Toward a Definition of the Bacteria
R . Y . ST A N I E R
I. H i s t o r i c a l B a c k g r o u n d of t h e P r o b l e m 445 I I . O r g a n i z a t i o n of F u n c t i o n a l S u b u n i t s in E u c a r y o t i c a n d P r o c a r y o t i c C e l l s . 449
I I I . N u c l e a r S t r u c t u r e a n d R e p r o d u c t i o n in P r o c a r y o t i c O r g a n i s m s 450 I V . T h e O r g a n i z a t i o n of R e s p i r a t o r y a n d P h o t o s y n t h e t i c F u n c t i o n in P r o
c a r y o t i c C e l l s 452 V. S t r u c t u r e s A s s o c i a t e d w i t h P r o c a r y o t i c Cellular M o v e m e n t 457
V I . T h e C h e m i c a l S t r u c t u r e of t h e W a l l i n P r o c a r y o t i c Cells 458 V I I . T h e C o m m o n D e n o m i n a t o r s of E u c a r y o t i c a n d P r o c a r y o t i c C e l l s 460
V I I I . E v o l u t i o n a r y I m p l i c a t i o n s 460
R e f e r e n c e s 462
T o conclude this treatise, it seems appropriate to consider the central question of bacteriology: W h a t are the bacteria, biologically speaking? I n other words, where do they belong in the hierarchy of life, and to which other biological groups, if any, do they show affinities? Logic would seem
ingly dictate t h a t an analysis of this problem should have prefaced, rather t h a n terminated, a treatise on the bacteria. I n fact, no satisfactory analysis was possible at the time when the treatise was originally planned ; only in the last five years has the general framework of an answer become evident.
I. Historical B a c k g r o u n d o f the P r o b l e m
T h e detailed exploration of the microbial world was one of the distinc
tive accomplishments of nineteenth century biology. As this last frontier of natural history was conquered, biologists came to recognize several major groups of microorganisms: algae, protozoa, fungi, bacteria, and—
after the turn of the century—viruses. They were therefore faced with the problem of classifying these groups. T h e solution adopted was arbi
t r a r y and illogical, t h a n k s to reliance on a false axiom and a stubborn refusal to look facts in the face.
T h e false axiom was the belief, essentially a prescientific judgment of common sense, t h a t the world contains only two, mutually exclusive, cate
gories of living organisms—plants and animals. Since this judgment had been accepted by the biological community as self-evident, the primary question to be decided was, apparently, to which kingdom the various microbial groups should be assigned. B y a judicious selective emphasis on certain characters, it proved possible to decree t h a t protozoa were
"animals," whereas fungi, algae, and bacteria were "plants." I n conse
quence of the primary taxonomic cleavage of biology t h a t existed in the
445
mid-nineteenth century, the protozoa accordingly became the domain of zoologists, and the algae, fungi, and bacteria, the domain of botanists.
Only one major biologist of the time, Haeckel,1 protested this arbitrary partitioning of the microbial world, and stressed the cardinal fact t h a t at this relatively simple level of biological organization the concepts of
" p l a n t " and " a n i m a l " lose their clarity. His taxonomic solution, to place all microorganisms in a third major category, the protists, was not adopted, despite its obvious merits.
The recognition of bacteria as important agents of disease led in the late nineteenth century to the establishment of bacteriology as a separate biological field; thereafter, bacteria became the domain of a new kind of biologist, armed with new, specialized techniques and a mental outlook quite unlike t h a t of the scientists who had previously worked with bac
teria. Insofar as bacteriologists thought about the matter at all, they tended to accept the dogma t h a t bacteria were non-photosynthetic plants, comprising a group cognate with the fungi. However, the question of bac
terial affinities was seldom seriously raised; in practice, it proved very easy to distinguish most bacteria from other microorganisms. T h e viruses were discovered and explored largely by scientists trained in the bacterio
logical tradition. At first, it seemed plausible to regard these entities as smaller, nutritionally highly specialized, cellular microorganisms of the bacterial t y p e ; this easy assumption was shattered only in 1935, with the crystallization of tobacco mosaic virus.2
T h e world of microbiologists has thus been split for historical reasons into three groups: one, with the prejudices of the zoological tradition, which studies protozoa; a second, with the prejudices of the botanical tradition, which studies algae and fungi ; and a third, with the very special prejudices of the bacteriological tradition, which studies bacteria and viruses. I t is an unfortunate fact t h a t the world of microorganisms is rather poorly served by this division of professional interest. Most algae, the protozoa, and the fungi—the higher protists—can be construed as belonging to one very large and diverse microbial assemblage, nonetheless united by a number of fundamental properties, and intergrading at m a n y different points, so t h a t in the final analysis no satisfactory and clear-cut distinctions between "algae," "fungi," and "protozoa" can be made. How
ever, it so happens t h a t the intergradations occur between algae and protozoa, and between protozoa and fungi, with the consequence t h a t the fundamental unity of the whole assemblage has never been evident to most botanists and zoologists. The broad affinities among higher protists were first clearly perceived by Pascher3 in terms of structural characters, and the Pascherian argument was later ably extended by Lwoff4 in terms of nutritional and physiological characters. However, one group of micro-
organisms traditionally assigned to the algae, the blue-green algae, stands apart. These organisms do not share the common properties which unite other algal groups with the protozoa and the fungi. We shall consider their affinities after discussing the various groups of organisms tradi
tionally assigned to the bacteria.
T h e establishment of the bacteria as a special microbial group antedated the foundation of bacteriology as a separate biological science. I t was, in essence, the work of one man, the botanist Ferdinand C o h n .5 - 7 With a few exceptions, the organisms united by Cohn as bacteria comprised a very homogeneous group : the unicellular non-photosynthetic eubacteria, which multiply by binary fission and show flagellar motility if they are motile.
However, as time went on, a whole series of microorganisms differing greatly in their properties from this original bacterial assemblage came, by common consent, to be accepted as bacteria. They included actinomy- cetes, the myxobacteria, the spirochetes, the photosynthetic bacteria, the rickettsias, and the pleuropneumonia group, with the viruses tentatively accepted (until their peculiar properties were understood) as a marginal addition. T h e extraordinary diversity of the organisms now generally placed among the bacteria (excluding viruses) is shown by a simple enumeration of the properties which they can possess. They can be uni
cellular, multicellular, or coenocytic; permanently immotile, or motile by any one of three distinct mechanisms; able to reproduce by binary fission, by budding, or by the formation of special reproductive cells, such as the conidia of actinomycetes ; photosynthetic or non-photosynthetic.
Despite this diversity, there has been remarkably little dispute among microbiologists about the assignment of any given organism or group of organisms to the bacteria. I n practice, an experienced microbiologist has no difficulty in distinguishing bacteria from protozoa, fungi, and most algae, even when (as in the case of actinomycetes and mycelial fungi) there are substantial similarities of gross form. T h e two groups from which it has appeared difficult to distinguish certain bacteria are the viruses and the blue-green algae.
Some bacteria—the rickettsias, and the psittacosis-lymphogranuloma venereum group—are obligate intracellular parasites, the structural units of which are so small as to be barely resolvable by the light microscope.
I n these two respects, accordingly, they resemble the larger animal viruses, and the view has often been expressed t h a t they are "transitional" be
tween other bacteria and viruses. I t was possible to express such a view, even as recently as 1955,8 only because the essential properties of viruses had not been clearly grasped. This important logical analysis was under
taken in 1957 by Lwoff,9 who for the first time formulated the constella
tion of properties which distinguishes the virus from the cell. T h e infectious
viral particle, or virion, contains only one kind of nucleic acid, enclosed in a coat of protein, formed by the polymerization of identical subunits. I t carries few, if any, proteins endowed with enzymic function. I t cannot divide. During its replication, which can take place only within a sus
ceptible cell, the only component of the virion t h a t is directly reproduced is its nucleic acid. Once the general properties of viruses had been formu
lated, it became evident t h a t the differences between viruses and cells are of such a nature t h a t no truly "intermediate" stage of biological organiza
tion could be envisaged. T h e problem presented by the rickettsias and the psittaeosis-lymphogranuloma venereum group can now be differently phrased : D o these entities have the fundamental properties of cells, or of viruses? All present evidence (summarized by M o u l d e r1 0) indicates t h a t they are cellular in nature, and hence belong to the bacteria sensu lato.
T h e other diagnostic problem alluded to above—namely, t h a t of dis
tinguishing bacteria from blue-green algae—is not so easily solved. As Ferdinand C o h n7 was the first to emphasize, there are close structural similarities between bacteria and blue-green algae; m a n y of the nonmotile, coccoid or rod-shaped, unicellular blue-green algae have counterparts among the unicellular bacteria. A clear-cut distinction between these two groups could, however, be made on a physiological basis, by defining blue- green algae in terms of their photosynthetic metabolism, which is of the oxygen-evolving type characteristic of other algae and higher plants, as well as in terms of their characteristic pigment system. More serious diffi
culties arose through Winogradsky's s t u d i e s1 1 on the sulfur "bacteria"
Beggiatoa and Thiothrix, which though indistinguishable in structure and mode of locomotion from filamentous blue-green algae, lack a photosyn
thetic pigment system. I n more recent years, m a n y other non-photosyn
thetic, filamentous, gliding organisms have been recognized.1 2 - 1 7 I n every case, these microorganisms could be defined either as blue-green algae (if one wished to emphasize their structural attributes) or as bacteria (if one wished to emphasize their mode of energy-yielding metabolism). I n evo
lutionary terms, there can be little doubt t h a t they represent non-photo
synthetic descendants of filamentous blue-green algae, analogous to the leucophytes4 so commonly found among the various groups of flagellate algae. This very instructive series of examples shows that, unless one invokes biochemical criteria, it is not possible in the last analysis to draw any sharp line of distinction between bacteria and blue-green algae.1 7
Since blue-green algae are the only group of organisms which pose this problem of differentiation from bacteria, it is an obvious inference t h a t the two groups must share some fundamental similarities of cellular construc
tion t h a t set them rather sharply apart from other organisms. Such a view has been put forward at intervals by microbiologists ever since the time
of Ferdinand Cohn; but the seemingly intractable problem has always been to characterize the distinctive common features of bacteria and blue- green algae in a biologically meaningful way. Simply to state, as m a n y authors have done, t h a t their cells are "primitive" does not serve any use
ful purpose; and the first attempts (e.g., Stanier and van N i e l1 7) to de
scribe the common group features in more specific terms were not particu
larly successful.
Since 1950, the development of the electron microscope as an effective instrument for biological research and the introduction of new analytical approaches to cytological problems have revolutionized our understanding of the organization of cells. For the most part, this rapid growth of knowl
edge has emphasized or brought to light fundamental homologies of cellular construction; the resemblances between the cells of plants, animals, and most microorganisms now seem much greater t h a n the differences. How
ever, the new cytological generalizations cannot be extended to the level of the bacteria and blue-green algae; their cells have proved to be con
structed on an organizational plan entirely different from the plan t h a t underlies the construction of the cell in other g r o u p s .1 8 , 1 9 W e shall term the cellular plan characteristic of bacteria and blue-green algae "pro
caryotic" ; and the plan characteristic of other protists, plants and animals,
"eucaryotic." I t is the procaryotic nature of their cells t h a t unites the bacteria and blue-green algae, and separates them from all other cellular organisms. I n the following sections of this chapter, the distinctive proper
ties of this kind of cell will be outlined.
II. O r g a n i z a t i o n o f Functional Subunits in Eucaryotic a n d Procaryotic Cells
One basic difference between the two plans of cellular architecture is the manner in which major subunits of cellular function are housed within the enclosing cytoplasmic membrane. I n the eucaryotic cell, the nuclear m a t e rial (at least during interphase) and the multienzyme systems which per
form respiration and photosynthesis are severally enclosed within indi
vidual unit membranes, distinct from the cytoplasmic membrane. These internal membranes accordingly serve to isolate physically the genetic system and the enzymic machinery of respiration and photosynthesis from other internal regions of the cell, each in a structurally distinctive organelle. I n the procaryotic cell, on the other hand, there is no equivalent structural separation between the major subunits of cellular function; the cytoplasmic membrane itself is the only bounding membranous element in the cell, and within its confines the separation between functional sub- units is maintained without the interposition of any obvious physical barriers. T h e maintenance of separate functional regions in procaryotic
cells without an interposed membrane is particularly evident with respect to the nucleus. Cytochemical methods define a discrete nucleus region, the unique site of the cellular D N A (deoxyribonucleic a c i d ) .2 0 Electron micrographs of thin sections fixed by the procedure of R y t e r and Kellen- berger2 1 show t h a t there is a sharp separation between the nuclear region, densely filled with fibrils of D N A , and the adjacent, ribosome-filled cyto
plasm; but at the same time these electron micrographs provide clear evidence for the absence of an interposed membrane. The continuous maintenance of this phase separation between nucleus and cytoplasm in the procaryotic cell is a very curious phenomenon; it is scarcely under
standable unless one assumes t h a t the contents of the cell have at all times the properties of a gel. I n fact, the immobility of the cytoplasm of procaryotic cells in the living state affords one of the most helpful clues for recognizing bacteria and blue-green algae by examination with the light microscope. Of course, the cytoplasm of some eucaryotic cells shows little if any internal movement; but there are a host of phenomena—ame
boid movement, cytoplasmic streaming, the formation, migration, and coalescence of vacuoles, the migration of nuclei, the light-directed orienta
tion of chloroplasts—which all attest to the internal mobility, actual or potential, of the cytoplasm in eucaryotic cells. None of these phenomena has a counterpart in bacteria and blue-green algae.
III. N u c l e a r Structure a n d Reproduction in Procaryotic O r g a n i s m s A second fundamental difference between the eucaryotic and procaryotic cell lies in the organization of the genetic elements. A clear description in classical cytological terms of the properties of the procaryotic nucleus be
came possible only 20 years ago, when R o b i n o w2 2 developed satisfactory procedures of fixation and staining. Ensuing cytological studies showed t h a t every kind of procaryotic cell contains discrete nuclear elements, the division of which is regularly correlated with cell division. The division of each nuclear element involves a broadening and splitting, unaccompanied by any gross change of form through the divisional cycle. Despite transient dissent,2 3 it is now universally agreed by bacterial cytologists t h a t nothing resembling a mitotic apparatus, or an organization of the nuclear sub
stance into discrete chromosomes, can be recognized during the course of division. The electron microscopy of bacterial thin sections (see Robi
n o w2 4) simply confirms these inferences, without adding any further information about the details of the divisional process.
The next step to the understanding of procaryotic nuclear organization came with the genetic analysis of Escherichia coli (see Wollman and J a c o b2 5) . The genetic data could be physically interpreted only by the assumption t h a t the genes are linearly arranged in a single, closed linkage
group. I n other words, E. coli appeared to have a single circular chromo
some, if we wish to employ classical cytogenetic terminology.
Recent physicochemical work has invested this model with structural reality. The radioautographic investigation by C a i r n s2 6' 2 7 on the chromo
some of E. coli has demonstrated t h a t it consists of a single piece of two- stranded D N A , approximately 1000 μ long, which duplicates by forming a fork, the new limbs of the fork each containing one strand of new material and one strand of old material. Furthermore, physical evidence for the circularity postulated for genetic reasons could be derived from these experiments. Equally striking electron microscopic evidence t h a t the bacterial chromosome comprises a single, continuous molecule of two- stranded D N A has been obtained by Kleinschmidt and L a n g2 8 for Micro
coccus lysodeikticus, using the ingenious technique of breaking proto
plasts by osmotic shock, and spreading the D N A molecules out with a film-forming protein.
T h e combined genetic and physical studies on the structure of the bac
terial chromosome accord excellently with the grosser picture of procary
otic nuclear structure and behavior established by cytological methods, both classical and modern. W h a t is perceived by the cytologist as the bacterial nucleus actually consists of a single, two-stranded D N A molecule, almost 1 mm. in length, and probably circular. This giant molecule is compressed into a compact mass some 0.2 μ in diameter. I t replicates by forking, new strands being laid down in strictly polarized sequence along each strand of the fork.* T h e physical mechanism for the segregation of the daughter D N A molecules is not y e t established. T h e absence of mitosis in procaryotic cells now becomes fully understandable. Mitosis is an elabo
rate biological device which permits the equipartition of the replicated genetic material at the time of nuclear division when, as in all eucaryotic cells, the sum of genetic determinants is dispersed over two or more units of structure, the eucaryotic chromosomes. A mitotic machinery could have no reason for existence in a cell where all genetic determinants reside in a single molecule.
Considering its traditional meaning, the word "chromosome" is ob
viously not a desirable designation for the D N A molecule which carries the genetic determinants of the procaryotic cell. The procaryotic "chromo
some" is functionally equivalent to the total chromosome complement of a eucaryotic nucleus ; it does not undergo the elaborate secondary struc
tural changes characteristic of eucaryotic chromosomes; and it does not even have the same gross chemical composition, being devoid of the basic proteins which are always associated with D N A in eucaryotic chromo-
* For a genetic confirmation of the m o d e of replication, see N a g a t a2 8* and Y o s h i k a w a and S u e o k a .2 8 b
somes. This terminological difficulty demonstrates in the most effective possible fashion the distinctiveness of the procaryotic cell. Since all our basic concepts about cells were derived historically from the study of eucaryotic cells, we now find ourselves saddled with a cytological termi
nology which cannot be easily applied to a different kind of cell.
As an appendix to this analysis of procaryotic nuclear structure, we m a y briefly summarize the consequences of the physical organization of the bacterial genome insofar as mechanisms of genetic transfer are con
cerned.2 5 All modes of gene transfer so far discovered in bacteria have certain common features t h a t distinguish them sharply from sexual and parasexual processes in eucaryotic organisms. Except in rare cases of bacterial conjugation, procaryotic gene transfers involve the introduction of a small fragment of the genome from a donor cell into a recipient cell with a complete genome. T h e recipient cell is consequently not genetically
equivalent to a eucaryotic zygote; it becomes a partial diploid, or merozy- gote. If recombination then takes place, the normal haploid condition is reestablished by the elimination of supernumerary alleles; there is never a production of reciprocal recombinants, a basic feature of the recombina
tional event in eucaryotic sexual processes. A further difference from the situation in eucaryotic organisms is t h a t modification of the procaryotic genome by genetic transfer does not necessarily require t h a t the introduc
tion of new genetic material into the recipient cell be followed by recombi
nation. I n m a n y instances, the newly introduced genetic fragment can maintain itself more or less indefinitely in the autonomous state, as an episome.
IV. The O r g a n i z a t i o n of Respiratory a n d Photosynthetic Function in Procaryotic Cells
One of the functional consequences of the internal compartmentalization characteristic of eucaryotic cells is t h a t two major energy-generating metabolic unit processes, respiration and photosynthesis, t a k e place en
tirely within the confines of special organelles, the mitochondrion and the chloroplast, respectively. Each of these organelles is in turn compart
mentalized. The systems directly responsible for A T P (adenosine triphos
phate) synthesis (oxidative phosphorylation and photosynthetic phos
phorylation) are localized in the internal membrane systems of the respective organelles, while associated biochemical processes (reactions of the Krebs cycle in mitochondria, of the Calvin cycle in chloroplasts) t a k e place in the adjacent, less highly structured regions. T h a n k s to the characteristic dispositions of the internal membrane systems associated with mitochondria and chloroplasts, each of these organelles can be readily identified by its distinctive profile as seen in electron micrographs
of thin sections. T h e electron microscopic studies of the past decade (see Novikoff2 9 for a summary) compel us to conclude t h a t despite minor structural variations from group to group, the mitochondrion is a basically homologous cellular element throughout the entire span of eucaryotic cellular organisms (higher protists, plants and animals). I t is also a well- nigh universal component of the eucaryotic cell, seemingly absent only in those rare eucaryotic protists t h a t are obligate anaerobes. T h e chloroplast is, of course, a cellular organelle of less universal occurrence ; but over the tremendous organismal span of eucaryotic photosynthetic organisms, when it does occur as a component of the cell, it likewise seems to be basically homologous (see G r a n i c k3 0 for a s u m m a r y ) .
Procaryotic organisms can also use photosynthesis and respiration as major energy-generating metabolic unit processes. Frequently, these proc
esses are biochemically almost indistinguishable from the same processes as they occur in eucaryotic cells (e.g., photosynthetic metabolism in blue- green and in eucaryotic algae). However, the structural basis for the per
formance of respiration and photosynthesis in a procaryotic cell is wholly different. No organelles homologous with mitochondria and chloroplasts exist. I n fact, the structural difference can be epitomized by the statement t h a t no unit of structure more simple than the procaryotic cell as a whole
(exclusive of wall and appendages) can be recognized as the site of either metabolic unit process.
A partial analysis of the organization of bacterial respiratory function became possible as a result of WeibulPs discovery3 1' 3 2 t h a t the cytoplasmic membrane of certain bacteria can be isolated in a structurally identifiable state, free both from wall material and from the internal constituents of the cell. This isolation is dependent on a total dissolution of the cell wall by lysozyme, followed by gentle osmotic lysis of the protoplast. I t can, there
fore, be applied only to those relatively few Gram-positive bacteria, the walls of which can be completely stripped from the protoplast by lysozyme treatment. I n two such organisms, Bacillus megaterium and Sarcina lutea, it has been s h o w n3 3 - 3 5 t h a t the whole machinery of respiratory electron transport is intimately associated with the cytoplasmic membrane, as are also a few enzymes of the Krebs cycle (notably, succinoxidase). If we m a y generalize from these two cases, it accordingly appears t h a t the cyto
plasmic membrane of aerobic bacteria, in addition to fulfilling the universal physiological function of such membranes (namely, regulation of trans
port) , has associated with it the enzymic machinery which, in eucaryotic cells, is built into the internal membranes of the mitochondrion. As a consequence of this organizational pattern, the physical integrity of the cell as a whole cannot be impaired without far-reaching effects on the biochemical integrity of the respiratory system. T h e "soluble" enzymes
required for respiration, located in the cytoplasm proper, flow out of the cell and become dissociated from the electron transport system as soon as the cytoplasmic membrane is breached. T h e effects with regard to respiration are entirely analogous to those which follow osmotic or me
chanical rupture of isolated mitochondria, and explain why bacteria proved unsuitable in the hands of the biochemists as sources of cell-free systems capable of full respiratory activity.
T h e original assumption of the cytologists, t h a t the cytoplasmic mem
brane of bacteria is a simple membrane following the contour of the sur
rounding wall, has recently proved to be an oversimplification. I n m a n y aerobic bacteria, the membrane shows a more or less extensive degree of in
folding, which m a y either be localized at certain points in the cell, or else extend over the entire cortical region of the cytoplasm. Localized com
plex infoldings, characteristically occurring in close association with sites of transverse wall formation, were first observed by electron microscopy of thin sections in Gram-positive b a c i l l i3 6 , 3 7 and actinomycetes.3 8 These are the structures for which F i t z - J a m e s3 6 has coined the name of "meso- somes." They have been found in m a n y other groups of Gram-positive bacteria, including Corynebacterium,39 Mycobacterium40 Micrococcus,40a and Listena.41 Mesosomes are, however, not confined to Gram-positive bacteria; typical ones occur in Caulobacter.42 The membranous infoldings m a y also assume other forms, such as the "simple" intrusions observed by M u r r a y4 3 in Spirillum. I n two nitrifying bacteria, Nitrosocystis and Nitrobacter, M u r r a y4 4 has found extremely elaborate lamellar intrusions, disposed with great regularity in a particular region of the cytoplasm. The lamellar stacks of Nitrosocystis form a closely packed band across the cell, of sufficient size to be detectable in stained cells by light microscopy, although of course the fine structure cannot be resolved with the light microscope. I n Nitrobacter, the lamellae are concentrated at one end of the cell ; it is this internal arrangement which appears to determine the char
acteristically pear-shaped form of Nitrobacter cells. A particularly in
structive study has been conducted by Pangborn et al.45 on the internal membrane system of Azotobacter. When cells of this bacterium are broken by "gentle" methods (ballistic disintegration, osmotic shock) and washed free of liberated soluble cytoplasmic components, thin sections of the residual cytoskeleton reveal an extremely extensive internal network of tubular and vesicular membranes, connected with the cytoplasmic mem
brane proper. Further mechanical treatment greatly reduces the internal membrane system, and at the same time liberates a considerable amount of the reduced diphosphopyridine nucleotide oxidase originally present in the cytoskeleton in a form t h a t is no longer readily sedimentable. From this, the authors conclude t h a t the internal membranes are the probable site
of the electron transport system in the cell. T h e internal membrane system can be detected in sections of whole cells, b u t is not readily visible, as a result of low contrast with the surrounding cytoplasmic materials.
T h e recent revelation of the structural complexity and variability of the cytoplasmic membrane in aerobic bacteria of course raises at once a new question: is the electron transport system evenly distributed through the cytoplasmic membrane as a whole, or is this system localized, as Pangborn et a L4 5 imply, in the internal intrusions from the membrane? At present, no clear-cut answer to this question can be given.*
W e must now consider the nature of the structures associated with the performance of photosynthesis in the cells of bacteria and blue-green algae.
A number of recent electron microscopic studies on thin sections of blue- green algae (e.g., Niklowitz and D r e w s ,4 6 S h a t k i n ,4 7 Ris and Singh4 8) re
veal t h a t much of the cytoplasmic region is traversed by an extended system of paired lamellae. As shown by Ris and Singh,4 8 the patterns of these lamellar systems v a r y considerably in different members of the blue-green algae. T h e lamellae seem to be structurally analogous to (and conceivably homologous with) the internal lamellae of chloroplasts ; how
ever, they are not enclosed within a common membrane which separates them from other regions of the cytoplasm. This point is particularly well shown in the electron micrographs of Ris and Singh,4 8 where the ribosomal matrix of the cytoplasm is clearly resolved ; in a number of cases it can be seen t h a t this matrix actually extends between adjacent pairs of lamellae.
Fragments of the lamellar system have been isolated from broken cells by P e t r a c k and L i p m a n n4 9; they contain the chlorophyll of the cell, and are endowed with photophosphorylative function.
I n photosynthetic bacteria, the picture is a much more variable one. As Vatter and W o l f e5 0 first showed, the cytoplasmic region of m a n y nonsulfur purple bacteria grown anaerobically in the light appears to be packed with spherical vesicles, about 500 A. in diameter; these vesicles were absent from cells of facultatively aerobic species grown aerobically in the dark, and thus rendered essentially free of photosynthetic pigments. Cohen- Bazire and K u n i s a w a5 1 have recently made a detailed study of the fine structure of one species, Rhodospirillum rubrum, grown under a series of well-defined conditions, the influence of which on the pigment content of the cell was known from previous physiological w o r k .5 2 They established t h a t the abundance of vesicles is correlated with the specific chlorophyll content of the cell. Each vesicle is defined by a unit membrane, indistin
guishable in cross-section from the cytoplasmic membrane, which encloses a region of very low electron density. I n sections of cells with a low con
tent of vesicles, it could be seen t h a t some of the vesicles represent simple
* See, however, Vanderwinkel and Murray.4 5*
intrusions of the cytoplasmic membrane, the transparent central region being directly connected with the space between the membrane and the cell wall. These observations suggest t h a t the vesicles characteristic of so m a n y purple bacteria, and shown by isolation5 3 to contain the photo
synthetic pigments of the cell, m a y in fact be an extremely extensive in
ternal membrane system formed from the cytoplasmic membrane, and a t all times in physical continuity with it.
I n at least two purple b a c t e r i a5 4 - 5 6 vesicles are absent, their place being taken by a system of paired lamellae resembling those found in blue-green algae. J u s t as in blue-green algae, the internal arrangement differs in the two purple bacteria in question. Giesbrecht and D r e w s5 7 have recently shown, by examining thin sections of osmotically lysed cells, t h a t the leaf- shaped lamellar bundles characteristic of Rhodospirillum molischianum arise from, and are attached to, the cytoplasmic membrane. There is probably no fundamental difference between the vesicular and lamellar systems of purple bacteria: if flattened, a vesicle would in effect become indistinguishable from a paired lamella. Indeed, F u l l e r5 8 has recently established t h a t the purple sulfur bacterium Chromatium m a y contain either vesicles or lamellae, depending on the conditions of cultivation;
and Cohen-Bazire5 9 has found t h a t another purple sulfur bacterium, Thiocapsa, contains a mixture of vesicular and lamellar elements.
I n the case of purple bacteria, a problem analogous to t h a t discussed in connection with aerobic bacteria presents itself: Is the photosynthetic apparatus evenly distributed throughout the cytoplasmic membrane as a whole, or localized in the vesicular and lamellar intrusions? Here also, no final answer can be given a t present.
One further point concerning the organization of photosynthetic func
tion in procaryotic cells deserves emphasis. As so conclusively demon
strated by Arnon and his collaborators,6 0 the eucaryotic chloroplast is the site not simply of the multienzyme system and pigment system required for photosynthetic energy conversion, b u t also of the entire array of solu
ble enzymes responsible for primary photosynthetic carbon assimilation and sugar synthesis. Although there is now abundant evidence t h a t the internal membranes of photosynthetic bacteria and blue-green algae contain the systems necessary for photosynthetic energy conversion,6 1 there is no evidence whatsoever t h a t the soluble enzymes responsible for the reactions of primary photosynthetic carbon assimilation are specifically associated with the photosynthetic apparatus.
I n concluding this analysis of the localization of respiratory and photo
synthetic function in procaryotic cells, we must attempt to see w h a t gen
eralizations emerge. I n the first place, it seems highly probable t h a t the cytoplasmic membrane of the procaryotic cell is functionally far more
complex than the equivalent structure of the eucaryotic cell. It must, of course, regulate the entry and exit of materials, but in addition to this, it (or its extensions) appears to be the sole site within the procaryotic cell of respiratory and photosynthetic ATP synthesis. Since the thickness of this unit membrane is fixed, the only way in which its mass can be varied, relative to that of the cell as a whole, is by a change of area. Since the total volume of the cell is established by its rigid enclosing wall, increase in the area of the membrane can be achieved only by infolding. We would therefore expect to find extensive infolding of the cytoplasmic membrane in any procaryotic cells that have a high specific level of respiratory or photosynthetic activity. The observations of Cohen-Bazire and Kunisawa
51on
R ho do spirillum rubrumprovide an experimental demonstration of the predicted correlation between photosynthetic activity and the extent of the internal membrane system. It is also no doubt significant that
Azotobacter,
which has the highest respiratory rate of any known organism, likewise has the most extensive internal membrane system so far found in an aerobic bacterium.
45The particular form which the membranous in- foldings take in different procaryotic cells may vary widely, and is prob
ably of secondary importance. From their structure alone, it cannot be deduced with certainty whether such infoldings are associated with a respiratory or a photosynthetic apparatus : the simple intrusions in
Spirillum
look very like the photosynthetic vesicles of
R ho do spirillum rubrum,and the lamellae of nitrifying bacteria mimic the regular lamellar stacks of
Rho do spirillum molischianum.
V . Structures Associated with Procaryotic Cellular M o v e m e n t
One of the greatest achievements of electron microscopy has been to demonstrate the basic structural homology of all eucaryotic contractile locomotor organelles. The flagella of algae, protozoa, and lower fungi, the ciliary apparatus of ciliates, the tails of male gametes, both plant and animal, are all constructed on the same fundamental pattern, and probably share the same ontogeny, although this is less certain.
62-
6 3Contractile locomotor organelles also occur in two groups of procaryotic organisms, the true bacteria and the spirochetes, but are not homologous with eucaryotic locomotor organelles. A single bacterial flagellum, which has the approxi
mate dimensions of one of the eleven internal fibrils of the eucaryotic flagellum, can serve as the complete unit of bacterial locomotor function (see Weibull
64for a general discussion). Until recently, electron micros
copy has not revealed any internal structure in the bacterial flagellum ; but
Kerridge
et al.65have now shown the presence of molecular subunits,and
have suggested alternate models, consisting either of three helical or of
five parallel strands of subunits. Unlike eucaryotic flagella, bacterial
flagella are not surrounded by an extension of the cytoplasmic membrane ; instead, they extend through it, and are chemically distinct from i t .6 5
T h e axial filament of spirochetes has been much less closely studied.
Electron micrographs by Bradfield and C a t e r6 6 and S w a i n6 7*6 8 suggest t h a t it m a y be structurally equivalent to a bundle of bacterial flagella, wrapped helically about the cell and anchored in its two poles.
M a n y eucaryotic protists, the cells of which are not enclosed within a wall, can move over solid surfaces by directed cytoplasmic streaming
("ameboid movement"). No procaryotic organism capable of such move
ment is known.
One other major type of cellular locomotion occurs in procaryotic or
ganisms: the "gliding movement" characteristic of myxobacteria and many blue-green algae. N o locomotive organelles have ever been demonstrated on cells capable of gliding movement, and its mechanism is still unknown.
Some groups of higher protists (desmids, gregarines, the simpler red algae) also move by mechanisms which have been described as "gliding," but here again the mechanism is as yet unclear. Hence we do not yet know whether the gliding movements of eucaryotic and of procaryotic cells are mecha
nistically identical.
V I . The C h e m i c a l Structure o f the W a l l in Procaryotic Cells A wall, structurally distinct from the cytoplasmic membrane, is a well- nigh universal component of procaryotic cells; the pleuropneumonia-like organisms ( P P L O ) are the only group in which it appears to be absent.
T h e obvious selective value of the wall in bacteria and blue-green algae m a y be correlated with the fact t h a t they possess no physiological mecha
nism for the maintenance of water balance in a hypotonic environment;
in eucaryotic protists without walls, the contractile vacuole provides a device for coping with this problem.6 9 Hence, loss of the wall in procaryotic organisms (as exemplified by the P P L O group) restricts possible habitats, by prescribing an osmotically buffered environment.
Although studies on the chemical composition of procaryotic cell walls were initiated little more t h a n ten years a g o ,7 0 a large body of information about them has now accumulated ; in fact, far more is known about their structure t h a n about the structure of the walls of eucaryotic protists. Here we shall ignore the structural complexity and diversity of procaryotic walls, which have been ably reviewed elsewhere,7 1"7 3 and discuss only their common chemical properties.
I n the Gram-positive bacteria which possess walls of relatively simple chemical composition, electron microscopy of thin sections reveals the wall as consisting of a single, apparently homogeneous layer. T h e chemical analysis of such walls first revealed the existence of a distinctive class of
biological heteropolymers, the bacterial mucopeptides,7 4 which in extreme cases are the sole wall constituents of some Gram-positive bacteria. The polysaccharidic backbone of these mucopeptides consists of alternating units of muramic acid and iV-acetylglucosamine. The carboxyl group of muramic acid affords a point of attachment, although peptide bonding, of short chains of highly characteristic amino acids. These amino acids in
variably include glutamic acid and alanine, both of which have in p a r t the
" u n n a t u r a l " D configuration, together with either lysine or diaminopimelic acid. A few other amino acids (glycine, serine, aspartic acid) m a y in some cases also be incorporated.7 2
I n Gram-negative eubacteria, which have chemically more complex walls, the electron microscopy of thin sections reveals the wall as a multi- layered structure, the various layers often differing from one another in thickness and profile. Once the fundamental chemical composition of the mucopeptides from the walls of gram-positive bacteria had been worked out, it became evident t h a t the same key constituents (notably muramic acid and diaminopimelic acid, which occur uniquely in this class of hetero
polymers) were also present in the welter of monomeric constituents ob
tainable by the hydrolysis of walls from Gram-negative bacteria. M a r t i n and F r a n k7 5 have recently demonstrated, by an elegant series of fractiona
tions, t h a t in two Gram-negative bacteria the basal layer of the cell wall, adjoining the cytoplasmic membrane, consists of pure mucopeptide. Ac
cordingly, the Gram-negative bacteria have, underlying additional layers of different chemical nature, a wall t h a t is chemically homologous with the simple walls of certain Gram-positive bacteria. Although this basal layer makes a very small contribution in Gram-negative bacteria to the total mass of the w a l l ,7 5 it is evidently in large p a r t responsible for main
taining the structural integrity of the cell, as shown by the susceptibility of Gram-negative bacteria to penicillin and lysozyme, which both affect the integrity of the mucopeptide l a y e r .7 6 , 7 7
T h e discovery of diaminopimelic acid in the cells of blue-green a l g a e7 8 provided the first indication t h a t they might possess walls with the same distinctive chemical constituents as those of bacteria. This has been beauti
fully confirmed b y F r a n k et al.,79 who have used a combined electron microscopic and chemical approach to the analysis of wall structure in a filamentous blue-green alga, Phormidium. T h e y were able to show t h a t each cell in a filament is completely enclosed by a layer of pure muco
peptide; the wall in addition contains a second, outer layer of different composition.
T h e wall structure of other major procaryotic groups has not yet been studied in detail. I t should be noted, however, t h a t mucopeptide con
stituents have been reported in m y x o b a c t e r i a8 0 and in rickettsias.8 1 T h e
absence of mucopeptide has been reported for only one procaryotic group, the P P L O organisms,8 2 which appear to be devoid of walls.
There are conceivably other chemical and biochemical properties which m a y prove distinctive of procaryotic organisms as a whole; but the pres
ence of a unique class of mucopeptides as the principal strengthening element of the wall is as yet by far the best established one.
V I I . The C o m m o n Denominators of Eucaryotic a n d Procaryotic Cells
The foregoing analysis of the organizational peculiarities of procaryotic cells makes it easy to understand in retrospect why the problem of defining concisely the common properties of bacteria and blue-green algae proved so intractable. A p a r t from the absence of chloroplasts, long recognized to be characteristic of blue-green algae, none of the distinctive features of the procaryotic cell could be fully resolved by the techniques of classical cytology. The difficulties were compounded by the fact that, at the level of gross structure and function, eucaryotic and procaryotic cells are very similar to one another. I n each case, growth and reproduction bring into play much the same characteristic sequence of events. Grossly considered as units of biochemical function, the two kinds of cells are likewise equiva
lent; all modern biochemistry attests to this fact. At the gross genetic level, there are likewise far-reaching homologies. Both kinds of cell can enter into the same modes of organismal structure: unicellularity, multi- cellularity, and the coenocytic state. The differences between them are, accordingly, expressed only to a very minor extent in terms of gross prop
erties. Essentially, the differences now revealed represent two different modes of detailed construction, each of which can serve effectively as a basis for the performance of the universal functions of a cell.
VIII. Evolutionary Implications
P r i n g s h e i m8 3 has presented convincing arguments for the belief t h a t the bacteria and blue-green algae encompass a number of quite distinct major groups, not closely related to one another, but nonetheless united (as we can now see) by the possession of procaryotic cells. The procaryotic cell has, accordingly, provided a framework for extensive evolutionary diversification. Diversification is expressed in both structural and physio
logical properties. Among the most important structural features m a y be noted: organismal structure (unicellular, coenocytic, and simple multi
cellular groups) ; manner of cell division (binary fission, budding) ; mecha
nism of cellular movement. The physiological diversification is most strik
ingly exemplified by the variety of patterns of energy-yielding metabolism:
there are three quite distinct photosynthetic groups, each with its unique
pigment system, as well as a vast number of specialized ehemotrophie groups.
In many of these respects, there are parallel modes of evolutionary diversification among the eucaryotic protists as a whole (protozoa, fungi, and eucaryotic algae). It thus appears that natural selection has operated in much the same ways on the two different lines of protists, eucaryotic and procaryotic, to produce specialized groups in each line which grossly mimic one another. There is a strikingly analogous case, at a much higher level of biological organization. This is the evolutionary diversification of the marsupials on the Australian continent, to produce a whole series of specialized groups which mimicked the adaptive radiations of the placental mammals on other continents. We can, however, infer from the much more restricted development of marsupial lines when they existed in free competition with mammals that the possibility for this parallel evolution in Australia depended on the geological accident of continental isolation.
The eucaryotic and procaryotic protists, on the other hand, are at all times in direct competition. The successful survival and diversification of pro
caryotic organisms in the face of active competition from analogous forms with more highly developed cells are, therefore, at first sight an evolu
tionary paradox. How can it be explained? In the case of certain physio
logically highly specialized groups, the answer is obvious. The photosyn
thetic bacteria can perform energy conversion with wavelengths in the solar spectrum that are not absorbed by any other phototrophs ; the chemo- lithotrophic bacteria employ chemical energy sources not utilizable by any other living organisms. For the many procaryotic organisms that are not so nutritionally specialized, the occupation of a unique ecological niche cannot, however, be invoked as a solution. In this more general case, it may be surmised that the primary selective advantage has been conferred by the capacity for rapid growth, a consequence of the relative cellular sim
plicity, and above all of the enormously greater simplicity of the genetic material and its mode of replication.
In the whole span of contemporary living organisms, the gap which
separates procaryotic from eucaryotic protists is without doubt the largest
single evolutionary discontinuity. So far, we know of no organisms which,
in terms of their cellular construction, could be considered transitional
between the two. It is, nevertheless, difficult to avoid the conclusion that
at some time in the past the transition from procaryotic to eucaryotic forms
of life must have taken place; despite the differences, there are too many
features shared by both kinds of cells for us to assume completely separate
evolution. Above all, the sharing of a distinctive mode of photosynthetic
metabolism by blue-green algae and the various groups of eucaryotic algae
suggests that this crucial step in the evolution of the cell took place after
the emergence of aerobic photosynthesis, and in a line which had acquired this physiological capacity. For the evolutionary biologist, the procaryotic protists thus provide a precious and unexpected body of evidence about one of the earliest stages in biological evolution, the evolution of the cell.
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