix (arrows Fig. 75). It is possible that the cells

are osmotically sensitive and a layer, if present, was

damaged during processing.

The sarcinal colonies of strain LYC will sometimes

completely dissociate over a period of 48 hours.

Usually though, degradation stops before the entire

colony is broken up and colonies, although reduced in

size, are still seen at the bottom of culture tubes.



Freeze-Fracture Analysis



Unfixed, propane jet frozen, sarcinal colonies of

Ms. mazei strain S6 and Ms. barker strain 227 show a

tendency to fracture through the cytoplasm rather than

the plasma membrane and membrane faces are only rarely

















Figures 76-81. Freeze-fracture analysis of the variant
strain of Ms. mazei strain S6 and of Ms. barker strain
227.

Figs. 76 and 77. Cytoplasmic fractures through Ms.
mazei grown in the limited cycle showing two types of
particles.


Fig. 76.


Fig. 77.


A high magnification of the smaller class
of cytoplasmic particles.

Low magnification with larger particles
(arrows) which probably correspond to the
polyphosphate bodies.


Figs. 78 and 79. Plasma membrane fractures through Ms.
mazei growing in the limited cycle revealing a high
number of intramembrane particles.


Fig. 78.


Fig. 79.


Fig. 80.



Fig. 81.


High magnification of intracytoplasmic
particles.

Low magnification of plasma membrane
fractures of several cells.

Cytoplasmic fracture through Ms. barker
showing irregular particles and a smooth
fracture surface through the matrix.

Plasma membrane fracture through Ms.
barker showing a high number of membrane
particles arranged in regular patterns.


M= matrix

Cy= cytoplasm


pf= protoplasmic fracture













observed. Replicas of the variant strain S6 growing in

the limited cycle show two classes of intracellular

particles (Figs. 76 and 77). One ranges in size from 10

to 20 nm and the other measures from 80 to 200 nm. The

latter number 1-2 per cell and are probably the

polyphosphate bodies. A dense arrangement of protein

particles 9.0 nm in size was found in the plasma

membrane (Figs. 78 and 79). Cultures of S6 examined at

5, 10, 14, and 21 days showed no difference in the

particle distribution in the fracture planes. Free

single cells of strain S6 were not observed to fracture

through the plasma membrane.

Ms. barker strain 227 possessed a heterogeneous

population of cytoplasmic particles ranging from 10 to

30 nm. with approximately the same distribution as

observed in Ms. mazei. The larger class of particles

corresponding to the polyphosphate bodies was not

observed (Fig. 80). The matrix is approximately 120 nm

thick and fractures with a smooth surface. The plasma

membrane contains a large number of particles 9.0 nm in

size that are commonly arranged in regular arrays in

many areas (Fig. 81).

Colonies of strain S6 growing in the complex life

cycle showed a similar tendency to fracture through the

cytoplasm rather than the plasma membrane. However

these showed a more complex structure than those of

colonies growing in the limited cycle. Fracture planes


























Figures 82-85. Freeze-fracture analysis of Ms. mazei
strain S6 grown in the complex life cycle.
Figures 83-85. Specialized areas found associated with
the plasma membrane.


Fig. 82.


Fig. 83.


Fig. 84.


Fig. 85.


Fracture through the cytoplasm and a large
polyphosphate body.

A fracture through a stack of laminated
material.

A fracture through several laminated areas
that curve around and back on the stack.

A fracture through a lamminated region
where individual layers can be clearly
observed. The layer folds back on itself
and may be one continuous layer.


P= polyphosphate


cy= cytoplasmic fracture








86























Ilk.
lto

















T i





4V


















4 40
tr








b
8 5h










at .









would frequently pass through polyphosphate bodies (Fig.

82). These are very large up to 0.8 pm in size with a

rough even fracture surface. Specialized laminated

structures are associated with the plasma membrane

(Figs. 83-85). These usually consist of stacks of

material in curved or convoluted forms with individual

layers approximately 12 nm thick. Sometimes the

structures appeared as tightly packaged folded layers

11.4 nm thick arranged in parallel coils (Fig. 85).

These areas may correlate to groups of tubules observed

at the cell surface in thin sections.



Cytochemical Analysis of the Cell Surface



Silver Stain



When thin sections of 12-day sarcinal colonies of

strain S6 are etched with 1% periodic acid for one min

and then stained with silver, the colonies show moderate

labelling of the matrix material (Fiq. 86) indicating a

general polysaccharide composition. The layer just

outside the plasma membrane does not label. When

sections of disaggregating colonies were etched for 15

min before staining, the matrix shows a much higher

level of staining (Figs. 87 and 88). The matrix

material is evident and fibers throughout the

extracellular space are prominent. Again, the layer























Figures 86-88. Siver methenamine staining of Ms. mazei
strain S6.


Fig. 86.






Fig. 87.






Fig. 88.


Sections of cells etched for 1 min show a
light labelling of the matrix
(arrowheads). The cell surface has a dark
appearance due to the etching but does not
label with silver (arrows). The
cytoplasmic granules show heavy labelling.

Thin section showing an intact colony,
disaggregating colony, and free cells.
The sections were etched for 15 min and
the staining is much heavier than in
figure 86. Only the matrix material
stains. The free cells do not stain.

Higher magnification of a cell with some
remaining matrix material. The cell
surface and associated tubules do not
stain while the matrix stains heavily
(arrowheads).


g= granules

t= tubules





89



4,o
















I 4


0. 5
rt 4


I pm -











close to the plasma membrane and the associated membrane

tubules do not stain. Free cells do not show any

specific labelling of the cell surface, indicating a

lack of matrix material. Controls not treated with

fixer after silver staining had the same level of

non-specific background labelling and this step was

subsequently eliminated. The other controls did not

label.



Fluorescein-Labelled Lectins



A strong fluorescence was observed at the colony

surface when strain S6 was treated with fluorescein-

labelled PNA, SBA, DBA, or RCA120 lectins (Fig. 89).

These lectins are specific for galactosyl groups.

Fluorescence was not observed when galactosamine was

present in the reaction mixture. Free cells generally

did not label with only an occasional cell showing

fluorescence (not shown). This is expected due to the

lack of matrix material. No fluorescence was observed

in samples treated with ConA, UEA I, or WGA lectins.

These are specific for mannosyl and glucosyl, fucosyl,

and glucosaminyl groups respectively.




























Figures 89-91. Lectin labelling of the matrix of
intact colonies of Ms. mazei strain S6.

Fig. 89. UV micrograph of fluorescein-labelled
soybean lectin binding to whole cells.
Peanut, Dolichos, and Ricinus lectins
showed a similar fluorescence (not shown).

Fig. 90. Unstained thin section treated with
colloidal gold-soybean lectin. The
labelling is heaviest over the matrix
material (arrowheads).

Fig. 91. Control section with the blocking hapten
sugar galactosamine added to the reaction
mixture. The labeling is non-specific.





92





ii'%^>-^ <












Gold-Labelled Lectins



Based on the results obtained using fluorescein-

labelled lectins, an attempt was made to stabilize

colloidal gold with PNA, SBA, and RCA. PNA and SBA

stabilizations were successful but RCA-gold would

flocculate during centrifugation and could not be used.

PNA and SBA-gold treated cells are labelled lightly over

the entire matrix (Fig. 90). These lectins are specific

for N-acetyl galactosamine. Only non-specific labelling

was observed on thin sections of single cells. The

controls that included galactosamine in the labelling

mixture (Fig. 91) show only non-specific labelling.

Sections receiving colloidal gold only did not label

(not shown).



Enzyme Extraction



The layer just outside the plasma membrane can be

removed when colonies are treated with protease before

fixation. Colonies treated for 5 or 10 min show

protoplasts enclosed by the intact matrix which is 20-70

nm thick (Fig. 92). At higher magnifications (Fig. 93),

the cytoplasm is bound solely by the plasma membrane,

and the region between the cell surface and the matrix

is free of particulate matter. Extensive cell damage