Control of Cell Division: Models from Microorganisms

[CANCER RESEARCH 28, 1802-1809,September 1968]

Arthur B. Pardee Program in Biochemical Sciences, Moffett Laboratory, Princeton University, Princeton, New Jersey 08540

Control of Cell Division of a few years ago on the bacterial division cycle are sum One approach to the discovery of a difference between nor marized (12). The reviews furnish a guide to the literature mal and malignant cells is to investigate the regulation of cell before 1966. division. Normal tissues are regulated so that their cells are in a steady-state balance between duplication and destruction. Bacterial Division Malignant cells appear to duplicate unceasingly and are not Most investigations of bacterial division have been carried out with the closely related Gram-negative staining organisms in balance with the rest of the organism; they appear to have lost a control mechanism for cell division. Our problem is to Escherichia coli and Salmonella typhimurium. These organisms are implied unless otherwise stated. Gram-positive Bacillus determine how normal control mechanisms function, how they are deranged in malignant cells, and how they can be restored. species have been used for some fundamental studies on chro The working hypothesis of this article is that the funda mosome replication and for morphologic investigations. It is mental biochemical events which regulate cell division are too early to say whether important differences of cell division similar in both bacteria and higher organisms. This hypothesis regulation exist between different bacteria. Major points of will be useful at present to the extent that bacteria provide bacterial division and chromosomal duplication are illustrated a logical framework for ideas and experiments regarding an schematically in Chart 1. imal cell division. Bacteria increase their mass and cytoplasmic components Research with microorganisms has frequently furnished val (total RNA and protein) approximately exponentially with uable models for workers with higher organisms. Well-known time if they are uncrowded and well nourished. A transverse examples are biochemical pathways, gene structure and func barrier or septum appears periodically at the middle of the rod-shaped cell. Light and electron microscopy show that in tion, and control mechanisms at the levels of both enzyme syn E. coli this is created by an inward-growing furrow of the cell thesis and catalytic activity. Even for hormone action, a phe nomenon which does not appear in bacteria, fundamental in membrane which lies just inside the more rigid cell well. In Gram-positive organisms the septum appears simultaneously sights have been provided by microbiology through ideas of metabolic control. Studies of cell division with microorganisms across the entire cytoplasm; wall material is formed on the might similarly provide a valuable source of concepts and a septum. Following this, the daughter cells separate. They are frame of reference for workers with animal cells. The obvious nearly of equal size (coefficient of variation Â± 10%) in a differences, morphologic and temporal, between the two sys constant environment. A compensation mechanism must re tems may well be only variations on a basic theme. store unusually sized cells to the average upon the following This article- will attempt to present an organized picture of division, since a negative correlation between the size of mother current beliefs regarding bacterial replication. Our knowledge and daughter cells is observed. The precise distribution of size of bacterial division is increasing rapidly, though it is far from suggests a close relation between total cell mass and the timing complete. Conflicting reports are published; some of these no and spacing of septum formation. The actual separation of doubt will prove important, but now they are interesting the cells is less precisely timed (Â±20%), probably because of randomness of the movements which shake the cells apart mainly to specialists. These differing results probably reflect (16). the complexity of cell division, which must depend on the in Nuclear bodies can be observed by light microscopy with fluences of poorly appreciated experimental variables originating staining or phase, or in the electron microscope. There are from all parts of the cells and from the environment. The author has tried to piece the data together into a sort of "best- often two or four to an organism, depending on nutrition. The guess" guide. In no sense is a critical review of the entire lit septum is formed between the central pair. These nuclear bodies contain bacterial DNA. They are believed to be attached to erature intended. This would obscure the main concepts in a the cell membrane, or in the case of Bacillus to complex mem mass of detail. No attempt will be made to give a historic brane structures named mesosomes (9). There is no evidence perspective, or even to give credit to individuals where it is of nuclear membranes or of any of the complex mitotic ap certainly due. References will be to a limited set of very recent paratus found in animal cells. In this connection, it should be articles which can provide the next level of understanding and remembered that an entire bacterium is scarcely larger than references to earlier work. an animal mitochondrion (1 to 3 cu /*). Fortunately, several of the most active groups have recently Freely growing bacteria divide as often as once every 40 summarized their efforts (9, 14, 16, 22). This author's views min in a synthetic medium which contains a single well-utilized

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BACTERIAL CYCLE

/ Ornimin = 40 min

Growth Membrane Replicase Attachment DNA Origin 30 min 20 min

Chart 1. The bacterial cycle, a schematic representation. carbon source such as glucose. They can divide twice as frer tion, and vice versa. DNA replication should be, in part, con quently in very well-supplemented media, or many times more trolled by the events of cell division. Cell components other slowly with inferior carbon sources such as acetate. The cell than DNA do not have to be so closely coordinated with cell size and number of nuclear bodies decreases several-fold as the division; they exist in numerous copies and can be distributed medium becomes poorer. The main requirement for division approximately equally by chance. is neither a constant time nor a critical mass that is the same Bacterial DNA is found in the nuclear bodies, as shown for under all conditions. A subtler control is suggested by the example by radioautography of bacteria which have incor differences in composition and the complex adjustments in porated thymidine-3H. In the resting state, each nuclear body macromolecular syntheses that bacteria undergo when they are consists of a single molecule of double-stranded DNA of length transferred from one medium to another (16). about 1.3 mm (about 1,000 times as long as the bacterium) Bacteria, unlike most cells of higher animals, do not reach and molecular weight about 3 X 10*. Radioautographs of care a limit of division even in colonics on solid media. Bacteria fully lysed E. coli show the DNA to be circular, at least part stop dividing only when they reach high concentrations in of the time. These morphologic studies are completely sup liquid media. The cells become smaller when they reach this ported by genetic mapping which shows the E. coli chromo terminal stage of their growth; they then resemble bacteria some to carry all of its over 100 known genetic markers in a which are growing on a poor carbon source. When they are single, circular order. Bacillus subtilis has a similar chromo resuspended in fresh medium, they start growing again only some; the evidence for a circular structure has so far been after a time lag. The basis for these changes, especially of found only in germinating spores (25). failure to divide, is not well understood, but in some instances A chromosome starts to replicate at a definite, heritable lack of oxygen or nutrients, or accumulation of toxic products origin. Replication is semiconservative, each of the two new including hydrogen ions is responsible. strands being base-paired by hydrogen bonds to an old strand of the opposite polarity. Recent studies using density labeling The Cycle of DNA Replication suggest that each new strand is covalently linked to the term Duplication of bacterial DNA must be coordinated with inus of an original strand (25). All three double strands re cell division so that each daughter cell obtains a full comple main together at the origin, forming a Y-shaped fork which ment of hereditary material. Timing of DNA duplication during opens toward the replication point. The replicase is thought to the bacterial cycle is represented schematically in Chart 1; be attached to the cell membrane; the old DNA moves into the DNA is illustrated at about VLOOOitsrelative length. One this replication point and the replicas emerge, so that a second anticipates some sort of coupling mechanism which permits the Y-shaped fork completes a loop within the larger circular chro cell division mechanism to sense the progress of DNA replica mosome (see Chart 1). The DNA at the replication fork seems

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1968 American Association for Cancer Research. Arthur B. Pardee to be more easily denatured than the bulk of the DXA (10). replication increases, as if building blocks become limited. The As the chromosome moves through the replication point, genes time of chromosome replication is not sufficiently long to double in number one after the other in the order of their occupy the entire division cycle but takes place only during the sequence on the chromosome. Finally, when the end of the some part (12). Similar results have been obtained with B. chromosome is reached, a new round of replication commences subtilis (18). In contrast to rapidly growing E. coli, and like after some special events of initiation. animal cells, synchronized cultures of Alkaligenes fecalis can be Evidence for sequential gene replication is of three main made to synthesize DNA during only part of the division sorts. First, the quantity of a given gene can be measured in cycle at rapid growth rates (13). 0. subtilis by transformation with free DNA, the number of transformants being proportional to the number of genes (after Partition of DNA between Daughter Cells applying suitable controls) (22). Genes at the origin exist in The replication of bacterial DNA does not require cell divi twice as many copies as genes at the terminus; intermediate sion. Under many conditions bacteria grow into long filaments genes are present at intermediate concentrations in an unsyn- when they fail to divide. These conditions include poor nutri chronized culture,; owing to the random location of the Y- tion, Mg+ + deprivation, presence of toxic substances such as shaped replication points. Second, in cultures undergoing syn penicillin or crystal violet, mutagens, inhibitors of DNA syn chronous replication, genes are shown by transformation and thesis, or very mild irradiation with ultraviolet light or X-rays density-labeling to double in a definite order (22). Third, as (2, 12). Septa are not formed; their synthesis is more sensitive each element on the chromosome is replicated, its maximum than almost any other process in the bacteria. If DNA syn ability (potential) for producing a corresponding enzyme (or thesis continues, the nuclear bodies are distributed along the lysogenic virus) doubles; this can be observed as an increased entire length of the filamentous cells in many cases. One con rate of enzyme (or virus) synthesis upon induction of a syn cludes that DNA initiation, synthesis, and nuclear body for chronously dividing bacterial culture (20). Furthermore, these mation require neither cell division nor septum formation. Also, increases in potential depend on DNA replication (4). These longitudinal membrane growth requires none of the events of results indicate a definite direction of replication around a DXA synthesis and replication. circular chromosome, but it is not clear whether the direction In spite of the ready dissociation of DNA synthesis from is the same in all substrains of an organism, nor indeed whether cell division, DNA is precisely partitioned between daughter the origin of replication is the same or different in all sub- cells under normal conditions. This is noted from the constancy strains. This question is stimulating some very active research. of both the quantity of DNA and the number of nuclear bodies DNA replication can also be initiated by bacterial conjuga per cell in each medium (1C). Furthermore, old and new strands tion, in which DNA is transferred to a recipient bacterium (ÃŸ, of DNA are not passed on at random to the daughter cells but 8). Here the origin and direction of replication are clearly in a definite order according to when they were synthesized fixed in any one strain, but both origin and direction differ (5, 14). Episomes (nonchromosomal DNA molecules that carry from one strain to another. Bacteria with different conjugation genetic information) are also partitioned in a definite way be origins also show different timing of enzyme potential changes tween daughter cells, and their number per cell remains con (20). stant (9). The rate of DNA synthesis appears to double at about 20 This separation of various DNA components, precise in time min before division in synchronized cultures. This is attributed antl quantity, cannot be arranged by chance, as with cyto- to initiation at this time of a new round of chromosome dupli plasmic contents. The most attractive model assumes that the cation, with a doubling of the number of replication points. mechanism for segregation of genetic material at bacterial di The rate of synthesis per growing point appears to be con vision is very like the one observed with cells of higher or stant, as if the limiting factor were the rate at which DNA ganisms, in which the chromosomes are physically attached to could move past a single replicating enzyme (7). This result a mitotic apparatus that separates them. There is no indication and the constancy of rate of DNA synthesis in a variety of of a mitotic apparatus in bacteria. However, bacterial mem media which permit fairly rapid growth suggest that neither branes replace parts of animal cells for other functions such the supply of nutrients nor their rate of conversion to the as sites for oxidative phosphorylation. The DNA molecules deoxynucleoside triphosphates is limiting under these condi are visualized as being attached to the longitudinal bacterial tions. However, several workers using other conditions of syn membrane (!)). The observed partitioning of DNA of various chrony have found an exponentially increasing rate of DNA ages can be accounted for by a model in which attachment of synthesis, as if the supply of immediate precursors were in a DNA molecule occurs at the time its replication commences creasing throughout the division cycle (12). (5). This membrane elongates as the bacterium grows, thereby The time required for chromosomal replication depends on separating the attachment points of the newly formed sister nutrition, but to a smaller extent than does the time required chromosomes. These attachment points would have to serve for cell division (14). The two events are not occurring in as loci around which the daughter DNA strands condense at parallel but are synchronized at division. DNA replication can the time of septum formation in order for the long DNA continue through the entire E. coli cycle when division times strands to be completely segregated by the short distance be are an hour or less. When the growth rate is slower (division tween the attachment points. But if the origins of daughter times of more than two hours), the time required for DNA chromosomes are connected during DNA replication, which can

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1968 American Association for Cancer Research. Models from Microorganisms occupy the entire division cycle, the points of attachment that amino acid starvation or adding chloramphenicol, 5-fluoro- separate during this cycle cannot be at these origins. One can uracil, or phenethyl alcohol suggest that DNA initiation re imagine how one of the new replicas passes through a new quires synthesis of two proteins with different sensitivities to attachment site and remains with this site at completion, while these inhibitors; these might be new membrane attachment, the other replica remains attached by the replicase (Chart 1). initiator, or replicator proteins (14). Linking of DNA to membranes was originally suggested by General nutritional supply, measured by growth rate, has a .studies of bacterial conjugation in which membrane contact marked effect on initiation. When a culture reaches the end between bacteria appears to trigger DNA replication (6, 8), of its growth, the chromosomes complete their replication but Evidence for attachment of DNA to membranes has been ob do not initiate the next round. Slowly growing, poorly nour tained by electron microscopy of B. subtilis; membrane bodies ished bacteria complete a round of DNA replication and then known as mesosomes appear to be the points of connection there is a delay before the next round is initiated. In succinate (9). These might also be the growing points of DNA replica medium alternate replications of the two chromosomes in one tion, with the replicase enzyme holding DNA at the position E. coli cell have been reported (14). At intermediate growth where it is being synthesized. As further evidence, presumed rates each replication of a chromosome is initiated very soon DNA growing points are found in a membrane fraction of after the previous one is completed; DNA replication appears disrupted bacteria. continuous. In very rich medium, a dichotomous replication In support of this spatial fixation, specific DNA strands are occurs in B. subtilis: about half way through the first round often conserved throughout many generations at the extreme a second round of replications commences at both origins, and ends of growing chains of B. subtilis cells (5). These same the growing chromosome has three forks and thus four copies regions conserve their membrane material (9). These two re of each genetic locus near the origin (22). sults taken together suggest a firm union between DNA and Studies with specific inhibitors have indicated that proteins definite membrane sites. Conservation of membrane and DNA are required for initiation, as mentioned above. Phenethyl al in the same progeny cells has now been demonstrated (3). cohol blocks initiation at the same point as does amino acid The relatively exact partition of cell mass between daughter starvation (14). This inhibitor seems to act by increasing per cells (16) might also be explained by the growth character meability of the bacterial membrane (21), again suggesting a istics of the longitudinal membrane. If the growing point of connection between membrane and DNA replication. Acridine this membrane is at the center of the cell and between two dyes appear selectively to inhibit replication of episomes rela points of DNA attachment, if growth is equally rapid toward tively more than chromosomal replication. These dyes "cure" both ends from this point, and if septum formation occurs the bacteria of their episomes (6). at this point, the cell would divide equally. Chemical activators of DNA initiation have not been reported, except for the influence of rich medium in initiation of dichoto Initiation of DNA Replication mous replication. However, bacteria whose DNA synthesis is Biosynthesis of a macromolecule requires a special initiation blocked by thymine starvation initiate a new round of DNA reaction in addition to the sequential attachment of building synthesis at only one of the two potential new origins when blocks that make up the bulk of the synthesis. This is as true thymine is restored. This differs from initiation at both origins of bacterial chromosome duplication as it is of RNA or pro following amino acid starvation. Initiation in thymine-starved tein synthesis. Initiation appears to be that part of replication bacteria is inhibited by chloramphenicol or 5-fluorouracil and, where regulatory influences determine the timing of macro- therefore, seems to require RNA-dependent protein synthesis. molecule synthesis and the quantity of completed macromole Cytosine arabinoside, which inhibits DNA synthesis, does not cule. The subsequent synthesis, so much more prominent in cause premature initiation, suggesting that some metabolic im quantity and duration, proceeds relatively automatically (1C). balance of thymine-deprived cells initiates, perhaps by inducing This concept is analogous to the regulation of small-molecule the essential protein (14). These effects strongly hint at some synthetic pathways by end-product inhibition, where regula process similar to enzyme induction-repression in the DNA initiation event. tion of the initial step determines the others. Thus, the key event in regulation of chromosome replication should be sought The ReplicÃ³n Hypothesis in initiation. This must be studied with intact cells at present, The most plausible current model of DNA initiation is based since DNA synthesis by extracts or purified enzymes is not on the represser-operator model for regulation of protein syn sufficiently physiologic to be significant to the problem. thesis through messenger RNA synthesis (8). An entire DNA The most significant finding is that protein synthesis must molecule (chromosome or episomal element) is considered to occur before each initiation (16). In bacteria deprived of an be a unit of DNA replication. This is named a "replicÃ³n." Initi essential amino acid, the round of DNA replication in progress ator and replicator genes on the replicÃ³nare thought to be in is completed but a new round does not commence. Reinitiation volved in initiation by analogy with the represser and operator upon amino acid addition starts up DNA synthesis. This occurs genes for regulation of enzyme synthesis on an operon. The after different times in individual cells; probably cytoplasmic initiator gene carries the information for structure of an initi events which occur prior to initiation were stopped at different ator protein which enters the cytoplasm after it is synthesized. stages in individual bacteria. The different consequences of When the replicator gene receives this initiator protein, DNA

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1968 American Association for Cancer Research. Arthur B. Pardee synthesis is initiated and is propagated down the chromosome. (23). Lysogenized phage also start autonomous replication after The effect of the initiator is positive in the sense that it is exposure to "induction" treatments such as irradiation. required for starting DNA synthesis. Other results suggest very definitely that episome replication The principal evidence for the replicÃ³nhypothesis is obtained depends on some property of the host, as would be expected with mutant episomes defective in DNA replication (8). Nor from its replication in rhythm with host division. The number mal episomes replicate independently of chromosomes, yet in of episomes that a bacterium can carry is limited, which sug rhythm with cell division. The mutant episomes replicate more gests a competition between episomes for sites in the cell. Mu slowly than the cell divides if the temperature is raised from tations of the bacterial host chromosome, as well as episomal 30Â°Cto 42Â°C.The bacteria are not killed because all of their mutations, can cause the higher-temperature elimination of essential genes, including those required for chromosome repli episomes (8). cation, are on the chromosome and are heat-stable. But loss of The replicÃ³nhypothesis is the most plausible and useful one, the episome can be detected by loss of the genes it alone carries. at present, because it suggests experiments to test it. It presents Thus, when an episome carrying the yS-galactosidase gene was major questions: What chemical change occurs upon initiation? lost from a host with a lac~ chromosome, lactose-negative bac Is it a change in DNA, such as local denaturation (10), or a terial colonies were easily identified on selective agar plates. scission of covalent bonds which connect terminal and original With such a system, conditions affecting replication of episomes DNA strands (25) ? Or is it the combination of an enzyme or could be investigated readily. membrane protein with a starting site? Is the protein which The main conclusion was that initiation of DNA replication is required for initiation (16) the product of the initiator struc requires the synthesis of a replicon-specific cytoplasmic protein. tural gene? Is the initiator protein identical to the membrane Involvement of a protein was inferred from the sharp heat attachment site? lability of replication (characteristic of protein denaturation). This protein has a positive role, since DNA replication stops Cytoplasmic Coinitiators in its absence. (By contrast, destruction of a heat-labile re- According to the replicÃ³n hypothesis, the initiator protein pressor permits enzyme formation.) That the protein is cyto made during one round of DNA replication activates the next plasmic was indicated by cooperation between an episome with round. One can imagine a short burst of initiator synthesis, a heat-stable, replication-controlling system and a heat-labile perhaps created by duplication of the initiator gene. The entire episome within the same bacterium. Specificity is shown by the chromosome, including the initiator gene, is thought to repli inhibition at increased temperature of episomal but not chro cate in the absence of protein synthesis when the bacteria are mosomal replication in the bacteria with a heat-labile episomal starved of amino acids. The initiator gene in its replicated form replicÃ³n. might produce initiator only after amino acids are again sup Observations on episomes and injected DNA fragments sup plied. This could account for the source of protein required for port the idea that DNA units must carry special structures in initiation. order to replicate independently. Episomes are thought to repli This simple model runs into several problems. The prema cate under the control of their own replicator and structural ture, dichotomous initiation that occurs in very rich medium, genes. Their transfer from one bacterium to another by con and also the long delay between termination and the next initi jugation requires DNA replication which is controlled by these ation observed in poor medium strongly suggest specific nu genes, also called sex factors (6). tritional requirements for initiation. The coordination of dupli In contrast to episomes, chromosome segments injected by cation in each cell cycle of all of the replicons in a cell suggests high-frequency recombination donor bacteria usually cannot some common cytoplasmic factor that interacts with all of the replicon-specific initiator proteins at the same time. Also, a replicate unless they are integrated into the recipient chromo some by genetic recombination. These segments cannot serve general cytoplasmic change, the degradation of 10% of the cell as templates for DNA synthesis in the same cell where the com proteins, has been noted at the time of DNA replication (19). plete chromosome is replicating. They appear to lack the repli Still consistent with the replicÃ³n hypothesis would be a replicon-specific initiator protein whose function or synthesis cator gene which is at the terminal end of a replicÃ³n (6). is activated periodically by a low molecular weight compound. The establishment of lysogenic phages as prophages in their The concentration of this compound, which we will name a co- bacterial hosts and subsequent harmonious replication requires inhibition of the phages' autonomous replication apparatus. initiator, could change during the cell cycle in a manner that depends on both chromosome replication and nutrition. This inhibition in lysogenized bacteria creates immunity to The only molecules so far proposed as co-initiators are DNA superinfecting phages of the same strain. However, when super- precursors (13). The concentrations in bacteria of purine infection is by related phage of a different immune type, both deoxynucleotides are low except when purine deoxynucleosides the superinfecting and the lysogenic phages replicate autono are supplied in the medium, or upon thymine starvation. Py- mously. The explanation is similar to the replicÃ³nmodel : a posi rimidine deoxynucleotides are found, but in amounts (3% of tively acting initiator substance is required for autonomous DNA) insufficient to suggest that DNA synthesis is initiated phage replication; production of this initiator is inhibited in by their availability as precursors. However, their role as co- the lysogenized situation; and the superinfecting phage pro initiators is possible because their concentration rises a few duces initiator that is used by both phages in the same cell minutes before stepwise DNA synthesis commences in synchro-

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1968 American Association for Cancer Research. Models from Microorganisms nized A. fecalis, as would be predicted. Periodic deoxyribo- the one-generation delay in increased rate of cell division follow nucleotide synthesis is still observed when DNA synchrony is ing a nutritional shift-up (15). An exception to this pattern is destroyed by adding excess of all four deoxynucleosides (13). found with a newly described mutant in which cross walls create The pool levels are therefore not determined by DNA synthesis, "minicells" containing about one-tenth the normal amount of and DNA initiation does not depend solely on variations of cytoplasm and no DNA (1). Apparently the positioning of these pools. The requirement of all four deoxynucleosides sug septa is deranged; nuclear bodies do not have to exist on both gests that the control of initiation might be dependent on a sides of the division point. balance or cooperation effect between these compounds, similar The coupling between DNA and septum formation is not a to multivalent repression of enzyme synthesis. Although no di tight one, since it can be perturbed by various nutritional con rect evidence is available regarding the chemical nature of co- ditions, inhibitors, and mutations. For instance, rapidly growing inducers, deoxy-compounds, or others, the data on nutritional cells have more than one nuclear body per cell; that is, septa effects and pools appear sufficient to retain this concept in the are formed regularly but less often between nuclear body di scheme of DNA initiation. visions. The observations on uncoupling of DNA synthesis from Co-initiators would have to rise and fall during the replication cell division fall into a similar pattern to those described above cycle in order to be effective periodic triggers of DNA initi for uncoupling initiation of DNA replication, and suggest a ation. One often-suggested basis for these changes depends on similar hypothesis. Perhaps DNA replication initiates septum the cells reaching a critical mass at the time of replication (this formation by creating some cytoplasmic change upon its own mass depending on nutrition). The concentrations of some in- completion. Transmission of this impulse could depend on tracellular metabolite could change as the cell grows because cytoplasmic conditions for its effectiveness in starting cell the mass increases more rapidly than the surface (more so in division. a spherical cell tlian in a rod-shaped one). If the rate of syn A relation between DNA and septum formation can be studied thesis of cell envelope precursors was proportional to the mass using lon~ mutants (2). These mutants fail to form septa after and their rate of use was proportional to the surface, their very mild ultraviolet- or X-irradiation. Irradiation does not concentration could increase through the cell cycle and trigger appear to act directly on the septum-forming membrane region, replication at a critical level. Other evidence regarding a con although it is quite specific in not noticeably altering DNA nection between cell envelope and DNA replication will be synthesis or other metabolism. The nucleic acid-type action discussed in the next section. spectrum and the higher sensitivity of these mutants when they A different basis for rising and falling pools of co-initiator also lack a DNA-repair mechanism suggest that DNA is the depends on periodic enzyme synthesis during the division cycle. target. This is shown clearly when bacterial division is made The doubling of a cell's potential (maximal ability) to form an more sensitive by incorporation of 5-bromouracil into DNA enzyme as the corresponding gene is replicated can be modified (24). A transient damage to DNA in these mutants seems to by enzyme induction, repression, and inactivation. Based on prevent septum formation for a half-dozen generations, where this, models for self-generating (autogenous) cycles of enzyme upon the filaments lyse. synthesis have been suggested (18, 20); this predicts that en The lon~ mutants also provide a clue to cytoplasmic events zyme oscillations could in turn create marked metabolite oscil involved in initiation of septum formation. The damage in lations with periods equal to the DNA replication cycle. In this irradiated mutants can be reversed by shifting the bacteria to way, co-initiator concentrations could periodically reach a maxi poorer media; some compounds in the rich medium seem to mum, the timing depending on nutritional effects on repression prevent repair of damaged septum formation. Increased tem mechanisms and also on the replication of a structural or regu perature or pantoyl lactone can also reverse the inhibition. In lator gene once per DNA replication. jection of an episomal lon+ gene into irradiated lon~ bacteria repairs the defect, indicating a dominant irons effect of lon+ Timing of Septum Formation (24). Most interesting, lon+ bacteria release substances into The periodic formation of cross walls is an essential prelude the medium which help irradiated lon~ bacteria to recover. to separation of daughter cells and their DNA. What mecha There are at least two of these two factors; they can be ex nism initiates septum formation periodically in time? tracted from lon+ cells (2). All of these results suggest that Cell division follows DNA completion by 20 min in a variety irradiation of lon~ DNA causes an imbalance of metabolism of media (7). This close coordination of DNA replication and which prevents septum formation. Depending on the genetic cell division suggests that completion of DNA replication might factor Ion and nutrition, the original balance is restored or the trigger septum formation (15). This is supported by many imbalance perpetuates itself, resulting in filament formation observations that, when DNA synthesis stops (because of in and eventual lysis. hibitors, irradiation, or thymine starvation), septum formation A clue to the kinds of metabolites that might be involved is and cell division also stop. Filamentous bacteria are formed. gained from the observation that lon~ bacteria are usually mu- The spatial separation of two nuclear bodies upon completion coid; that is, they overproduce cell wall components (17). Possi of DNA replication might in some way trigger septum forma bly cell envelope precursors which are in excess in the Ion- tion. The requirement of two nuclear bodies has been suggested bacteria inhibit the recovery of the irradiated cells (24). Further on the basis of electron micrographs which show that the sep indications that cell wall metabolism plays a role in septum tum is between them (12). It has also been invoked to explain formation is gained from observations that inhibitors of cell

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1968 American Association for Cancer Research. Arthur B. Pardee wall synthesis, such as penicillin or crystal violet, cause filament lated facts are brought into suggestive arrangements. It is hoped formation. Septa also do not form in spheroplasts, which are that this organization of the simpler, more readily perceived bacteria in hypertonic, osmotically protecting medium whose phenomena of bacterial division will serve initially to guide cell wall has been partly dissolved by lysozyme or malformed some who work with more complex systems. by exposure to penicillin. Perhaps the most profitable working hypothesis is that peri REFERENCES odic DNA completion in turn drives an oscillation of metabolites in the cytoplasm which triggers septum initiation. Alternatively, 1. Adler, H. I., Fisher, W. D., Cohen, A., and Hardigree, A. A. the attachment of DNA to the longitudinal bacterial membrane Miniature Eschcrichia coli Cells Deficient in DNA. Proc. Nati. might, upon completion, transmit a signal from one of the repli- Acad. Sei. U. S., 57: 321-326, 1967. cons directly via the cell membrane to the septum forming site. 2. Adler, H. I., Fisher, W. D., and Hardigree, A. A. Repair of Radiation-Induced Damage to the Cell Division Mechanism Summation and Prospect of Escherichia coli. J. Bacteriol., 01: 737-742, 1966. The main concepts regarding bacterial division are the follow 3. Chai, X., and Lark, K. G. Segregation of Deoxyribonucleic Acid ing. Bacteria divide periodically at intervals precisely deter in Bacteria: Association of the Segregation Unit with the Cell mined by their environment as well as their genetic composition. Envelope. J. Bacteriol., 94: 415-421, 1967. 4. Donaehie, W. D., and Masters, M. Evidence for Polarity of Unlike animal cells, intracellular or intercellular controls do not Chromosome Replication in F" Strains of E. coli. Genet. Res., cause division to stop. Cell division is in coordination with DNA 8: 119-124, 1966. replication so that the genetic material is exactly partitioned 5. Eberle, H., and Lark, K. G. Chromosome Segregation in Bacil between daughter cells. The spatial separation of daughter lus subtilis. J. Mol. Biol., S3: 183-186, 1966. chromosomes appears to depend on their connection to the 6. Hayes, W. Sex Factors and Viruses. Proc. Roy. Soc. London, longitudinal bacterial membrane. Growth of this membrane Ser. B, 164: 230-245, 1966. might move apart the DNA attachment points, and its extent 7. Helnistetter, C. E., and Picrucci, O. Cell Division During In of growth might be a regulating factor. Formation of an inter hibition of Deoxyribonucleic Acid Synthesis in Eschcrichia vening septum can account for partition of genetic material coli. J. Bacteriol., 95: 1627-1633, 1968. between daughter cells. 8. Jacob, F., Brenner, S., and Cuzin, F. On the Regulation of Although DNA synthesis appears to be continuous through DNA Replication in Bacteria. Cold Spring Harbor Symp. the cell cycle under some conditions, it is a periodic event whose Quant. Biol., 28: 329-347, 1963. end can be separated in time from the beginning of its next 9. Jacob, F., Ryter, A., and Cuzin, F. On the Association between round. The single bacterial chromosome is conceived of as a DNA and Membrane in Bacteria. Proc. Roy. Soc. London, unit of replication which has been named replicÃ³n. A special Ser. B, 164: 267-278, 1966. initiation event which requires protein synthesis starts each 10. Kidson, C. Deoxyribonucleic Acid Secondary Structure in the Region of the Replication Point. J. Mol. Biol., 17.â€¢1-9,1966. cycle of replicÃ³nsynthesis, which then continues automatically down the chromosome. Each replicÃ³n is proposed to contain 11. Kubitschek, H. E., Bendigkeit, H. E., and Loken, M. R. Onset of DNA Synthesis during the Cell Cycle in Chemostat Cul genetic elements that create a positive feedback loop, which tures. Proc. Nati. Acad. Sei. U. S., 67: 1611-1617, 1967. together with oscillations of cytoplasmic co-initiators is sug 12. Kuempel, P. L., and Pardee, A. B. The Cycle of Bacterial gested to control the timing of DNA initiation. Duplication. J. Cellular Comp. Physiol. Suppl. 1, 62: 15-30, DNA initiation does not depend on cell division. Rather, the 1963. timing of cell division depends on the DNA cycle. Septum for 13. Lark, K. G. Cellular Control of DNA Biosynthesis. In: J. H. mation, which is the first morphologic step in cell division, might Taylor (ed.), Molecular Genetics, Vol. 1, pp. 153-206. New be initiated by a signal from the completion step of DNA repli York: Academic Press, 1963. cation, which probably occurs on the membrane. Transmission 14. Lark, K. G. Regulation of Chromosome Replication and Segre of this signal is modified by nutrition and inhibitors, as is the gation in Bacteria. Bacteriol. Rev., 30: 3-32, 1966. signal for DNA initiation. Events initiated by the DNA cycle 15. Maal0e, O. The Relation between Nuclear and Cellular Di indirectly trigger both a new round of DNA replication and vision in Escherichia coli. In: J. Cairns, G. S. Stent, and initiation of cell division via septum formation. J. D. Watson (eds.), Phage and the Origins of Molecular Many parallels can be found between those observations and Biology, pp. 265-272. New York: Cold Spring Harbor Labora the mass of information regarding division of animal cells (13). tory of Quantitative Biology, 1966. 16. MaaI0e, O., and Kjeldgaard, N. 0. Control of Macromolecular The fundamental difference, aside from cell structures, seems Sj-nthesis. New York: W. A. Benjamin, Inc., 1966. to lie in a regulation by which the impulse that initiates DNA 17. Markovitz, A., and Baker, B. Suppression of Radiation Sensi replication is blocked in mature, normal animal cells. The in tivity and Capsular Polysaccharide Synthesis in Escherichia hibition seems to originate mainly from contact with neighbor coli K-12 by Ochre Suppressors. J. Bacteriol., 94: 388-395, 1967. ing cells. Its absence in malignant cells suggests both a role of 18. Masters, M., and Donaehie, W. D. Repression and the Control cell membrane in regulation, similarly to bacteria, and differ of Cyclic Enzyme Synthesis in Bacillus subtilis. Nature, 209: ences in cell membranes of normal and cancer cells. 476-479, 1966. A major step in originating and developing any hypothesis 19. Nishi, A., and Kogoma, T. Protein Turnover in the Cell Cycle is to collect and analyze the available information. For analysis, of Escherichia coli. J. Bacteriol., 90: 884-890, 1965. data must be divided and subdivided into a scheme where re 20. Pardee, A. B. Periodic Events in the Bacterial Duplication

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Cycle. Japan Protein, Nucleic Acid, Enzyme, 11: 829-836, 23. Thomas, R. Control of Development of Temperate Bacterio- 1966. phages. I. Induction of Prophage Genes Following Hetero- 21. Silver, S., and Wendt, L. Mechanism of Action of Phenethyl Immune Super-Infection. J. Mol. Biol., S3: 79-95, 1966. Alcohol: Breakdown of the Cellular Permeability Barrier. 24. Walker, J. R., and Pardee, A. B. Evidence for a Relationship J. Bacteriol., 93: 560-566, 1967. between DNA Metabolism and Septum Formation in 22. Sucoka, N. Synchronous Replication of the Chromosome in Escherichia coli. J. Bacteriol., 95: 123-131, 1968. Bacillus subtilis. In: I. L. Cameron and G. M. Padilla (eds.), 25. Yoshikawa, H. The Initiation of DNA Replication in Bacillus Cell Synchrony, pp. 38-53. New York: Academic Press, 1966. subtilis. Proc. Nati. Acad. Sei. U. S., Ã¶S:312-319, 1967.

SEPTEMBER 1968 1809

Arthur B. Pardee

Cancer Res 1968;28:1802-1809.

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