Comparative ultrastructure of selected aerobic spore-forming bacteria: a freeze-etching study.

33 Pages·2000·20.76 MB·English

BACTEMOLOGICALREVIEWS,June 1969,p. 346-378 Vol.33,No.2
Copyright ©1969 AmericanSocietyforMicrobiology Printedin U.S.A.
Comparative Ultrastructure of Selected Aerobic
Spore-forming Bacteria: Freeze-Etching Study1
a
S. C. HOLT AND E. R. LEADBETTER
Department ofMicrobiology, UniversityofMassachusetts, andDepartment ofBiology,
Amherst College, Amherst, Massachusetts 01002
INTRODUCTION............................................................ 346
MATERIALS AND METHODS ............................................ 347
Organisms................................................................. 347
Growth.................................................................. 348
ElectronMicroscopy......................................................... 348
RESULTS................................................................... 348
VegetativeCells............................................................. 348
Cellwalls................................................................ 348
Plasmamembranes ..................................................... 350
Mesosomes............................................................... 350
Spores.................................................................... 360
Coats................................................................... 360
Exosporia................................................................ 360
DISCUSSION................................................................ 370
VegetativeCells............................................................. 370
Cellwalls................................................................ 370
Mesosomes............................................................... 372
Spores.................................................................... 372
Exosporia ............................................................. 372
Sporecoats............................................................... 372
Nomenclature............................................................ 374
COMMENTS ............................................................ 376
LITERATURE CITED..................................................... 376
INTRODUCTION morphology of the vegetative cells and spores
"What, then, are the basically important was naturally explored with the advent of the
morphological characters clearly para- electronmicroscope;eventheearlieststudiesindi-
mount is the structure o.f. t.he individual cated that, compared to the vegetative cell, the
vdeugcettiavteivoercerlelssti.ng. .ce[lalnsd]. .. .." o[fSttahneierreparnod- spoOrneewaaspparsotarcuchtutroalluyndceormsptlaenxdienngtittyhe(33d,o3r4m)a.nt
van Niel (74)]. state of the mature spore and its tolerance to
severe chemical and physical extremes has in-
That bacterial endospores differ in their size, volved an attempt to correlate and integrate the
shape, surface morphology, thermal stability, presence, or development, of ultrastructural
siteofformationin the parent cell, as well as in features of the spore with physiological aspects
other characteristics, has been recognized for ofsporeformationandgermination (51,75).
some time (51, 63, 71). So also has been the Electron microscopic examination of thin
diversity of vegetative cells ofBacillus and Clos- sections ofspores revealed, as Robinow (63) has
tridium species with respect to temperature and observed, that spores ofdifferent Bacillus species
pHoptima,nutritionalversatility,and soforth. differ primarily in the nature and organization
Striking differences between bacterial spores oftheirexteriorlayers, whereasthesporeinterior,
and the vegetative cells, in which they were from which the emerging vegetative cell will
formed and which appear again following spore develop, is basically similar from species to
germination, were readily apparent to early species.
students who examined stainedor unstained cells Although a comparative study of the surface
in the light microscope, as well as to those who anatomy of spores of an immense variety of
later applied phase-contrast microscopy (63). Bacillus species was undertaken a decade ago
The anatomical basis for these differences in the (3, 4), there are essentially no truly comparative
IDedicatedtoBacillusfastidiosusandtoitsdiscoverer. studies of the surface, or internal, details of
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VOL. 33, 1969 AEROBIC SPORE-FORMING BACTERIA 347
Bacillusvegetativecellsorsporesusingthenewer, scopic examination ofcells and their parts might
improved methods of tissue preparation com- now reflect more accurately their structural
bined with examination in electron microscopes details and arrangement. This usefulness of the
capable of higher resolution. The use, for freeze-etching approach to study and compare
example, of the Ryter-Kellenberger procedures the anatomy of spores and vegetative cells of a
and their modifications (23, 39) for cell fixation, few Bacillus species (40, 57, 58) prompted us to
improved techniques ofshadowing (55), negative broaden our study to include representatives of
staining (14, 49, 79, 84), and of surface replicas the three classic cytological groups of aerobic
(20, 65) indicatedevenmoreclearlythestructural spore-forming bacteria (6, 71): those species
complexity of cells and their components. The which form ellipsoidal or cylindrical spores
paucity of comparative information of this sort without swellingofthesporangium, those species
became apparent to us (i) when we attempted to which form ellipsoidal or cylindrical spores but
integrate our examination (39) of the fine struc- which swell the sporangium, and those species
tureofvegetativecellsandspores ofB.fastidiosus withsphericalsporesandswollensporangia.
with the ultrastructure of other Bacillus species, Wehopedthatsuchacomparativeexamination
(ii) when the globular wall surface revealed by of this group would establish and clarify the
Nermut and Murray's (55) elegant analysis of
extent of similarity or diversity in the structural
B. polymyxa was contrasted with the apparently featuresofthesebacilliandspores.
nonglobular surface of B. megaterium (54), and
(iii) when the apparent differences in the wall
MATERIALS AND METHODS
structure of B. cereus examined by different
meansbecameknown (14, 57). Organisms
The introduction and refinement ofthe freeze-
etching (5, 7, 44-46, 48) approach for tissue With the exception ofB.fastidiosus (9), which
preparation made it clear that electron micro- wasreisolated byPaulButler, theorganismsused
TABLE 1. Organisms and their sources
Organism Strain Source
Bacillus cereus.7004 R. A. Slepecky, Syracuse Univ.
B. cereus.7004 C. B. Thorne, Univ. of Massa-
chusetts
B. cereus.4342 R. A. Slepecky, Syracuse Univ.
B. megaterium.6459 R. A. Slepecky, Syracuse Univ.
B. megaterium.899 R. A. Slepecky, Syracuse Univ.
B. megaterium QM 1551 (ATCC 12872) H. Levinson, U.S. Army, Natick
......................
Laboratories
B. megaterium QM 1584 (ATCC 19213) H. Levinson, U.S. Army, Natick
......................
Texas Laboratories
B. megaterium QM 1605 (ATCC 14581) H. Levinson, U.S. Army, Natic
......................
Laboratories
B. polymyxa UC R. A. Slepecky, Syracuse Univ.
........................
B. licheniformis 9945A C. B. Thorne, Univ. of Massa-
.....................
chusetts
B. subtilis W23Sr C. B. Thorne, Univ. of Massa-
...........................
chusetts
B. sphaericus ATCC 12300 J. M. Larkin, Louisiana State
.......................
Univ.
B. stearothermophilus 10 L. L. Campbell, Univ. of Illinois
...............
B. macroides........................A and P E. Canale-Parola, Univ. ofMassa-
chusetts
B. cereus var. alesti E. Canale-Parola, Univ. ofMassa-
chusetts
B. anthracis ......................... SterneC. B. Thorne, Univ. of Massa-
chusetts
B. psychrophilus.W..W16A..16AJ. M. Larkin, Louisiana State
..
Univ.
Sarcinia ureae.....................860 J. M. Larkin, Louisiana State
Univ.
348 HOLT AND LEADBETTER BACTERIOL. REV.
in this investigation were provided by the indi- microscope (28). Electron micrographs were
vidualsindicatedinTable 1. takenonKodakfinegrain,positive, 35-nmfilmor
Growth. Vegetative cells and spores of B. on Kodak contrast projector slide plates. Photo-
fastidiosus were grown as previously described graphic reversal of micrographs offrozen-etched
(39). Other organisms were grown in nutrient specimens did not lead to any different inter-
broth, on nutrient agar (Difco), or on Thorne's pretation or enhancement of quality. Therefore,
potato agar (77). Increased sporeformationoften theoriginalnegativeimagesarepresented.
occurred when NaNO3 and MnCl2.4H20 were
added, at final concentrations of 2 mg/liter, to RESULTS
nutrient broth or nutrient agar. Incubation was
Vegetative Cells
at 30 C, except for B. stearothermophilus (56 to
60C) andB.psychrophilus [22C (38)].Vegetative Cellwalls. Frozen-etched preparations reveal
cells grown on solid medium nearly always pre- that the surface of the cell wall of B. fastidiosus
sented more pronounced surface detail(s) than (Fig. 2, 3) isahighlyorderedstructureapparently
did those grown in liquid medium, although the composed of interwoven fibers or strands
basicfeatures wereseenincellsfromboth growth (approximately 0.5 nm in diameter), which form
conditions. a mesh or weave with apparently square inter-
stices of approximately 13.5 nm. This structure
Electron Microscopy of the cell wall fits well with the expectations
For thin sections, cells were fixed chemically based on the examination of thin sections (39;
with OSO4 and embedded in Epon (28). Samples Fig. 1), where a mesh-like fine structure may be
for freeze-etching were prepared as previously seen. In these chemically fixed preparations, the
described (29), except that cells grown on solid space between strands is approximately 11 nm,
media were placed directly on copper grids for a result not inconsistent with the shrinkage ex-
freezing. pectedfromchemicalfixationanddehydration.
The effect of glycerol as a cryogenic agent An apparently identical pattern (Fig. 5, 8-10)
(5, 45) was regularly examined. Such treatment may also be seen in the wall of two strains, A
yielded results essentially identical to those ob- and P, of B. macroides (2), although in this
tainedwithnonglycerinatedsamples. organism this pattern seems to be clearer and
Samples for negative staining were treated sharper than in B. fastidiosus. The underlying
with 1% (w/v) sodium phosphotungstate (pH inner wall layer is often observed in strain A
6.8), 0.5% (w/v) uranyl acetate (pH 4.4), or (Fig. 9) of B. macroides, apparently as a result
with 1% (w/v) ammonium molybdate (pH 7.0). of the ease with which the mesh-like layer is
Staining was done directly on 300-mesh carbon- removed. In these organisms, the dimensions of
coatedgrids (28). the interstices seem to be between 5 and 6 nm,
Carbon replicas were prepared by allowing a and the diameter ofthefiber or strand is 3 to 3.5
cell suspension placed on 3-mm copper grids nm. In addition to this interwoven layer, there
(solid) to dry for 12 to 18 hr in a desiccator is present, in strain P, an external globular layer
containing CaSO4. The dry grids were placed (Fig. 8) composed of particles 9 to 10 nm in
onthecoldtable (-150C) oftheBalzarsappara- diameter. Examination of thin sections likewise
tus, immediately shadowed, at an angle of reveals that in strain A (Fig. 6) the cell wall
approximately 30 to 32°, with platinum-carbon, appears incomplete, and portions of the outer
and were then coated with carbon. The replicas layer areabsent. Thecell wall ofstrain P (Fig. 7)
were removed from the copper grids by scoring is more electron-dense and thicker than that of
the surface and floating off the specimen in dis- strain A; these findings, then, are consistent with
tilled water or in 70% H2SO4. Organic material thoseoffrozen-etchedcells.
was removed as described for frozen-etched The mesh or screen pattern thus far described
samples (29). Allsamplesforelectronmicroscopy is not observed in the cell wall of B. polymyxa
were examined in a Philips EM 200 electron (1, 55, 56). Here,theouterlayerregularlyappears
FIG. 1. Tangential thin section ofchemically fixed B. fastidiosus vegetative cell. The fine structure ofthe
cell wallappearsasamesh. Abbreviations: W,cellwall; PM,plasmamembrane;S, cellwallseptum. Lead-stained.
Barrepresents 0.25 jm.
FIG. 2-4. Freeze-etch preparations ofvegetativecellsofB.fastidiosus. The meshedouterlayerofthe cell wall
is shown. Smallarrows indicate connections between the wallandthe underlyingouterlayeroftheplasma mem-
brane.InFig.3,note thelocalizedorderedarrayofparticles (whitearrows).Note thecompletedseptuminFig. 4.
Barrepresents0.25 ,um. Theencircledarrowinthecornerofeachmicrograph ofafreeze-etchpreparationindicates
the direction ofshadow; shadowsappear whiteagainstadarkbackground.
3.
11 "NOW
aI m
349
350 HOLT AND LEADBETTER BACTERIOL. REV.
asanarray ofparticles orglobules approximately surface structure apparently identical to that
7 nm in diameter (Fig. 11-13) with a center-to- observedinB.fastidiosusandB.macroides.
centerspacingof9to11 nm. Plasma membranes. In all the organisms
Thisglobulararrayisalsofoundonthesurface examined, the plasma membrane is clearly visible
ofvegetativecellsofB.anthracis (Fig. 15, 16),but beneath the cell wall, and fibrous materialappar-
here this outer layer seems easily fragmented. ently linking the inner layer of the wall and the
The center-to-center spacing for these particles outer surface ofthe membrane is usually evident
is7to 10nm,whereasthediameteroftheparticles (e.g., Fig. 3, 4, 9, 16, 17, 22). These "connecting
is 6 to 8 nm. The outer globular surface appears strands," whichvaryfrom 5to 15nmindiameter
to be composed of two (possibly three) layers, (Fig. 2, 4, 22) often seem considerably larger
each layer in turn being composed of parallel than the fibrous material composing the mesh
strands ofglobular particles to form the sheet or orscreen-typeouterwalllayers.
layer. The direction ofthesheets is randomfrom The outer surface of theplasma membrane is
layer to layer. Whether this apparently multiple covered with particles approximately 12 nm in
layering is an accurate representation of the B. diameter (Fig. 3, 4, 9, 11, 15, 20, 22) although
anthracis wall or the result of the slippage or these particles seem not to be present in such
translocation of wall fragments during freeze- profusion on the inner surface ofthe outer mem-
etchingis not known. brane (Fig. 23).
The surface ofB. psychrophilusvegetative cells Mesosomes. In addition to these membrane-
appears granular (Fig. 17), although there is boundparticles, thereareareaswithmoreordered
some suggestion of fine structural detail. A structure. These areas we willcollectively refer to
globular wall layer becomes strikingly evident, as mesosomes (12, 17, 32). In Fig. 24 to 27, the
however, when sporulating cells are examined mesosome takes on arrangements suggestive of
(Fig. 18, 19). The regularly arranged globules the vesicular mesosomes seen in thin sections.
are about 14 nm in diameter and have center-to- These mesosomes are found not only in small
center spacing of 15 to 16 nm. Whether this localized areas (Fig. 24, 26), including areas of
globular layer, which we have never succeeded cross-wall formation (Fig. 24, 28), but may oc-
in detecting in vegetative cells, appears on or in cupylarge regions ofthe membrane surface (Fig.
the wall as a result ofspore-associated syntheses 25, 27). Such alarge area may be seenin Fig. 27,
orissimplyunmaskedasaresultofwalldegrada- inwhichthemesosomeisrevealedasanextensive
tionis unknown. honeycomb structure beneath the inner layer of
B. sphaericus, which like B. psychrophilus thecellwall.OfinterestinFig. 27isthelargearea
forms spherical spores which swell the spo- ofthe plasma membrane which is neither honey-
rangium, has the same wall characteristics: the combed nor covered with the typical 12-nm
vegetative wall lacks structural detail, whereas particles; the texture of this region is similar to
the sporulating cells possess a globular wall layer that of the adjacent mesosome itself. This rela-
(see Fig. 55). tively smooth, particle-free plasma membrane
Stillanothertypeofcellwallsurfaceisfoundin surface is found also in areas of septum forma-
B.megaterium (Fig.20,21)andinB. licheniformis
tion (Fig. 29), with which mesosomes have often
inwhichwehavebeenunabletodiscernapattern
or texture on the outer wall layer in vegetative been associated (17, 32). The uneven, rippled
or sporulatingcells. surface oftheinnerlayer ofthecell wall (Fig. 14,
The aerobic spore-forming coccus Sarcina 27,28, 31)probablyreflectsthepresenceofunder-
ureae (Fig. 22) and the thermophile B. stearo- lyingmesosomes ofatleastmoderate complexity.
thermophilushaveanoutermeshedorinterwoven Still another type of mesosome, suggestive of
FIG. 5. Freeze-etch preparation ofvegetative cellofBacillus macroides strain A showing the woven or mesh-
likecellwall. Thecylindricalstructuresoverlayingthe wallareflagella (F). Barrepresents 0.25um.
FIG. 6and 7. Thinsection ofB. macroides strainsAandP, respectively. Note (Fig. 6) that the wallofstrain
Aappearsincomplete (arrows) whencomparedto that ofstrain P (Fig. 7). Lead-stained. Bar represents0.1 ,um.
FIG. 8. Freeze-etch preparation ofB. macroidesstrainP. Note the globularsurfaceexternal to the interwoven
ormesh layer. Barrepresents 0.5 Mm.
FIG. 9 and 10. Freeze-etch preparations of B. macroides strain A showing connections between the inner
layer ofthe wallandplasma membrane (dark arrows). In Fig. 9, the outer layer ofthe wall has been fractured
to reveal the underlying inner layer (IL) ofthe cell wall. In Fig. 10, the interwoven nature ofthe wall isshown
athighermagnification. Barrepresents0.25,um.
351
352 HOLT AND LEADBETTER BACTERIOL. REV.
FIG. 11-14. Freeze-etch preparations ofvegetative cellsofB. polymyxa. Thefine textureoftheouterlayerof
the cell wall is shownasalayer ofparticles6 to 7nm in diameter. These particles appearto becomposedofsub-
units (Fig. 12a, 13a). In Fig. 11 what traybea vesicularmesosome is visibleandin Fig. 14 theinnerlayerofthe
cell wall is clearly visible. Note the wallstructure in Fig. 14 (arrows). In Fig. 11, 12, and 14, thebarrepresents
0.25Am; in Fig. 13, 1 ym; andin Fig. 12aand 13a, 0.1 sum.
FIG. 15 and 16. Freeze-etch preparations ofvegetative cells ofB. anthracis. The outer layerofthe cell wall
iscomposedofparticlesapproximately 8nmindiameter, underwhichthenonstructuredinnerwalllayerisvisible.
InFig. ISa,atleastthreestructurallayers (arrows)areapparentlyingabovetheinnerlayer. Inthe high magnifica-
tion micrograph (Fig. 16a), the individual particles are evident. Note the directional change in the outer layers
(arrows). In Fig. 15, thebarrepresents 0.5Am;in Fig. 16, 0.25Am;andinFig. 16a, 0.1 jim.
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VOL. 33, 1969 AEROBIC SPORE-FORMING BACTERIA 355
-.%-.Iz,.- "VA
4f1
i.
FIG. 20 and21. Freeze-etch preparations ofvegetative cells ofB. megaterium 899. The cell wall lacks ap-
parentfine structure. In Fig. 21, a completedseptum beneath thepartially constrictedportion ofthecell wallis
visible. Note thepoly-g-hydroxybutyrate (PHB) granule. Barrepresents 0.5,m.
FIG. 17-19. Freeze-etch preparations ofB. psychrophilus. The vegetative cell wallsurface (Fig. 17) appears
granular. However, duringspore development a layer ofregularly arrangedparticles, approximately 14 nm in
diameter, becomesevidentin oron the wall. Thesphericalnatureofthese globulesisevidentin Fig. 18a and19a
In Fig. 17-19 the bar represents 0.5,m; in 18aand19a, 0.1 ,um.