THE ANATOMY OF THE BACTERIAL SURFACE' its usageno longer mystifies or amuses those ...

23 Pages·2002·5.63 MB·English

THE ANATOMY OF THE BACTERIAL SURFACE'
M. R. J. SALTON
Department of Bacteriology, University ofManchester, Manchester, England
I. Introduction.......................................................................... 77
II. Surface Appendages.................................................................... 78
III. Bacterial Capsules...................................................................... 80
A. Chemical Composition.............................................................. 80
IV. The Cell Wall....................................................................... 83
A. Chemistry of Cell Walls............................................................. 83
V. Protoplast Membranes................................................................. 86
A. Chemical Composition of Membranes................................................ 89
VI. LocalizationofEnzymesinBacterialCellsandaSummaryoftheComparative Chemicaland
Biochemical Anatomy of Gram-positive and Gram-negative Bacteria.................... 90
VII. TheGramStainandtheBacterial Surface................................................ 91
VIII. References............................................................................ 95
I. INTRODUCrION which have accompanied this period. Thus bac-
terial anatomy has been concerned not solely
Despite its minute dimensions, the bacterial
with the microscopic appearance of the various
cell has been quite amenable to dissection into
morphological entities of the cell, but also with
itscomponentpartsand, duringthepasttenorso
the more exciting and broader problem of inte-
years, a great deal has been learned about the
grating both the structural and functional
chemical and biochemical anatomy of micro-
properties of the bacterial cell.
organisms. The term "bacterial anatomy" has
The student of human anatomy is only too
become firmly entrenched in our literature and
well aware that shape is a most important ana-
its usage no longer mystifies or amuses those
tomical attribute. The general importance of
biologists who are used to handling the "coarse"
shape is amply illustrated by the anatomical and
structures of higher animal and plant cells. The
aesthetic qualities of the female form (unfor-
transition from the older study of bacterial
tunately, for technical reasons, the slide illus-
cytologytothemodernfieldofbacterialanatomy
trating this point in the lecture could not be
has really dated from the introduction of the
reproducedhere). Sowithbacterial anatomy, the
electron microscope, the shadow-casting tech-
shapeofthecellisofgreatinterestand,asrealized
nique, and the development of elegant methods
long ago by Leeuwenhoek, it is the surface struc-
for slicing up the bacterial cell into incredibly
ture which determines the characteristic shape of
thin sections. These advances have occurred at
the time of a tremendous expansion of our bio- the cell. The elucidation of the surface structure
chemical knowledge and the emergence of a of bacterial cells has attracted much attention in
wealth ofbiochemical and biophysical techniques recent years and for diverse reasons. Thus the
immunologist has beeninterested in the presence
1The Office of Naval Research Lecture at the ofspecific antigenic groupings onthe cell surface.
Society of American Bacteriologists Annual
The biochemist has concerned himself with the
Meeting at Philadelphia in 1960 with the above
transport of substances into and out of the cell
title formed the basis for this contribution. I
should like to express my sincere thanks to the across the permeability barriers and those inter-
Office of Naval Research and the Program Com- ested in the control of microorganisms by anti-
mittee of the Society for making this lecture biotics have become intrigued with the possi-
possible. This review of a rapidly growing field is bilities of killing bacteria by interfering with the
not intended to be comprehensive but has been biosynthesis of surface structures. The results of
prepared in the hope that it may focus attention
all these diverse investigations have begun to
on certain aspects and perhaps stimulate some
interest, to eliminate thelarge gaps in ourknowl- focus attention on the bacterial surface and we
edge. cannowbegintotalkaboutitsdetailed anatomy.
77
78 M. R. J. SALTON [VOL. 25
Figure 1. Spirillum serpens cells autolyzed and trypsin digested, showing flagella attached to basal
granules inside the cell (unpublished electron micrograph by R. W. Horne and M. R. J.
Salton). X60,000.
The surface structures of the bacterial cell can term fimbriae was first proposed by Duguid and
be conveniently classified (88) as surface append- his colleagues (27, 29) as the Latin equivalent
ages, surface adherents, rigid cell wall, and of "threads," "fibers," and "filaments" used in
protoplasmic membrane. earlier descriptions of these structures (3, 49).
This term has gained wide acceptance although
II. SURFACE APPENDAGES
Brinton (13) has suggested the word pili (Latin
Two main surface appendages ofbacterial cells
for "hair") as asynonym. The only other surface
are the flagella, the organs of locomotion of the
appendage that should be mentioned is that en-
cell (121), and the fimbriae (27, 29). The flagella
countered in the iron bacterium, Gallioniella
have now been well characterized chemically
(58, 121) and there is generally no difficulty in ferruginea, which possesses long strands of
distinguishing between the appearance of material that are obviously very different from
flagella and fimbriae in electron micrographs of flagella and fimbriae (113). All these surface
bacteria, as illustrated in figures 1 and 2. The structures may have intracellular origins;
1961J ANATOMY OF BACTERIAL SURFACE 79
Figure 2. Fimbriae arranged around the cell surface of Shigellaflexneri (Duguid and Gillies (28)).
X45,000.
flagella apparently pass through the wall and are responsible for the adhesive properties of
membrane and terminate in a spherical organelle certain bacteria (28), the functions they may
located in the bacterial protoplasm, as illus- confer on the cell have not been clearly estab-
trated in figures 1 and 2. Although the fimbriae lished.
80 M. R. J. SALTON [VOL. 25
III. BACTERIAL CAPSULES was brought to light when Ivanovics and Hor-
vath (55) detected fairly regular indentations
Capsules were among the first surface struc-
along the surface of the capsule of a Bacillus
tures to be removed from bacterial cells and
megaterium strain. There is probably little doubt
chemically characterized. The classical investiga-
tionsofAveryandHeidelberger(seeHeidelberger thatthe capsules ofmanybacteria arephysically
homogeneous structures of one type ofpolymeric
(43)) on the chemistry and immunochemistry of
the pneumococcal capsular polysaccharides were substance, but with refined methods of studying
the first extensive studies givingussome concept the anatomy ofthe bacterialsurface, some ofthe
more complicated structures, illustrated dia-
of the variety of compounds forming the surface
layers of certain bacterial cells. gramatically in figure 3, have now become well
established.
Until comparatively recent times, the bacterial
The relationship of the capsules to the rigid
capsule hasgenerallybeen regarded asahomoge-
neous accumulation of viscous material around cell wall has been of great interest and the isola-
the cell. However, Tomesik's (110) studies com- tionofspecific capsule-degrading enzymes (6, 23)
bining phase-contrast microscopy and antigen- hasbeenoftheutmost valueinstudying the sur-
faceanatomyof bacteria. There is now a variety
antibody reactions have shown that the capsules
of enzymes or enzyme systems available for the
of Bacillus anthracis and certain strains of
selective removal of bacterial capsules, leaving
Bacillus megaterium are by no means of simple
physical structure. Indeed, these investigations the viability of the decapsulated cells unim-
have revealed the presence of localized patches paired as well as the ability to synthesize the
of a capsular polysaccharide in a gel of capsular capsular material. Encapsulated pneumococci
(6, 23), klebsiellae (1), streptococci (63, 67), and
glutamyl peptide. The electron microscopic
Bacillus spp. possessing y-glutamyl peptides
studyby Labaw and Mosley (59) has established
a very complex physical state for the capsule of (112) have been enzymically decapsulated, thus
establishing the anatomical relationships of the
the Lisbonne strain of Escherichia coli. Macro-
capsules and wallsofthese organisms.
molecular components were embedded in a
structureless gel forming the capsule of this
A. Chemical Composition
organism. Stained preparations usually give the
impression that the capsular surface is smooth Chemically, bacterial capsules are polymeric
and continuous butafurtherphysical complexity substances of either polysaccharide or polypep-
I
a b c d
Figure 3. A diagrammatic representation of the types of capsular structure (taken from reference
88): (a) capsule forming continuous layer around cell; (b) capsular layer with banded fibrils (59); (c)
complex capsule with localized patches of polysaccharide and polypeptide (10); (d) discontinuities in
capsularsurface (55).
19611 ANATOMY OF BACTERIAL SURFACE 81
TABLE 1
Chemical nature of bacterial capsular substances
Organism ClassofSubstanceandProductsofAcid Hydrolysis
Gram-positive:
Bacillus anthracis Polypeptide: -y-D-glutamyl peptide
Bacillus megaterium Polypeptide: -y-glutamyl peptide
Polysaccharides: amino sugars; sugars
Bacillus circulans Polysaccharide: glucose, mannose, uronic acid
Pneumococci Polysaccharides:sugars, aminosugars,uronicacids,ribitolphosphate
Streptococci
Groups A and C Polysaccharide: hyaluronic acid-glucosamine, glucuronic acid
Rumen species Polysaccharide: galactose, rhamnose, uronic acid
Gram-negative:
Acetobacter capsulatum Polysaccharide: dextrin-glucose
Haemophilus influenzae Polyribophosphate
Aerobacter-Klebsiella group Polysaccharides: complex polyuronides-glucose, fucose, glucuronic
acid
Escherichia coli Polysaccharide: fucose, galactose, hexuronic acid
Data summarized from references 88 (giving original references), 42, 78, 127.
tidenature. Sofar asI am aware, heteropolymers structures, there being distinctive compounds in
containing polysaccharide and peptide residues the wall enabling us to differentiate one from the
covalently linked (as inbacterial walls) have not other. However, Nature has prevented us from
been encountered as capsular substances. Of all putting everythingintotightlittle compartments
the bacterial capsular polysaccharides studied, andtherearenowtwoinstancesinwhichcapsular
those of the pneumococci have been investigated substances have been found to contain com-
in greatest detail and the chemical structure of a pounds that we have come to regard as exclu-
number of different types has been determined sively wall substances. An extremely interesting
(43). Both amino sugars and uronic acids are example of this situation has arisen from the
widely distributed in capsular polysaccharides discovery of ribitol phosphate in the specific
from both gram-positive and gram-negative polysaccharide of type 6 pneumococcus by
bacteria. The capsular polysaccharides of gram- Rebers and Heidelberger (78). Up to the time of
negative bacteria must not of course be confused this report, ribitolphosphate compounds inpoly-
with the polysaccharide moieties of the protein- meric form had been found only in the teichoic
lipid-polysaccharide complexes constituting the acidsofthebacterialwall,discoveredbyBaddiley
macromolecular components of the wall or and his colleagues (4, 9). The second example of
envelope. The variety of materials forming bac- a material of capsular origin containing a wall
terial capsules (briefly summarized by Salton substance, probably the cell-wall amino sugar,
(88)) is illustrated in table 1 and includes hyal- muramic acid, isthepolysaccharide derived from
uronic acid, polyuronides, various polysac- BacillusmegateriumbyGuex-HolzerandTomcsik
charides, andthe y-glutamyl peptides. Thelist of (42). This material appeared to be immunologi-
capsular substances presented in table 1 is by no cally identical to the cell-wall substance and
means comprehensive. It does not include, for upon isolation the capsular polysaccharide was
example, other interesting surface polymers such found to contain glucosamine, galactosamine,
as the amino uronic acid forming the Vi antigen and an unknown amino sugar presumed to be
(16), the M proteins of streptococci (60), and muramic acid. Whether this material should be
other components that are not demonstrable by regarded as a true capsular polysaccharide, or
the methods usually employed for detecting whether it represents a local accumulation of
capsules (26). wall material being "over-produced" by the
In general, there is little chemical relationship dividing cell, could only be decided by further
between capsular substances and the cell-wall investigation. At least these two cases illustrate
82 M. R. J. SALTON[o[VOL. 25
1.0 7
0.9
w
0.8
w
0.7
0.6
U
w 0.5
0.4
0.3 a A
0.2 ll-..
A A A 4"A
0.1 m = - -A A A A
0
400 450 500 550
600
WAVELENGTH (MP
)
Figure 4. Absorption spectra of the products of Dische (22) reactions on Aerobacter (Klebsiella)
aerogenes strain A3 untreated walls (0-0); walls after extraction of capsular polysaccharide
(A A); and the hot-water extractable polysaccharide (E ). E, cm for 0.45 mgeachfraction
(89).
the point that there may be a greater chemical polysaccharide is retained and, on hydrolysis of
overlap between capsular substances and walls the "wall" fractions, monosaccharides character-
than we had hitherto suspected. Although both istic of the capsular substance are also present.
the cell walls and the capsular polypeptides from Fortunately with this strain it is possible to dis-
Bacillus anthracis and Bacillus megaterium con- tinguish between constituents of the capsule
tain the D-isomer of glutamic acid (52, 54, 84), (127) and the wall polysaccharide components,
the capsularpeptideisasimplepolymer,whereas for Dudman and Wilkinson (25) had shownthat
in the wall the D-glutamic acid is chemically the polysaccharide could be extracted from
linked in a more complex mucopeptide. intact cells with hot water. When the "wall"
The retention of capsular and surface sub- fractions were extracted in this way, the fucose
stances on the cell wall during its isolation pre- and uronic acid of the capsular polysaccharide
sents a problem in establishing the chemical werefoundinthewater-solublefractions, leaving
anatomy of the surface structures. Thus the M the insoluble wall fraction devoid of these sugars
protein was retained on isolation of the walls of (89). TheDische (22) reactionformethylpentose
a group A streptococcus and it could be removed can be used to follow the distribution of these
enzymatically from the walls by digestion with sugars in "wall" and capsular polysaccharide
trypsin (80). Further studies with a capsulated fractions as illustrated in figure 4. The monosac-
strain of Aerobacter (Klebsiella) aerogenes have charide constituents detected in "wall" fractions
also shown that during wallisolation some ofthe of encapsulated, slime-producing, and nonen-
1961] ANATOMY OF BACTERIAL SURFACE 83
TABLE 2 than those of gram-positive organisms (90). The
Monosaccharide constituents of "walls" of en- presence of spherical macromolecules in a cell
capsulated,slime-producing,andnonencapsulated wall was first reported by Houwink (48). Since
strains of Aerobacter (Klebsiella) aerogenes thenanumberofspirillaandothergram-negative
bacteriahavebeenshowntopossessfinestructure
Strain Preparation Monosaccharides
in the walls (see Salton (90)). Layers of hexag-
onally packed spherical macromolecules have
A3 capsulated Cell walls Galactose, glu-
been encountered most frequently. Although the
cose, fucose,
isolated wall of Escherichia coli has a homoge-
uronic acid
Wall extracted Galactose, glu- neous appearance on examination of shadowed
with hot cose preparations in the electron microscope (48, 93),
water it is evident from the beautiful thin sections,
Extracted Galactose, glu- prepared by Kellenberger and Ryter (57) and
capsular cose, fucose, presented in figure 5, that the wall is multi-
polysac- uronic acid layered. The studies ofWeidelandhis colleagues
charide
are now beginning to correlate the physical and
A3 (S) slime- Walls Galactose
chemical complexity of the wall of this organism
producing
(125). Atypeoffinestructurediffering fromthat
A3 (0) non- Walls Galactose
slime, non- commonly found in many of the gram-negative
capsular bacteria has been observed in the complex wall
or "envelope" of the organism, Lampropedia
Reference 89. hyalina. This macromolecular structure observed
independently by J. A. Chapman and M. R. J.
capsulated strains of Aerobacter (Klebsiella) Salton (unpublished observations) and R. G. E.
aerogenes are summarized in table 2. These Murray (unpublished observations) is illustrated
resultsemphasizetheneedforspecific removal of in figure 6.
capsular substances when a differentiation of
wall and capsular polysaccharides is being A. Chemistry ofCell Walls
sought. The first attempt to discover the chemical
composition of a bacterial cell wall was made as
IV. THE CELL WVALL
long ago as 1887 by Vincenzi (116). Apart from
The major structural component of the bac- finding substantial amounts of nitrogen and
terial cell is the rigid wall, which may account deducing that the wall was not composed of
for about 10 to 40 per cent of the weight of the cellulose, Vincenzi (116) was unable to suggest
cell (88). The excellent studies of Shockman, anything of a more definite nature. However,
Kolb, and Toennies (100) have demonstrated duringthepasttenyearsagreat dealofinforma-
how the wall contribution to the weight of the tion on the chemical composition of bacterial
cell depends on the growth phase, rising from 27 cell walls has become available and several out-
per cent in the exponential phase to 35 per cent standing features have emerged. The walls of
in the stationary phase of the organism, Strepto- gram-negative bacteria were found to be chemi-
coccus faecalis. Furthermore, the nutritional cally more complex than those of gram-positive
status of the organism can also influence the organisms (81). Walls of gram-positive bacteria
amount of cell-wall substance formed, as shown contained a small variety of amino acids, amino
inthe amino acid depletion studies byShockman sugar, and sugar components (81). It was thus
(99)- apparent thatthewallsofgram-positive bacteria
The isolation and characterization of bacterial were made up of a new structural class of poly-
cell walls has in recent years presented us with mers differing from the structural polysac-
some fascinating details of the comparative charides commonly encountered in the walls of
anatomy and chemistry of gram-positive and fungi andhigher plants.
gram-negative bacteria. From the variety of The study of the chemistry of bacterial cell
walls examined in the electron microscope, it is walls has attracted many investigators in the
now apparent that, ingeneral, thewalls ofgram- past decade and onlythe briefest account can be
negative bacteria are physically more complex given of some of the essential features. Several
84 4M. R. J. SALTON [VOL. 25
;..;;r"..:. *0.......
."_~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~IFAf.
Figure5. Thinsections ofEscherichiacolistrainBinfectedwithT2phageshowingmultilayered cell
wall and a separate electron-dense layer, the cytoplasmic membrane (Kellenberger and Ryter (57)).
X63,000.
substances not generally encountered in the the wall mucopeptides. If we can endow Nature
cells of higher organisms have been found in with purpose, it seems eminently sensible of Her
bacterial cells and shown to be localized in the to have designed walls containing D-amino acids
rigid wall structures. Thus muramic acid (104), in their peptides, thus providing structures
discovered first in spore peptides by Strange and resistant to many of the commoner proteolytic
Powell (106) and later in walls by Cummins and enzymes and peptidases.
Harris (19), Strange and Dark (105), and Salton The walls of many gram-positive bacteria
(83), and a,e-diaminopimelic acid, isolated and may be made up entirely of the mucocomplex
characterized by Work (128), are two substances substances, mucopeptides and mucopolysac-
confined to bacteria and closely related micro- charides, or both. The mucopeptides (64) invari-
organisms such as blue-green algae (90, 130). ably contain the amino sugars glucosamine and
Anotherfascinating featureofcell-wall chemistry muramic acid, and peptides composed of a
has been the widespread occurrence of the variety of 3, 4, or 5 principal amino acids. In
D-isomers of the amino acids, alanine, glutamic addition to mucopeptides, some walls contain
acid, and aspartic acid (52, 53, 84, 99, 109), in polysaccharide or oligosaccharide residues (46,
19611 ANATOMY OF BACTERIAL SURFACE 85
Figure 6. Fine structure in a wall layer obtained from disintegrated cells of Lampropedia hyalina
(unpublishedobservationsbyJ. A. ChapmanandM. R.J. Salton). X110,000.
68, 79) covalently linked to the mucopeptides. and ribitol-teichoic acid) do not occur together
A new class of cell-wall polymer differing from (5) andtheyseemto beabsent fromanumber of
the mucocomplex substances was discovered gram-positive bacteria. One interesting feature
severalyearsago byBaddiley and his colleagues of the teichoic acids is the presence of ester-
(4, 5, 9). Following the characterization of two linked alanine in both types. The relationship of
nucleotides, cytidine diphosphoglycerol and the glycerol-teichoic acid to the polyglycerophos-
cvtidine diphosphoribitol (8), asearch for a bio- phate compound found in a number of gram-
synthetic functionforthesenucleotidesledtothe positive bacteria by McCarty (69) has not been
detection of ribitol and glycerol-phosphate established. The latter could conceivably arise
polymers in bacteria and ultimately to the from the glycerol-teichoic acid ifthe labile ester-
localization of the teichoic acids (from Greek linked alanine was lost during isolation and
teichos = wall) intheisolatedcellwalls. Itwillbe purification.
recalled that in an earlier study Mitchell and Studies on the molecular structure of wall
Moyle (72) had found polyolphosphates in their mucopeptides have advanced rapidly in the past
"envelope" fractions of Staphylococcus aureus. In few years. Much information has been gained
general the two types of teichoic acid (glycerol- from several sources, including the elucidation
86 M. R. J. SALTON [VOL. 25
BACKBONE STRUCTURE TABLE 3
Chemical composition of bacterial cell
(1-6) (1-4) (1 6) (1 4) (1 6) (1-4) (1 6) walls
AG- AMA AG AMA AG -AMA. AG AMA.
PrincipalClassesofConstituentsand
Peptide Peptide Productsof AcidHydrolysis
L sozyme
Sensitivebonds Gram-posi- 1. Mucopeptides-glucosamine;
Figure 7. Typeofmolecularstructure proposed tive bacte- muramic acid; 3, 4, or 5 amino
for the wall of Micrococcus lysodeikticus (91), ria acids.
showing the arrangement of peptide side-chains 2. Mucopolysaccharides-amino
on an acetyl amino sugar backbone possessing sugars, monosaccharides
alternating 1-44, 1-46 bonds between N-acetyl- 3. Teichoic acids-ribitol, phos-
muramic acid (AMA) and N-acetylglucosamine phate, glucose or glucosamine,
(AG). alanine; glycerol, phosphate,
alanine
of the structure of the nucleotides accumulaitng Walls may have compositions
in the presence of penicillin and other antibiotics with the following combina-
(77, 107) and investigations of the products of tions:1,1 +2,1 +3,1+2+3.
enzymatic hydrolysis of isolated cell walls (83). Gram-nega- Mucopeptides as above. (Teichoic
One type of structure emerging from the early tive bacte- acids not as yet isolated from
work on the products of lysozyme digestion of ria this group.)
IProbably
walls (83) was suggested by Salton (85) and Protein
Lipid (present as com-
further expanded from the knowledge of the
Polysaccharides plexes of all
nucleotide structure by Brumfitt, Wardlaw, and
three classes
Park (14). The isolation of di- and tetrasac-
charides and peptide-amino sugar complexes References 88, 90.
(mucopeptides) in lysozyme digests of walls (35)
and their chemical characterization (34, 91) Some of the principal features of the chemical
enableustosuggest (92) thetypeofstructure for composition of bacterial walls are summarized in
thewall of Micrococcus lysodeikticus illustrated in table 3. More extensive accounts of cell-wall
figure 7. chemistry are available in earlier reviews by
The cell walls of gram-negative bacteria are Cummins (18) and WVork (129) and in more
much more complex. In addition to containing recent contributions by Salton (88, 90).
mucopeptides in common with gram-positive
bacteria (87, 124) they also possess major pro- V. PROTOPLAST MEMBRANES
tein, lipid, and polysaccharide constituents. The Oneofthemostimportant advances contribut-
mucocomplex or mucopeptide components of the ing to the further study of the anatomy of the
walls of gram-negative bacteria may account for surface structures of the bacterial cell was made
only 10 to 20 per cent of the weight of the wall, when Weibull (118) isolated and characterized
but it is apparent that it is this class of chemical the protoplasts of Bacillus megaterium in 1953.
constituent which isresponsible forthe structural This enabled a direct examination of the func-
rigidityofthewall (87, 88, 90, 124, 125). Itseems tional and chemical properties of the bacterial
likely that the protein, lipid, and polysaccharide membrane to be made. That the isolated proto-
components of the wall are present as a macro- plast presented a surface different from that of
molecular complex, with the mucopeptide form- the original intact cell was demonstrated in a
ing links in a rigid layer (125) or a reinforcing number of ways. It could not be infected with
network throughout the entire wall (90). The bacteriophages (118), which require a specific
various antigenic substances isolated as the 0, receptorinthe cellwall (94). Theantigens onthe
or somatic smooth-phase, antigens are probably surface of the protoplasts differed from those of
derived from the macromolecular complexes of theisolated walls and intact cells (111, 114, 115).
the cell walls (89). It is not known at present Unlike walls and intact cells, the protoplasts of
whether the wall is made up of a variety of Bacillusmegaterium and Micrococcus lysodeikticus
chemically and immunologically different macro- and their membranes are extremely susceptible
molecular subunits. to disaggregation with sodium dodecyl sulfate