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J. clin. Path. (1958), 11, 483.
THE BACTERIAL CELL
BY ROBERT CRUICKSHANK From the Bacteriology Department, University of Edinburgh
Our approach to the bacterial cell depends on our particular interests. The geneticist finds it a most useful plastic tool for studying mutations and variations, transductions and transformations; the chemist is interested in it as a source of enzymes of bewildering variety ; he also helps in identifying
the various components of the cell by fractiona-
tion and detailed chemical analysis^ of^ bacterial^ cell
masses; the cytologist is mainly concerned with
the anatomy of the cell, using modern staining and
microscopic methods, including phase-contrast and
electron microscopy; the bacteriologist is in-
terested in^ the cell's^ interaction^ with^ its^ immediate
environment, whether^ that^ be^ living^ tissue^ or^ dead
and decaying matter.
For clinical pathologists gathered together to^ do
honour to Virchow, it^ would^ seem^ most^ important
to discuss, if^ only in^ outline, our^ modern^ concepts
of the anatomy and^ physiology of^ the bacterial
cell.
Definition
Bacteria belong to the kingdom of Protista,
which also includes plant and animal forms and
has many resemblances to^ the^ blue-green algae.
According to^ majority opinion at^ the^ present time,
the bacterium may be unicellular^ or^ multicellular,
with haploid nuclear or chromatin material^ occur-
ring as a single body and dividing by transverse
fission. Cell division depends on constrictive
ingrowth of the cell wall and cytoplasmic mem-
brane.
Let us start with a^ simple diagrammatic repre-
sentation (Fig. 1) of^ the^ bacterial^ cell.^ It is^ en-
closed in^ a^ cell wall^ from^ which^ there^ may^ be
extruded flagella and^ fimbriae^ and^ may be^ sur-
rounded by a^ capsule. The main function of the
cell wall^ is to^ give form^ and^ rigidity and^ some
protection to the functional cell or protoplast. The
cytoplasm has a lining or membrane which acts as
a selectively permeable osmotic barrier and con-
tains within it many granules varying in size from
10 to 20 mI,u composed mainly of ribonucleic acid
(R.N.A.). Various^ inclusion^ bodies,^ such^ as
volutin granules, lipid granules, etc., may be con-
tained within the cytoplasm, and we shall refer
to these later. The nuclear or chromatin material
has the chemical and staining reactions of desoxy-
ribonucleic acid (D.N.A.), but cannot be regarded as a^ nucleus in the^ sense^ that^ we use^ the^ term^ for animal cells, since there is no nuclear membrane,
no centriole, and no^ unequivocal^ evidence^ of^ divi-
sion by mitosis.
Cell Wall
Although a rigid lining membrane for the bac-
terial cell, similar to that for the fungus mycelium,
was postulated by Cohn in 1875, it is only since
the introduction of electron^ microscopy that cell
walls have been clearly demonstrable. From^ bac- terial suspensions which have been mechanically
or sonically disrupted, the cell walls can be separ-
ated from the electronically more opaque cyto-
plasm by high-speed centrifugation at 8,000 to
10,000 r.p.m., after first removing any intact cells at 2,000 to 3,000 r.p.m. It is less easy to get pure
cell walls with Gram-negative than with Gram-
positive cells, and mechanical disruption gives
better results than sonic disintegration. The cell
wall constitutes about 20%^ of^ the^ dry^ weight^ of
Gram-positive cocci and as much as 45 % for
C. diphtheriae. The thinner walls of Gram-
negative bacteria probably account for consider-
ably less than 20 % of the total weight. The thick-
ness of the staphylococcal cell wall has been esti-
mated at 15 to 20 mr,, while that of Gram-negative
bacilli, like^ Bact.^ coli^ and^ salmonellae,^ is around
10 to 15 m,u and of Mycobacterium tuberculosis
23 mju. The selective rigidity of the outer casing
is demonstrated by the finding that the cell walls
of rod-shaped organisms when specially prepared
for (^) electron microscopy retain their cylindrical
form, whereas the protoplast assumes a spherical
form when the cell wall is dissolved (^) by lysozyme.
Cell wall suspensions have a milky white opales-
cent appearance, and in the case of chromogenic
bacteria there is no contained pigment which is
associated with the small particles in the cyto- plasm. There is considerable variation in the chemical composition of the cell walls in different bacterial
species, the main constituents^ being peptide-poly-
ROBERT CRUICKSHA NK
MEMBRANE
%1NCLUSION GRANULES
'CELL WALL:
EXTRA- PROTOPLASTIC PROTOPLASt STRUCTURES FIG. 1.-Diagrammatic representation of a bacterial cell (extracellular structures omitted at bottom of figure).
saccharide complexes. Certain major differences occur between the Gram-positive and Gram- negative organisms, the former having a limited range of amino-acids, whereas the Gram-negative bacteria have the same full range of amino-acids
as have most proteins. The lipid content of the
cell wall in Gram-negative bacteria is usually much
greater than that of Gram-positive organisms
(around 20% compared with 2-4%), and this high
content of^ lipid may be related^ to^ the^ lipo-poly-
saccharide of the 0 antigen or endotoxin. Poly-
saccharides and hexosamine are present in about
equal amounts in both Gram-positive and Gram-
negative bacteria. In the past few (^) years more detailed (^) analyses have been (^) made of the chemical content of the cell wall and its possible precursors (see Park, 1958).
Thus, analysis of staphylococcal cell walls has
shown that muramic acid, D-glutamic acid, lysine
and DL-alanine^ are^ present in^ a^ ratio^ of about
I: 1: 1: 3, while examination of penicillin-inhibited
staphylococci revealed an accumulation of a
uridine-5'-pyrophosphate complex which^ could be
a cell wall precursor, since it contained muramic acid and the amino-acids in the same ratio (Park
and Strominger, 1957). These findings may be linked
to the early suggestion by Duguid (1946), follow- ing observed morphological changes in penicillin- inhibited bacteria, that penicillin interferes (^) speci- fically with the formation of the cell wall while
allowing growth to proceed until the organism
bursts its defective envelope and so undergoes
lysis, and to the^ recent observations by Cooper
(1954, 1955) that penicillin is specifically bound to a bacterial lipid fraction which could be the cyto- plasmic membrane. At last we seem to be learning something about the mode and site of action of
penicillin and perhaps of other antibiotics.
Another application of^ our^ knowledge of^ the
chemistry of^ the cell^ wall^ concerns^ the action^ of
lysozyme, which acts on a mucopolysaccharide with the release of N-acetyl hexosamine. When
resistant variants of the susceptible M. lysodeik-
ticus are^ obtained, chemical^ analysis shows^ no
change in^ their sugar or^ amino-acid^ content^ but
a 100-fold increase in the 0-acetyl group. These
ROBERT CRUICKSHANK
Space does not allow any discussion of the in- teresting studies of Stocker and others on the genetic transductions of flagella and motility.
Finbriae
Before we pass inside the cell wall mention must
be made of another type of appendage, the short, slender and very numerous fibres, called fimbriae
by Duguid, Smith, Dempster, and Edmunds (1955)
and Duguid and Gillies (1957) to denote a fringe which surrounds many species of Gram-negative
bacteria (Bact. coli, Bact. cloacae, Salmonella,
Shigella flexneri, but not Sh. sonnei or shigae.
They are about half the thickness of^ flagella,^ and
fimbriation is enhanced by frequent passage in
broth culture but has no relationship to motility. These fimbriae have strong adhesive properties and are responsible for agglutination of the red cells of various animal species. The fimbriae from different serological types of Shigella flexneri are antigenically similar and may be responsible for some of the non-specific agglutinins present in the blood in many individuals. It is still uncertain what function, if any, they serve.
The Protoplast
Separation of the cytoplasm from the cell wall
was observed many years ago by Fischer (1900),
who described as " plasmoptysis " the ejection of
the cytoplasm from organisms like B. anthracis
and Vibrio proteus. The empty casing either ad-
hered to or was separated from the cytoplasm,
which then assumed a spherical form. This pheno-
menon was recently rediscovered by Stahelin
(1953, 1954) in weak-walled anthrax bacilli. The
term protoplast was, however, first used by Wei-
bull, who dissolved the cell wall of B. megaterium
by lysozyme and maintained the spherical naked
cell in a stabilizing solution of sucrose or poly-
ethylene glycol for at least 24 hours. Attached
flagella can^ be^ demonstrated in the^ intact^ cell al-
though motility does^ not^ occur, probably for^ phy-
sical reasons. The^ protoplasts disintegrate rapidly
in non-stabilizing solutions or if the suspension is
aerated and the lysate is seen to contain "^ ghosts "
or empty vesicles and small granules. The^ ghosts,
which probably represent the^ cytoplasmic lining,
are composed of delicate^ membranes^ and^ contain
most of the^ pigmented material^ of the cell.^ The
metabolism of protoplasts and whole bacterial
cells is remarkably similar, so that, besides the
cytochrome system, most^ of the cell's^ enzymic
activity is^ contained^ within^ the^ protoplast. Phage production and^ spore formation^ also^ occur, pro-
vided these phenomena have been initiated before
dissolution of the cell^ wall.^ To^ quote McQuillen
(1956), who has carried out many interesting studies with protoplasts: " Intact bacteria and the protoplasts derived from them have^ closely parallel capabilities. Both forms respire; both^ synthesize proteins and nucleic acids and form adaptive enzymes; both can support the multiplication of virulent and temperate bacterio- phages; both can support the development of endo- spores; both grow in appropriate^ media; and both can divide. Moreover, many of these activities are carried out at approximately similar rates and to similar (^) extents by the two forms, the whole and the part. And yet the part is not the whole ; there are differences in behaviour.^ To what extent some of these differences are due to the use of unsuitable conditions is not yet known." Protoplasts cannot build a cell wall, possibly because a starter of cell wall material is needed
before more can be laid down. Similarly, the
failure to sporulate unless the process has already been initiated probably means that cell wall is needed to complete spore formation. Again, al- though phage will multiply inside the protoplast, this body cannot be infected with virulent phage, which seems to need the cell wall for initial pene- tration.
The Nuclear Material
Fierce arguments have been raging for some
years on the nature and interpretation of the
changes that are demonstrable in the chromatin
bodies or nuclear material of the bacterial cell
during the process of growth and division. The
argument largely turns on whether the chromatin
bodies which^ grow in number^ with the^ growth of
the cell^ are^ composed of^ chromosomes^ which
divide like the chromosomes of animal and plant
cells, i.e., by mitosis, or whether they divide
directly or amitotically, as happens with many of
the higher bacteria and Protista. The opposing
views are set out by Robinow and DeLamater re-
spectively in their chapters in the Symposium on
Bacterial Anatomy (1956). Robinow, using Feul-
gen and acid Giemsa stains, has studied parti-
cularly the development of the single chromatin
body in germinating spores, and^ his^ interpretations
of the observed phenomena carry convincing evi-
dence for^ direct^ amitotic^ division.^ DeLamater
has used^ the^ actively growing cell^ for his^ studies,
and, having perhaps too^ readily assumed that bac-
teria divide by mitosis, interprets his findings to fit
this hypothesis. He^ claims that both B.^ mega-
terium and Bact. coli have three chromosomes
which can be^ shown^ pictorially to^ divide simul-
taneously by a^ complex mitotic mechanism inside
the nuclear membrane. He and his colleagues also
describe mitochondria and^ a^ centriole, which
Robinow, on the other^ hand, regards as an acces-
THE BACTERIAL CELL
sory chromatin granule. It is impossible for the layman to adjudicate on the merits of these diver- gent stories by highly specialized cytologists, but support can be^ given^ to^ DeLamater's^ viewpoint that the truth will best be elicited by the simul- taneous application of other techniques, such as those of biochemistry and bacterial physiology
along with the cytochemical methods of the cyto-
logist.
If we turn for a moment to the evidence of the
microbial geneticist,^ it^ is^ obvious^ that^ genetic^ ex- change of nuclear material takes place as a result of sexual pairing in certain species; in addition,
transfer of genes occurs^ to cause^ the^ phenomena
of transformation in^ pneumococcus and haemo-
philus types and^ of transduction of^ toxigenicity in
C. diphtheriae or motility in^ salmonella. The^ cyto-
logists have so far been unable to contribute to a
better understanding of these strange happenings,
although there^ has been^ some^ correlation between
cytological changes^ and^ genetic^ exchange^ in^ higher
organisms like^ Drosophila and^ maize.^ There^ is
little doubt that D.N.A. is the^ active^ principle in
the genetic mutations of bacteria, mediated^ some-
times through bacteriophage. It^ has also^ been
shown that genetic differentiation of the D.N.A.
material is linear, i.e., the "bacterial^ chromo-
some" may be regarded as a^ long, thread-like
D.N.A. molecule, carrying the genes in an orderly
sequence like the inscription on a tape. The
D.N.A. molecule, pictured by Watson and Crick
(1953) as a cylindrical double helix with a diameter
of 2.5 m,u, would correspond to a thread about
1,000 times^ the^ length^ of^ the^ parent^ bacterial cell.
Attempts at^ cytological study of the^ D.N.A.^ mole-
cule are now being made^ by electron^ microscopic
examination of ultra-thin sections^ of bacteria. It is suggested that the^ thread is^ coiled up^ to^ form^ a
tube, and in the^ photomicrographs of^ Maal0e^ and
Birch-Andersen (1956) tube-like^ structures^ cut^ in
all different directions can be demonstrated. It may be possible in the future to learn more about the nucleus and its mode of division by detailed studies of this kind.
Cytoplasm and Inclusion Bodies
In electron photomicrographs of sectioned bac- teria (staphylococci, streptococci, paracolon bacilli,
aerobacter, mycobacteria) the^ cytoplasm seems^ to
consist of fine granules of 10 to 20 m,u with some- times larger granules, the total number in a single
cell being of the order of 104 to 105. Chemically
the cytoplasm is composed mainly of ribonucleic acid and protein and^ contains^ most of^ the^ enzymic activity of the cell, partly in its membrane. The
small size of the granule and the multiplicity of enzymes in many bacteria suggest that there will be numerous enzymically different types of granules. They are probably derived from the nucleus, growing by the formation or accumula- tion of enzyme proteins. In^ discussing the^ rela- tionship of bacterial cytoplasm to that of^ other
lowly creatures, Bradfield states that:
" the most striking structural difference between bacterial cytoplasm and the cytoplasm of animals and plants is the absence from bacteria of all^ kinds of intra-cytoplasmic membranes; the oxidative granules are not wrapped in^ membranes to form mitochondria; the^ RNA-rich^ granules^ are^ not arrayed on membranes to^ form^ endoplasmic^ reticu- lum; and^ finally^ the^ cytoplasm as a^ whole^ is^ not separated from the nucleus by a nuclear mem- brane." Among inclusion bodies, the most commonly seen are the Babes-Ernst metachromatic bodies,
often called volutin granules. These meta-
chromatic granules, seen best in C. diphtheriae
and ranging in size up to 0.6 (^) /u, are very electron
opaque and chemically contain polymetaphosphate
and probably other phosphorus compounds.
Smith, Wilkinson, and Duguid (1954) found that,
although volutin granules might account for 20%
of the bacterial volume, only 1 % of the dry weight
was polymetaphosphate.
Wilkinson and his^ colleagues (Williamson^ and
Wilkinson, 1958; Macrae^ and^ Wilkinson,^ 1958)
have also studied the lipid inclusions found^ parti-
cularly in the larger varieties of the^ Bacillus
species. These lipid granules, which are composed
mainly of a polymer of f8 hydroxybutyric acid, are
most numerous in cells about to sporulate or when
grown in semi-synthetic culture media containing
an excess of glucose, pyruvate, or ,B hydroxy-
butyrate as the^ main^ carbon^ sources^ for metabol- ism. They are^ regarded by the^ Edinburgh^ workers as (^) stores of carbon and energy and are probably used up in the process of sporulation. Whether inclusion bodies like volutin and^ the^ lipid
globules serve a^ specific function in cellular
metabolism, or whether they represent an ab-
normal or pathological state of the bacterial cell,
is a debatable point. As already mentioned, the
pigments of chromogenic bacteria are found pre- dominantly within the cytoplasm, and Schachman,
Pardee, and^ Stanier (1952) have^ observed many
well-defined chromatophores of approximately
40 m,u diameter in the cytoplasm of Rhodo-
spirillum rubrum. They were absent from organ-
isms grown in the dark. Probably many other
photosynthetic bacteria possess similar chromato-
phores.
