Control of Cell Division Bacteria · directly involved in cell division). Thepresence orabsenceofa morphologically distinct septum may be a real difference between species and mayreflect

BAnrEmoIwOOIcAL REVIEWS, June 1974, p. 199-221Copyright 0 1974 American Society for Microbiology

Vol. 38, No. 2Printed in U.S.A.

Control of Cell Division in BacteriaMARTIN SLATER AND MOSELIO SCHAECHTER

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston,Massachusetts 02111

INTRODUCTION .......................................... 199A Digression into Yeast ......... ................................. 199

PHYSIOLOGICAL ASPECTS OF CELL DIVISION ......... .................... 200Division Process .......................................... 200Site of Envelope Synthesis ........... ............................... 202Sensitivity of Cell Division to Interfering Conditions ......... .................... 204

GENETIC STUDIES OF CELL DIVISION ....................................... 204General Comments .......................................... 204DNA Metabolism .......................................... 206Protein Synthesis .......................................... 209"Division Potential".......................................... 210

Placement of the Site of Division ........................................... 210Cell Wall .......................................... 212Small-Molecule Metabolism .............. ............................ 213

CONCLUSIONS.................................................................. 215

INTRODUCTIONBecause in one sense bacteria are structurally

and genetically simple and amenable to study,it might be expected that their division dependson relatively few events which are interrelatedin simple patterns. Our principal conclusion isthat this expectation is essentially wrong.We will concern ourselves with the regulation

of division, rather than with the process itself. Arecent review by Higgins and Shockman (52)deals extensively with the present knowledge onthe process of division in bacteria. Here we willdiscuss briefly the operational definitions ofbacterial division, the sequence of events lead-ing to its initiation, and how these events areinterrelated. Our emphasis will be on geneticstudies.

In our minds, there are major operationalproblems in working with bacterial cell division.These are: (i) determining if inhibitors or muta-tions which affect division are specific; (ii)deciding whether closely linked mutations arein different sites of the same cistron or indifferent cistrons; (iii) recognizing the existenceof bypasses and possible artifacts produced nearrestrictive conditions; and, (iv) finding bio-chemical means of studying the synthesis of thecross wall and membrane separately from thesynthesis of the peripheral envelope. Recentwork indicates that these problems appear to beless troublesome in the yeast Saccharomycescerevisiae (47). We will describe some of thiswork and use it as a guideline for determining

the kind of questions that may be asked of celldivision in bacteria.

A Digression into YeastTwo facts about yeast indicate some of the

advantages of this system. First, the primarywall septum seems to consist of a single specificsubstance, chitin, which is not found in the restof the cell wall (18). Thus, the study of thebiochemistry of chitin synthesis may constitutea direct study of the regulation of cell division inyeast. Second, the regulation of division seemsto be more specific and less sensitive in yeastthan in bacteria. In bacteria a wide range ofinhibitors, many of which stop growth at highconcentrations, preferentially stop division atlow concentrations. In yeast, the spectrum ofpreferential inhibitors of division is both morenarrow and more specific (89).A remarkably rapid and straightforward

study of the genetic control of the yeast cellcycle is being conducted by Hartwell's group(47). From an array of temperature-sensitivemutants, "cell division cycle" (cdc) mutantswere selected for study initially on the basis oftwo defining properties: (i) when shifted from 23to 36 C, all of the cells of an asynchronouspopulation accumulate with a uniform mor-phology characteristic of a block in one step ofthe cell cycle (this is called the terminal pheno-type). (ii) After a discrete point during the cellcycle, the remainder of the cycle can be carriedout to completion even at 36 C (this is called the

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SLATER AND SCHAECHTER

execution point). Cells which have passed theexecution point at the time of the shift completethe ongoing cycle, divide once, and accumulateterminal phenotypes in the next cell cycle.Because mutations affecting the synthesisrather than the activity of proteins would notnecessarily confer a relevant execution time onthe mutant, in some cases cdc mutants areselected on the basis of uniform terminal pheno-types. However, the execution point remains animportant criterion in selecting and groupingspecific mutants. Blocks at each of the majorobservable events in the "deoxyribonucleic acid(DNA)-division" cycle in yeast are found amongthese mutants. It has also been possible todetermine the time at which cells "decide" tostart a mitotic cycle rather than enter meiosis orconjugation. This is essentially a differentiationstep subject to regulation (in this case bymating factors) which changes one type of cellcycle to another type (in this case from haploidcycles to mating and diploid cycles).The mutations are recessive and, by com-

plementation tests and tetrad analysis, werefound to fall into 35 nuclear cistrons. In allcases, different mutations in a given cdc cistronresulted in the same phenotype (same terminalphenotypes and execution points). Apparent,exceptional cases were shown to be doublemutants. The terminal phenotypes and execu-tion points are, then, cistron rather than allelespecific. These results purport to show that acdc gene specifies a function which is requiredat a particular time to mediate one and only onestep in the cell cycle. These studies have per-mitted determination of the sequence and rela-tionship between all the observable events inthe cell cycle of yeast.

PHYSIOLOGICAL ASPECTS OF CELLDIVISION

Division ProcessGram-positive bacteria divide by forming a

cross-septum consisting of both membrane andwall (22) (Fig. 1). In some gram-negative bac-teria such a structure is usually not apparent(as an example, see 46a). Dividing Escherichiacoli cells, for example, appear constricted at thesite of division, showing a gradually increasinginward curvature of both membrane and wall(Fig. 2). (Morphologically speaking, it is errone-ous to refer to this configuration as "septum."Nonetheless, we are forced to make a generaluse of this term since there is no other conven-ient way to refer to the wall and membranedirectly involved in cell division). The presence

or absence of a morphologically distinct septummay be a real difference between species andmay reflect possible differences in the timing ofvarious steps in the division process (123) (seeFig. 2 and 3). Thus, a septum would exist onlyfleetingly if the inward growth of envelope isfollowed very rapidly by cell separation. On theother hand, it is possible that the reason whysepta are usually not seen in gram negatives isthat they are destroyed during cytological fixa-tion. This seems to be the more likely case,because septa can be seen when E. coli are fixedwith 5% Acrolein and 0.5% glutaraldehyde (I.D. J. Burdett and R. G. E. Murray, personalcommunication) (Fig. 4). They are also seenwhen other fixation procedures are carried outat certain temperatures or when cells are grownat 45 C (138). This question needs furtherattention because precise knowledge on themode of division of E. coli is required to definethe phenotypes of mutant cells.These uncertainties make it difficult to esti-

mate the time required for various steps in thedivision process. The only step whose timinghas been determined with precision is the inter-val from the initiation of production of a septumto its expression in a division in Bacillussubtilis. Paulton showed that the time for thisstep is constant over a wide range of growthrates and is 138 min long at 30 C (109). At rapidgrowth rates, this step is initiated repeatedlybefore the original cells separate, thus produc-ing multiseptate cells. This is analogous to theconstancy of the time required for a round ofDNA replication at various growth rates andwith the multifork pattern of chromosome re-plication which exists at rapid growth rates(e.g., 48). It must be emphasized that 138 min isthe time from the inception of a septum to itsparticipation in division and not the time re-quired to make the septum. In the case ofgram negatives, comparable studies have notbeen carried out, but there are suggestions thatthe time required for division is also not a fixedfraction of the generation time. The proportionof cells with a visible constriction was measuredin cultures of E. coli growing at different rates(84). This proportion was found to be greater incultures growing at faster growth rates. Incultures growing with a doubling time of 30 min,it reached a value of 25% of the total cellpopulation. Because cells with visible constric-tions have obviously initiated "septum" synthe-sis sometimes before, this measurement is anunderestimate of the fraction of the cell cyclerequired for "septum" synthesis. The magni-tude of this underestimate is unknown, but can

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BACTERIAL CELL DIVISION CONTROL

FIG. 1. The septum in gram-positive rods. Electron micrographs of thin sections of Bacillus cereus showingsepta (A) and their thickening by exposure to chloramphenicol (B); reproduced from reference 23 by permissionof K. L. Chung and the National Research Council of Canada.

be approximated from another study. Whencultures of E. coli or Salmonella typhimurium,also doubling every 30 min, were transferred tostarvation media, they continued to divide untilthe cell number nearly doubled (128). If, byanalogy with DNA replication, starvation pre-vents the initiation, but not the actual forma-tion of the "septum," then this extent of resid-ual division indicates that "septum" synthesiswas initiated in the previous cell cycle. It shouldbe noted, however, that the assumption con-cerning the effect of starvation on initiation ishitherto unproven.The most important physiological step in cell

division may be the functional partitioning ofthe cell, that is, the process whereby the twodaughter cytoplasms become separate compart-ments. The timing of this step has been studiedby using physiological criteria for the partition-ing of daughter cells. The principal method hasbeen to determine if the kinetics of killing ofcells vary during the cell cycle by using phageinfection or sonication of synchronized cultures(25). Clark (25) found that within a defined timein the cell cycle, before a morphological parti-

tion is seen, the number of hits required to kill acell changed from one to two. This wouldcorrespond to a time of "physiological" ratherthan physical separation between sister cells.Extensive work on this subject was carried outby Onken and Messer (108), who studied severalactivities expected to be affected by separationof sister cytoplasms. They found the followingsequence of events in synchronously dividingcells whose doubling time was 45 min: DNAreplication ends at 25 min, the synthesis ofmurein increases at 30 min, sensitivity to colicinE2 increases between 30 and 35 min, inactiva-tion by phage T4 changes from one to two hits at35 min, and the ability of two mutant phages torecombine and complement each other contin-ues until shortly before division. They favoredthe interpretation that some of these results aredue to changes in properties of the cell envelopeand not to compartmentalization of the cyto-plasm. They concluded that cells become bothphysically and functionally separated onlyshortly before they actually divide.

Little is known about the mechanisms in-volved in the physical separation of divided

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Site of Envelope SynthesisIt has been proposed repeatedly that cell

division is under the control of the activities of a"membrane growth site" which is responsiblefor both the extension and the division of thecell. According to this model, the site feeds wallin the peripheral direction for cell extensionand in the centripetal direction for cell division.We will consider the evidence for the exist-

ence of a "membrane growth site," as well asthe available data regarding its location. Ingram-positive cocci the existence of such a siteis firmly established, thanks to a detailed analy-sis of the morphological changes associated withthe function of new cell wall. This work hasbeen recently reviewed and will not be furthertreated here (52).

In rod-shaped bacteria, the existence of amembrane growth site rests on two types ofevidence. (i) New cell wall appears to be depos-ited in cells of all ages at the center of the rod, as

FIG. 2. Constriction in gram-negative rods: celldivision in E. coli D21. All layers of the envelopeparticipate in the concentric invagination process.x62,400; reproduced from reference 104 by permissionof S. Normark and the publishers ofActi PathologicaMicrobiologica Scandinavica.

bacteria. On the level of single cells, it isthought that the variability in the timing ofseparation is mainly responsible for the differ-ences in generation times of individual cellsobserved in many species (e.g., 82, 112, 129). InB. subtilis and pneumococci there is evidencethat hydrolases function in the separation step(34, 38a, 142a). The extent of cell separation iscorrelated with the hydrolase activity of variousmutants and of cultures growing under differentconditions (34, 35). There are mutants of E.coli, called envA, which form chains (104-106).Their murein cross wall is completed withoutthe outer lipopolysaccharide-containing layer,which later is placed between the layers of themurein septum. A mutant of S. typhimurium,4a, forms filaments at 42 C, but at 25 C in thepresence of high concentrations of yeast extract FIG. 3. The septum in mutants of gram-negativeit forms chains of completely partitioned cells. rods: cell division in the E. coli envA mutant D22. AIn these chains, the lipopolysaccharide layer is septum separates individual cell units. Centrallyincluded in the cross walls between cells (7). In located in the septum is a thin structure of moderately

electron-dense material. Close to one invaginationthis mutant, in otheraoniss point this structure is split into two components.the variability in the time of separation of x60,000; reproduced from reference 104 by permis-individual cells may be due to the sticky proper- sion of S. Normark and the publishers of Acta Patho-ties of a slime layer. logica Microbiologica Scandinavica.

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A

r.

FIG. 4. The septum in wild-type E. coli seen as the result of special fixation. Cells grown in glucose minimalmedium were fixed with 5% Acrolein and 0.25% glutaraldehyde in 0.05 M sodium cacodylate buffer, pH 7.5. A,E. coli B, x60,000; B, E. coli Bir, x 121,000. Micrographs were provided by courtesy of I. D. T. Burdett and R.G. E. Murray (submitted for publication to J. Bacteriol., 1974).

shown radioautographically by the location of peripheral bulges produced by weakening of thediaminopimelic acid newly incorporated into wall. It has been proposed (30) that these bulgesmurein (126). (ii) E. coli treated with low correspond to the areas of murein synthesis andconcentrations of penicillin shows characteristic to the sites where division would have taken

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place but for the inhibition by penicillin. Thelocation of these bulges was determined in cellswhose size was altered by growth at differentrates (30). The bulges were always formed at aconstant distance from one of the poles of thecells. Thus, in small cells growing at a slowgrowth rate, this distance is smaller than one-half of the cell length. In such cells the site istherefore asymmetrical and closer to one pole.The contradiction between this result and thatobtained by radioautography has not yet beenresolved, but in either case it seems that themurein component of the cell wall is synthe-sized at a unique site.

After being synthesized, murein is distributedrapidly over the cell surface (88). This pointsout at least one of the difficulties in the inter-pretation of studies of segregation of parentalwall or membrane material. Thus, the findingthat wall or the membrane subunits are dis-tributed randomly among progeny cells does notpermit differentiating between synthesis atmany sites in each cell or synthesis at one sitefollowed by rapid randomization of the newlymade material.

It is not known with certainty how membranegrowth sites originate. One study with mutantsof B. subtilis indicates that new sites mayoriginate de novo and are not formed by theduplication and segregation of preexisting sites(98). The converse mechanism has been pro-posed for E. coli (30).

Sensitivity of Cell Division to InterferingConditions

An unusual feature of cell division in bacteriais that it can be selectively inhibited by a greatvariety of chemical or physical agents. Manysubstances which stop growth at high concen-trations will, at lower concentrations, not in-hibit growth, but will stop cells from dividing(61, 89, 120). The result is that rod-shapedbacteria continue to grow and form nonseptatefilaments. The list of agents capable of this isremarkably varied. It includes antibiotics, dyes,detergents, disinfectants, antimetabolites (e.g.,114), high and low temperature (133), ultravio-let light (75), the presence of ind--X prophage(142), deprivation of certain essential nutrients,and excesses of other nutrients (7, 147). Al-though some of these substances or physicalagents selectively inhibit the synthesis of con-stituents of cell envelopes (e.g., penicillin),others are not known to do so.Few cases have been studied in detail, and it

is not possible to make definite statementsconcerning the reasons for this unusual sensitiv-

ity of bacterial division. However, some tenta-tive generalizations concerning conditionswhich favor either filament formation or divi-sion can be made. Generally, filament forma-tion is: (i) caused by a low dose of an agentwhich inhibits growth; (ii) enhanced by fastgrowth in rich media (e.g., 2, 40, 59, 144, 149,152); (iii) reversed by suddenly slowing growth(chloramphenicol treatment; 17, 43, 152), nutri-tional shift down (144), or "liquid holding" (73,74). In fact, it seems possible that any chemicalat some concentration, whether attainable inthe laboratory or not, could cause filamentformation. (When this was told to Rollin Hotch-kiss, he muttered: "How depressing!")Agents that restore the ability of filaments to

divide make up another varied list. Thus, fila-ments of various species which result from theexpression of various mutations or nutritionalconditions divide after the addition of sodiumchloride (43, 66, 93), pantoyl lactone (3, 44),spermine (46), dimethyl sulfoxide (63), lysoleci-thin (63), sodium oleate (63), or short-chainalcohols (63). Cell division also requires mag-nesium in Aerobacter aerogenes (78) andClostridium (146) and calcium in Lactobacillusbifidus (83). A division-promoting, lipase-sensi-tive particulate fraction has been isolated fromE. coli (3, 4). Small-molecular-weight sub-stances isolated from the blue-green alga Ag-menellum quadriplicatum stimulate the divi-sion of filamentous mutants (63). Althoughsome of these agents can be expected to act onthe cell membrane, others influence a largevariety of cell functions. Some recent findingsemphasize the need for caution in interpretingthe reversion of division mutants by nonspecificagents such as salt. Thus, 80% of randomlycollcted temperature-sensitive mutants of E.coli can be reversed at the restrictive tempera-ture by raising the osmotic pressure of themedium (12, 119a, 125). Salt dependence of adivision mutant of B. subtilis was traced to itseffect on enzymes of glutamine metabolism. Infact, the osmotic protection of temperature-sen-sitive mutants may be explained by a phasechange of membrane constituents at 40 C (72,95). At this temperature wild-type cells may bein a precarious position which can be upset byany of a number of mutations and affect themost sensitive event in the cell cycle-cell divi-sion.GENETIC STUDIES OF CELL DIVISION

General CommentsBefore discussing genetic studies on cell divi-

sion, we must introduce several notes of cau-

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tion. In order to make such studies meaningful,three types of operational criteria should bemet. (i) It is necessary to determine whethercomplex phenotypes are due to pleiotropic ef-fects of a single mutation or to multiple muta-tions. (ii) It must be determined whether twomutants with the same apparent phenotypehave a lesion in the same cistron. This can bedefinitely established only by complementationtests. Unless this test is carried out, it is notpossible to decide whether in closely linkedmutants allele- or cistron-specific differencesaccount for different phenotypes. Alleles ofgenes regulating division or DNA synthesis mayelicit different phenotypes. Mutations affecting,say, allosteric properties, might result in pheno-types different from those resulting from muta-tions in the same gene affecting catalytic sites.Complementation tests have not been reportedextensively with bacterial division mutants,and the interpretation of work done with themis limited. (iii) It is necessary to determine thespecificity of the mutation for cell division. Aspointed out by Mendelson and Cole(96 (97), allconditional lethal mutants are, in some man-

ner, division mutants. In order to study celldivision, one must choose from a wide spectrumof conditional lethals those believed to be in-volved specifically in cell division. Generally,mutants that under some conditions continue togrow without dividing are considered to bedivision mutants. The fact that division ispreferentially inhibited by a great many dispa-rate agents indicates the need for caution.Many mutations controlling functions relatedto growth may appear to be "division mutants"because they are just "leaky" enough to allowexponential growth without division. Manysuch filament formers may have a primarygenetic defect that is reflected in many meta-bolic processes other than those directly relatedto the division process. It is not obvious thatmuch will be learned from their study before thecomplexities of their causal relationships are

known.Mutations relevant to studies on cell division

may affect (i) functions in the division processitself (i.e., septum synthesis), (ii) functionspreceding the division process, but specificallyrelated to the preparation or triggering of theinitiation of division, or (iii) functions related tothe separation of sister cells. Besides the criteriadiscussed above, there are other difficulties inclassifying division mutants into the first two ofthese groups. At first glance, conditional divi-sion mutants may be grouped (in a fashionanalogous to that used with DNA mutants) into

those which cannot initiate division (initiationtype) and those defective in the process ofseptum formation (elongation type). Mutants ofthe first type should continue to divide if theyinitiated the process before being placed at therestrictive condition, whereas mutants of thesecond type should stop division immediately.Elongation-type mutants should be frozen atwhatever stage in division they were at the timeof the conditional shift. Mutants that stopdivision immediately have, in fact, been found.Unfortunately, they have not been shown topossess incomplete cross walls (6, 24, 101, 115,117). One must conclude that either the time forseptum synthesis is very short or that incipientsepta are resorbed (possibly by hydrolases con-centrated near their site of synthesis) or de-stroyed during cytological fixation (139).To predict the quantitative behavior of "ini-

tiation-type" mutants one must know the timerequired for division. The extent of residualdivision at the nonpermissive condition de-pends on the time in the cell cycle whenformation of the septum was initiated. Becausethis is not known with precision, it is notpossible to define initiation-type mutants onthe basis that they carry out some residualdivision (8, 15, 97). In the absence of any otheruseful criteria, we may say with assurance thatno satisfactory assay for initiation of bacterialdivision exists as yet.The difficulties of this field are compounded

by the fact that there is no known biochemicaldifference between the envelope componentsinvolved in cell division and those of the rest ofthe cell. This makes it very difficult to describevarious division steps at the biochemical leveland to assign mutants to these steps. We canexpect progress in this area because differencesin structure have been reported between thewall at the ends and at the sides of B. subtilis(37). However, these differences may arisesubsequent to completion of the septum (35a).

In 1968, Hirota et al. (58) summarized workdone with a wide spectrum of temperature-sen-sitive mutants of E. coli. Many of the mutantsshowed alterations in the normal relationshipsbetween events in cell division, thus permittingdissection of this process. At that time, thevarious components of the cell division cyclecould not be arranged in an ordered sequence.In the years that have followed, little progresshas been made in formulating a causal order forthese components. In 1973 the temperature-sen-sitive filament formers were genetically classi-fied in seven groups, ftsA through G, and physi-ologically separated according to reversibility

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and the effect of chloramphenicol on reversibil-ity (119a). Again, no ordering was attempted.Accordingly, in this article we will not attemptto do so, but will present separate data on therelationship between various physiological proc-esses and cell division. We thus manifest theopinion that the formulation of causal connec-tions in bacterial cell division seems premature.

DNA MetabolismIn this section we will present evidence that:

(i) the timing and the relationships within theDNA-division cycle are complex and subject tochange; (ii) some alternative mechanism isrequired for cells to divide in the absence ofDNA synthesis; (iii) the control of cell divisionis affected by several facets ofDNA metabolism(synthesis, repair, and recombination) ratherthan just DNA synthesis; the DNA repaircapacities of the cell act as negative controls ondivision; (iv) DNA synthesis is related to prop-erties of the cell envelope; the factors linkingDNA synthesis and the envelope are possiblyalso involved in linking DNA synthesis to celldivision; and (v) different mutations in thesame "gene" affecting DNA synthesis give riseto different phenotypes.Point (i): the timing and the relationships

within the DNA-division cycle are complexand subject to change. The normal temporalorder and how it can be changed. On a grosslevel, the sequence of events in the DNA divi-sion cycle has been worked out for E. coli. Overa wide range of growth rates at 37 C, a round ofchromosome replication begins 60 min before adivision and ends 20 min before that division(48). Division is correlated with the attainmentof a cellular "unit of mass" (28). The orderlysequence of morphological changes, division,and segregation of nuclei in living cells has beenstudied by several authors, notably Adler andHardigree (3). There is considerable variation inthe timing of those processes in individual cells(129). In rapidly growing E. coli, division of thenuclear bodies takes place, on the average,about halfway through the cell cycle, before anysigns of wall constriction (5, 129).The timing of these events can be altered. For

instance, lowering the temperature increasesthe time required for a round of DNA replica-tion (C period), but does leave the 20-min lagbetween termination of the round of replicationand completion of division (D period) relativelyunaffected (91). The D period in E. coli p245apparently is shorter than in E. coli B/r. Thelength of the C period in E. coli 15T - can bevaried by changing the thymine concentration

(151). Heat shocks synchronize division, but notDNA synthesis, thus changing the D period(135). Division takes place 5 to 10 min aftertermination of rounds of replication (short D)when E. coli is subjected to alternate blocks ofribonucleic acid (RNA) and DNA synthesis,followed by reversal of the DNA block. The Cperiod is not changed (71).

Increasing the generation time beyond 60 minalters both C and D, but, according to Pierucci(110), does not change the relationship C = 2D.On the other hand, according to Kubitchek andFreedman, the age at initiation of a round ofreplication varies with the growth rate, but Cand D do not (85). This contradiction has notbeen fully resolved, but may be attributable todifferences between strains (C.- Helmstetter,personal communication).

In conclusion, the time between initiation ofrounds of replication can be changed withoutaffecting C, D, or the mass per new growingpoint, the C period may be changed withoutaffecting D, the D period may be altered with-out affecting C, and both may be altered.Normal relationships and how these can be

changed. In E. coli, cell division does not takeplace in the absence of DNA synthesis. Addinginhibitors of DNA synthesis (25) or transferringmost conditional mutants defective in DNAsynthesis (dnai) to restrictive conditions (58)leads to filament formation. Although severalmodels for this connection will be discussedbelow, it is not known if the signal or signals actpositively or negatively, or if the biochemicalsteps involved are closely or distantly related.Under special circumstances, this normal con-nection is changed and cells can divide withoutDNA synthesis. To date this bypass has beenobserved only with specific mutations, and nonutritional conditions or chemicals or physicalagents have been found which allow wild-typecells to divide without synthesizing DNA. In B.subtilis the situation is different, because wild-type cells can divide in the absence of DNAreplication (31).

In E. coli, temperature-sensitive mutations inDNA synthesis have been mapped in sevenchromosomal positions (designated A throughG). The dna- A and C mutants are defective ininitiation of DNA replication, whereas the oth-ers are involved in the elongation of the mole-cule (e.g., 55). E. coli T46 (a dna- A mutation)and S. typhimurium 11G (probably dna- C) areexamples of DNA initiation mutants that havebeen studied intensively in connection with celldivision. The presence of a mutation, divA,allows E. coli T46 (54) to divide at the restric-

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tive condition, whereas S. typhimurium l1Gdivides without any known "div" mutations(138). E. coli T43 (56) and ts27 (42, 64, 66) aredna-B mutants, defective in chain elongation.DivB allows E. coli T43 to divide, whereas E.coli T27 divides in the absence of known "div"mutations. These examples show that the re-quirement of DNA synthesis for division ofgram-negative rods is not absolute and thatnearly all possible combinations have beenfound: usually division stops if DNA synthesisis stopped; sometimes division continues inmutants defective in initiation or continuationof DNA synthesis; some dna - mutants defectivein initiation or continuation of DNA synthesisrequire additional "div" mutations, whereas inothers such a requirement is not evident.Point (ii): some alternative mechanism

seems necessary for division to occur in theabsence of DNA synthesis. The bypass mu-tants just described do not divide if DNA syn-thesis is blocked by means other than the ef-fect of the mutation. In all cases tested. (E. coliT46-divA [54], S. typhimurium 11G [131,136-138], and E. coli ts27 [64, 66]), wheninhibitors of DNA synthesis were added at thepermissive temperature, the cells did not di-vide. Similarly, when DNA synthesis in dna-mutants is blocked by thymine starvation, cellsdo not divide (66). These results suggest that inthese mutants the relationship between DNAsynthesis and cell division has been altered andoperates by a different mechanism than innormal cells. We use the term "mechanism" inthe broadest sense, because we do not know if itreflects new biochemical steps or new insensi-tivity to the consequences of inhibiting DNAsynthesis.

Further evidence for an alternate mechanismis obtained from studies on conditional DNAinitiation mutants. Cell division in these mu-tants has a lag of about 1 h when cells are placedunder the restrictive condition. This lag isrelated only to the time spent at the restrictivecondition and is not related to the terminationof rounds of replication or events triggeredthereby (56, 132).Thus, division in the absence ofDNA synthe-

sis requires the specific expression of certainmutations. How this is done is as yet unknown.Point (iii): DNA metabolism, not just syn-

thesis, affects the control of cell division.There is evidence that the DNA repair mech-anisms are involved in the regulation of celldivision. This is based on studies of a certainclass of mutants such as E. coli tif, which donot divide at 42 C (20, 21, 80). At the restrictive

temperature these cells do not show defects inDNA or DNA synthesis, but have increasedrepair capacities. Suppressors of tif- are defec-tive in repair abilities. The tif gene may be aregulator for the repair systems, because at therestrictive temperature tif causes an increasein these systems, even when DNA synthesis isnot inhibited (20, 21). This suggests that whenDNA synthesis is inhibited in the wild type,lesions are produced in the DNA which "in-duce" an increased level of repair. This, in turn,stops cell division.A similar case was uncovered when studying

revertants of an E. coli DNA repair mutant,lex -. A suppressor of this mutation, tsl, leads toinhibition of cell division (99). This was alsointerpreted to mean that the tsl mutationcauses a gratuitous increase in repair enzymesynthesis which inhibits cell division. Thesemay be examples of two separate mechanismssince the tif- and tsl- mutations map at differ-ent sites, and the tsl- mutant does not exhibitthe same responses to added purines as tif- (80,81, 124). In this connection we may mention aclassical, but poorly understood, observationthat after conjugation the recipient cells show alag in division. Perhaps the DNA fragmentintroduced conjugally induces repair systemswhich inhibit division.RecA - mutants of E. coli continue to divide

when DNA synthesis is inhibited chemically orby the expression of conditional dna - muta-tions. Here the normal relationship betweenDNA synthesis and division appears to dependon the recA gene product, which mediates arecombination function (65). RecA - also sup-presses tif (99) and lon- (41) mutants. Thelon - mutant, which will be discussed in detailbelow, is not altered either in DNA synthesis orin the repair functions tested. This mutant isdefective in the recovery of division from tran-sient unbalanced growth during which theDNA-mass ratio is low. (It also appears to bedefective in the response to other regulatorysystems such as the gal operon control and Xrepressor). Because tif- is involved in the cou-pling of DNA synthesis to division, the suppres-sion of tif- mutants by recA- also implicatesthe recA gene product in the link betweensynthesis and division.Point (iv): the role of DNA synthesis in

division and properties of the cell envelope.To begin with, several envelope properties havebeen found to be altered by inhibiting DNAsynthesis. Hirota et al. (57) and Inouye andPardee (68) have reported that when DNAsynthesis is inhibited, certain membrane pep-

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28SLATER AND SCHAECHTER

tides are missing when analyzed by acrylamidegel electrophoresis. Conditional DNA mutantsas well as wild-type cells exposed to chemicalinhibitors of DNA synthesis have increasedsensitivity to sodium desoxycholate at hightemperatures, suggesting increased permeabil-ity of the outer membrane (57, 137). A variety ofdna - conditional mutants also exhibit general-ized changes in permeability (49, 57). The shapedetermination of cells is also affected by DNAsynthesis, because spherical cells of mutants ofE. coli (103) and B. subtilis (118) elongate whenDNA synthesis is inhibited. This is also true forwild-type Streptococcus faecium (52). Thesefindings are consistent with the proposal thatduring inhibition of DNA synthesis the synthe-sis of peripheral wall is favored over centripetalgrowth of the wall or thickening of the wall (52).Another possible connection between DNA me-

tabolism and properties of the envelopes can bederived from studies on the partial inhibition ofDNA synthesis. In E. coli decreasing the rate ofDNA chain elongation by lowering the thymineconcentration results in change in volume dueto changes in cell width rather than in celllength (151).Because the envelope is the agent of division

it would not be surprising to find that therelationship of DNA synthesis to the envelope ismediated by the same factors mediating thelink of DNA synthesis to division. There are, infact, indications that this is the case. The lon-mutant in E. coli cannot recover the ability todivide after transient, unbalanced growth whichlowers the ratio of DNA to mass. It is notdefective in DNA synthesis itself, but is be-lieved to be altered in the production of a factor

linking DNA synthesis to division (69, 75, 87,145, 152). It is not yet known whether thisalteration is due to a decrease in a divisionactivator, or an increase in an inhibitor, or a

change in a receptor site for an activator or

inhibitor specified by other genes. There are

some indications that favor the notion that themutant is defective in a receptor site and thatthis receptor site is located on the envelope (69).Lon mutants are unusually sensitive to peni-cillin and other wall synthesis inhibitors (87),they have increased permeability (38), and theyexhibit other envelope-related abnormalities(see Table 1). As yet, no defects in structure or

synthesis of the wall were found in exhaustivestudies. The Ion mutation is suppressed by thelex (33) (called exrA in E. coli B) and sul (32)mutations, which are themselves involved inthe control of envelope properties (69).Point (v): different mutations in the same

"gene" may result in different phenotypes.In most E. coli dna- A mutants, a shift to therestrictive condition allows rounds of replica-tion to terminate and cells to divide until eachcompleted genome segregates into a daughtercell. However, in one mutant in this gene,CRT83, division stops immediately whilerounds of replication continue until termination(79). If the dnaA gene product affected onlyinitiation of rounds of replication, it would bedifficult to explain why its inactivation stopsdivision immediately. It is possible, then, thatthe gene product also changes the activity ofother proteins involved in control of division.Increasing the temperature of CRT83 wouldthen inactivate its ability to initiate a round ofreplication and its ability to affect division-

TABLE 1. Envelope-related characteristics of Ion- mutant'

Osmotic Wall suscep- Accumulation Division KnownOsmotic tibility to of uridine recovery in gross

Mutant Penicillin pressure- sodium do- diphosphate Division presence of chemicalswelling decyl sulfate muramyl chlor- wall

plus trypsin pentapeptide amphenicol defects

Wild type, control Normal Yes Normal No N/A N/A NoIon-, control High No _ No N/A N/A NoPenicillin-induced

filamentsWild type N/A - _ No Normal Yes NoIon N/A - __ Normal No No

UV-induced lon-filaments - __ -_ No

Growth in FIMWild type - _ Normal _ _ - NoIon- High Yes No

a N/A, not applicable;-, not done; FIM, filament-inducing medium in Ion- cells; does not induce filamentsin the wild type.

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related protein. When CRT83 is returned topermissive conditions, division resumes after 10min. The recovery becomes insensitive to chlor-amphenicol after 5 min, but remains sensitiveto reshift to 42 C for the full 10 min. A similarsituation in the temperature-sensitive divisionmutant E. coli ts2O (to be discussed later) wasinterpreted as suggestive of complex proteininteraction.

Difference in alleles of dnaA mutants havebeen found with respect to the time at whichdivision stops after shift to restrictive condi-tions, recovery of division in the presence ofchloramphenicol at the time of reshift to per-missive conditions, reinitiation of rounds ofreplication in the presence of chloramphenicol,the number of growing points introduced uponreversal of inhibition, susceptibility to salt sup-pression, and response to integrative suppres-sion (42, 56).Much caution must be exerted in interpreting

the existence of different phenotypes in variousdnaA (or other dna-) mutants, because it hasnot always been proven that they are mutantsin a single locus. Nitrosoguanidine, the com-monly used mutagen, induces multiple closelyspaced mutations, which may lead to misinter-pretations of apparent pleiotropic effects (e.g.,11).

Protein SynthesisAlthough protein synthesis is, of course, re-

quired for the continued progress through thecell cycle which leads to division, division cantake place in its absence (15, 71, 96, 101, 111,141). In fact, in some cases division may bestimulated by blocking protein synthesis (43,152).

Pierucci and Helmstetter (111) found thatthere is a transition (execution) point in the cellcycle after which division can occur in theabsence of protein synthesis. They found that 40min of protein synthesis (normally coincidentwith period C) is required for division and thatcells in the D period can divide in the absence ofprotein synthesis. The required 40-min period ofprotein synthesis can be dissociated from thenormally concurrent DNA synthesis. It is notknown if the equal length of both periods ismore than a coincidence. Protein or RNA syn-thesis appears to be needed for some finalprocessing step of the chromosome which isrequired for cell division (23). Jones and Dona-chie (71) recently suggested that special pro-teins may be involved in connecting DNAsynthesis to cell division. They found that thesynthesis of certain proteins cannot be dis-

sociated from DNA synthesis. These appear tobe triggered by termination of rounds of replica-tion and have been termed "termination pro-teins." Their results also suggest that some ofthe events in the normal D period are triggeredby the 40-min period of protein synthesis. Theseevents require neither previous termination ofrounds of replication nor concurrent proteinsynthesis. Jones and Donachie proposed amodel in which DNA synthesis and the synthe-sis of "division proteins" occur in parallel path-ways which are normally concurrent. When theround of replication terminates, "terminationprotein" synthesis is triggered. Normally at thistime enough "division proteins" have beenmade to trigger a series of events needed fordivision which do not require concurrent pro-tein, RNA, or DNA synthesis. At a late stage ofthis series of events the preformed "terminationproteins" are utilized and division follows.Marunouchi and Messer (91a) reported that

a short terminal segment of the chromosome isnot replicated during amino acid starvation.(This segment is replicated when a conditionaldna A- mutant is placed under restrictiveconditions.) When amino acids are added, cellsdivide provided the terminal segment is allowedto replicate. This result does not disprove theconclusion reached by Jones and Donachie (71),that termination is required for the synthesisof proteins needed for division, but it does con-tradict some of their data. Both groups in-hibited protein synthesis and then inhibitedDNA synthesis after relieving the inhibition ofprotein synthesis. Marunouchi and Messer(91a) found no cell division, which is the basisof their report, whereas Jones and Donachie(71) found that such cells divided. However,these differences may be attributed to the factthat Jones and Donachie (71) inhibited DNAsynthesis by thymine starvation and Maru-nouchi and Messer (91a) by adding nalidixicacid. Dix and Helmstetter (27) reported that,in analogous experiments, the two treatmentsdiffer in that thymine starvation, but not nali-dixic acid treatment, allows cells to divideafter relieving the inhibition of protein synthesis.The matter, therefore, awaits further clarifica-tion.Evidence that protein synthesis is required

for the initiation, but not for the continuation ofthe division process, has been obtained fromstudies with mutants of B. subtilis 35ts (97) andtms 12 (15), which are thought to be defective inthe initiation of septum formation. If proteinsynthesis is inhibited at the time of reshift topermissive condition, recovery of division is

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blocked. If a short time elapses before proteinsynthesis is ihibited, division occurs some timelater. Apparently a brief interval of proteinsynthesis is required to initiate division butonce it is initiated, it continues in the absence offurther protein synthesis. Similar observationshave been made with temperature-induced fila-ments of E. coli ts2O (101), where they havebeen interpreted to be due to interactions be-tween protein subunits, and with penicillin-induced filaments of E. coli B (Ion-) (141),where they are thought to be due to alterationsin properties of the envelopes.

"Division Potential"We will now consider a speculative notion

regarding the activity of proteins directly in-volved in division. Let us imagine that some ofthese proteins are under a special regulatorycontrol, that they are concentrated at or nearthe site of division, and that conditions maydictate their utilization later than in the normalcycle. We will call these concentrations ofrelevant substances the "division potential."This is an extension of the use of that term asproposed by Reeve and Clark (115, 116). It isstimulated in good part by their studies on thekinetics of division of a temperature-sensitivefilament former, E. coli BUG 6, by the proposalsfor the involvement of the membrane in initia-tion of cell division by Ingram and Fisher (62,63) and by the pioneering studies of Adler et al.(2).The case for "division potential." There are

indications that some membrane and periplas-mic proteins are specifically related to celldivision. Examples of these indications are asfollows: it was found by Shen and Boos (134)that E. coli BUG 6 is deficient in some periplas-mic proteins and that there is a correlationbetween cell division and the synthesis of sev-eral of these proteins. Inhibition of cell divisionhas been correlated with changes in membraneproteins (46, 57, 68, 140) and membrane-boundenzymes (107). The appearance of several pro-teins in the membrane is more resistant todifferent antibiotics than protein synthesis ingeneral (53). In addition, several membraneproteins are related to DNA metabolism (57,68).When filaments are placed under permissive

conditions, division is sometimes very rapid,suggesting that "division potential" may haveaccumulated while division was inhibited (29,87, 116).The case for "division potential" is obviously

weak, and we believe that it rests more on itsplausibility than on actual data.

What "division potential" may explain.The concentration mechanism involved inbuilding up "division potential" may help ex-plain two puzzling aspects of cell division: (i)the general sensitivity of division to differentstresses, and (ii) the fact that slow growth tendsto favor cell division.The concentration mechanism of "division

potential" may be a cellular feature unique todivision and may be sufficiently delicate to bethe target of many unrelated stresses. Anyalteration of the organization of the cytoplasmor the cell membrane may affect it, lowering itsconcentration at its site of action and prevent-ing cell division.The formation of filaments occurs more read-

ily in cells growing rapidly than in culturesgrowing at slow rates. Filaments of severalmutants divide when shifted to media support-ing slow growth rates. It has been proposed thatfilament formation in these mutants is due tolowering of production of division promotingsubstances (2). Rapid growth, by greater pro-duction of cell mass, may lower the chances ofachieving their required concentration at thedivision site. Slowing growth by treatmentsaffecting the synthesis of cytoplasmic proteinsselectively may increase the chance of achievingthe proper local concentration of division-related proteins.

In certain permeability mutants, substancescomprising "division potential" may be lost byleakage through membranes. An example ofthis may be filament-forming mutants of theblue-green alga Agmenellum quadruplicatumwhich leak a division-related effector (61, 62).Erwinia sp. filaments produced by ultravioletlight or addition of fl-alanine and D-serine leakproteins into the medium (45). These proteinsoriginate from the periplasm and perhaps thecell wall, but not from the cytoplasm. Thefilaments also have decreased amounts of mu-rein. Adding pantoyl lactone or Carbowax pre-vents leakage and inhibition of cell division, butdoes not affect the decrease in the amount ofmurein. These results suggest that division isprevented by leakage of proteins from the pe-ripheral area of the cell. This phenomenonwould explain the observation that cell divisionis impaired in some E. coli mutants whichrequire high sucrose concentrations for survival(90).

Placement of the Site of Division

(i) Loss of control. A mechanism whichplaces sites for division at regular cell lengthintervals continues to operate whether or not

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divisions actually occur. In several cases (e.g.,E. coli BUG 6 [116] and S. typhimurium DivA[24]) shifting filaments to permissive conditionsleads to rapid expression of all of the divisionsmissed at the restrictive condition. Normal-sized cells are eventually produced. As men-tioned earlier, addition of low concentrations ofpenicillin prevents division and causes forma-tion of bulges at potential division sites. Theseresults indicate that during filament formation,the ability to measure normal cell lengths forplacement of the division site is not lost. Thisability can be abolished or altered by othermutations or treatments. Thus, penicillin-induced bulge formation can be prevented with-out affecting general sensitivity to penicillin(130) when DNA synthesis is inhibited bynalidixic acid or when an E. coli dna - B mutantis placed at the nonpermissive temperature(130).

In E. coli BUG 6 it was shown that placementof the site of division in filaments depends oncompletion of synthesis or segregation of chro-mosomes (115, 117). Cells produced in theabsence of DNA synthesis in the dna - B.subtilis mutant ts151 (95) and the E. coli dnaBmutant ts27 (64) are not of uniform size. In B.subtilis div 355ts (96), nuclear bodies are irregu-larly spaced in filaments, and irregularly sizedcells are produced when they are transferred topermissive conditions. These results suggestthat placement of the division site and thecompletion or segregation of chromosomes arerelated. However, as we have discussed above,this relationship can be bypassed by specificmutations because uniformly sized cells areproduced by E. coli mutants defective in chro-mosome segregation or in the initiation orcontinuation of DNA synthesis, provided thatsecondary "div" mutations are present (54, 56).In S. typhimurium 11G, where no div mutationsare found (138), uniformly sized cells as well asuniformly spaced penicillin-induced bulges(131) are produced in the absence of initiation ofDNA synthesis. Nutritional shifts-up at therestrictive temperature cause increases in therate of division, but do not change the size of thecells (131, 132). Apparently, the uniform place-ment of the division site is intact, but themechanism which alters the cell size in responseto nutritional conditions is not. It is not clear ifbulge formation is related to DNA initiation. InS. typhimurium, bulge formation occurs in theDNA initiation mutant 11G (131), which di-vides at the nonpermissive temperature, but notin the E. coli DNA elongation mutant, CR34-43(130), which does not. Bulge formation has notbeen tested in the dnaA or C (initiation) mu-

tants which do not divide, or in the dnaB(elongation) mutants, which do divide.

In B. subtilis the loss of control of properplacement of the septum occurs with or withoutalterations in the structure of the septa. B.subtilis divB mutants do not form proper septaand have irregularly spaced membranous pro-trusions (143). In B. subtilis div 355ts and divA,septa are misplaced, but their structure appearsto be normal in ultrathin sections (97, 119, 143).Thus, the control of septum placement can belost without disturbing the division processitself.The results discussed above indicate that: (i)

the proper placement of potential division sitescan occur under some conditions where divisionis prevented, (ii) other division-inhibiting con-ditions abolish the control of placement, (iii)abolishing the placement control does not ne-cessarily alter the proper orientation or struc-ture of the misplaced septa, (iv) normally,inhibition of proper chromosome synthesisand/or segregation inhibits proper placement,but this dependency can be overcome, and (v)the determination of cell size by the divisionprocess can be dissociated from the mechanismthat alters that size in response to nutritionalconditions.

(ii) Alteration of control. We have con-cerned ourselves with the production of cells ofvariable length, indicating that they have lostthe control in the placement of division site. Inother cases, cells of abnormal but uniform sizeare produced, indicating that this control is notdestroyed but is altered.

Cells of E. coli P678-54 exhibit both normal(median) and "minicell"-producing (terminal)divisions at an average ratio of 1: 2 (1, 5).Extensive studies with this strain suggest afairly explicit model, that a division site close toa pole becomes available, in addition to thenormal median site. Either site can be activatedby the cell's normal signal to initiate division.The choices are random, but mutually exclu-sive. Once it is triggered, the division process ateither site is normal. Because minicells lackchromosomal DNA, in this mutant the normalposition of DNA relative to the division site isnot required for the synthesis of a properlyoriented and structured septum. Thus far, thesurface features of the minicell are indistin-guishable from those of normal cells (2). This isan important fact because minicells, by theirgeometry, contain a high percentage of septummaterial.The production of minicells in this strain

involves mutations in at least two genes. Theirrole is not yet known, but this situation may be

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compared to that seen in other cases such asdna- div (58) or dna- recA- mutants (65),where one mutation blocks DNA synthesis andthe other uncouples division from its normalrelationship with DNA synthesis. In the mini-cell former, one mutation may increase thefrequency at which division sites are made andthe other may destroy a mechanism whichnormally ensures that accidently (or prema-turely) made extra sites are never used in onecell.The phenotype of B. subtilis div IV-B1 (119)

is intermediate between the two types of sep-tum-placement mutants discussed so far. Thedivision site is neither completely randomizednor is it as strictly controlled as in the E. coliminicell producer. The spectrum of cell sizes inthis B. subtilis mutant ranges from tiny spheri-cal minicells to short rods to normal-sizedcells. Some anucleate short rods divide, aunique finding which suggests either that apotential division site may occasionally be trap-ped in a short rod or that a new site is made bythe anucleate cell. In this mutant, short rodsand minicells are produced throughout thegrowth cycle, but their frequency increasesduring late log to stationary phase. Thus, theirproduction is dependent on the physiologicalstate of the cells (119). Stationary phase cul-tures of wild-type B. subtilis often contain verysmall "mini"-type cells (102). The mutant maybe defective in the mechanism that prevents theabnormal divisions, which occasionally occurwhen nutritional conditions become subopti-mal. Recent results (S. L. Coyne and N. H.Mendelson, personal communication) indicatethat minicell divisions occur one-third of thetime, instead of one-half of the time as in E.coli. The probability of a minicell division isgreater at the site of the oldest cell pole. (Thephenotype of this mutant may be due to anabnormal activation of genes for sporulation,which also takes place at the pole of the cell.) Inaddition, not all minicell divisions are normallyoriented. For those reasons, the E. coli and B.subtilis minicell producers may be fundamen-tally different.

In an earlier section it was stated that nearlyall possible combinations for the relationshipbetween DNA synthesis and division have beenfound. This also appears to be true for divisionand the placement of the division site. Certaintreatments which pevent division do not affectthe uniform placement of potential divisionsites, whereas others do. In some cases themechanism is destroyed, whereas in others theplacement is changed from one controlled stateto another. The same type of statements can be

made with respect to placement of the divisionsite and DNA synthesis or segregation. In somecases, but not others, interference with DNAsynthesis or segregation affects proper divisionsite placement. The proper placement of divi-sion sites has also been dissociated from themechanism which relates cell size to the growthrate.

In all of the cases discussed in this section,the misplaced septa are properly oriented andstructured. The site, but not the process, ofdivision is affected in all of the mutants dis-cussed so far. We turn now to cases where bothsite and process are affected and find that this isassociated with generally defective wall synthe-sis.

Cell Wall

Logically, there must be a direct connectionbetween cell division and the proper synthesisand functioning of the cell envelopes. We willnot discuss here the mechanisms of wall synthe-sis, the association of envelope components witheach other, or the structural role of the compo-nents in wall or cellular structure. We will dealhere only with the dependence of division on theelastic properties of the wall and on its shape-determining properties.

Elastic properties of the wall. It has beenproposed that division is initiated by the invagi-nation of the cell membrane, as a result ofchanging the direction of the membrane synthe-sis from the peripheral direction to a bidirec-tional (peripheral and septal) direction. Suchan invagination could result from a transientincrease in membrane synthesis relative to wallsynthesis and would depend on the properrigidity of the peripheral wall (10, 52). Suchdependence may be inferred from studies on therequirements for protoplast reversion (86, 127),on the effect of sudden changes in osmoticconditions on swelling and division (90), and onthe effects of inhibiting murein synthesis ondivision (130). It is also indicated by studieswith specific mutants. Four out of five E. colimutants selected for dependence on high os-motic conditions (sud mutants) grow as fila-ments in the absence of high sucrose, whereasthe fifth mutant lyses (90). Addition of wallconstituent D-alanine allows one of the fila-ments chosen for study (sud 25) to divide inlow-sucrose medium (90.)We discussed elsewhere the possibility that

the E. coli Ion- might be defective in the controlof wall synthesis and that the resulting alteredenvelope might decrease the affinity of anenvelope receptor for a signal linking DNA

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synthesis to division. E. coli B (lon-) is amongthe mutants which fail to swell upon suddenshift in osmotic properties (148), indicating analteration in the elastic properties of the wall.The studies with lon- and sud- mutants suggestthat those wall characteristics which promoteproper elasticity (and rigidity) are required forthe initiation of division.Determination of cell shape and its re-

lation to division. It may be expected thatdistortions in cell shape may affect the properorientation or formation of the cell septum andthus affect cell division. Studies with variousmutants of B. subtilis and E. coli have shownthat different changes in cell shape affect celldivision to different degrees.This type of relationship between the struc-

tural properties of the wall and cell division maybe particularly fruitful because something isknown about the chemical determinants of theshape of bacilli. Thus, in E. coli it has been longheld that the shape is determined primarily bythe murein sacculus (e.g., 113 and 120). Now, itappears that the outer membrane may also playa role in this process because "ghosts" lackingmurein, but containing outer membrane, main-tain the cell shape (50). A particularly propi-tious case for study may be Bacillus brevis,whose wall contains a protein whose purifiedsubunits, it is claimed, reassociate to formcylinders with the diameter of the normal cells(16, 19, 51).

Certain mutants of B. subtilis, called rod-,can change reversibly from a rod to a sphere.The spheres show morphological defects in thestructure of the wall and especially of the crosswalls which appear "chewed," misplaced, andmisoriented. There is defective splitting of thesepta, resulting in the formation of clumps ofcells. Correcting the defect in wall synthesis byplacing the cells at the permissive conditionsusually restores proper cross wall formation (13,14, 26, 36, 76, 77, 118, 120, 121, 122). However,during recovery some of the rod forms havenormal-looking peripheral walls, but "moth-eaten" cross walls (122). Thus, defective wallsynthesis can cause obstructions in formation ofsepta. Correcting the defect usually, but notalways, restores septa to normal. The converseof this is also true, when the rod- mutant isplaced in a different genetic background. Herethe ultrastructure and shape of the wall remaindefective, but properly placed and orientedsepta are made (Fig. 5). Studies with severalgram-negative mutants have shown that cellswith altered cell shapes can divide (50, 92, 103)(Fig. 6).

In none of the cases discussed here is the

biochemical site of the lesion known with preci-sion. However, important hints on the processesinvolved have been obtained from studies with amutant of Bacillus licheniformis (39). Thismutant provides a poignant example of theability of cells to bypass normal causal connec-tions. When cells are grown in low-phosphatemedia, teichoic acid in their walls is replaced byteichuronic acid. When teichuronic acid synthe-sis is blocked by a mutational change in theenzyme phosphoglucomutase, cells exhibit aspherical phenotype. The requirement for phos-phoglucomutase is lost when glycerol and galac-tose are added to the medium. Under theseconditions, lytic activity increases and the rod-shaped morphology is maintained (39). Notonly does this study exemplify the possiblealternate means and bypasses which bacteriause but, in an operational sense, it illustratesthe difficulties in assigning primary regulatoryroles to biochemical effectors.

In conclusion, it is evident that septum syn-thesis depends on definable properties of thecell wall. Of these, the proper determination ofcell shape is not absolutely required and can bebypassed in certain mutants.

Small-Molecule MetabolismWe will list here a variety of somewhat

unrelated observations which indicate that in-terference with cell division can be traced to themetabolism of some small molecules. In keepingwith previous discussions, it is not possible toinfer the biochemical relevance of these connec-tions.Amino acids and amines. Filaments induced

by high temperature in E. coli ts 52 (152) or byD-serine in Erwinia (43) divide when treatedwith chloramphenicol. The proposed interpreta-tion of this finding is that when protein synthe-sis is blocked, small molecules accumulatewhich, conceivably, stimulate division (152).Because rapid growth increases the tendency toform filaments, it may decrease the level ofaccumulation of these molecules.

In some species, the proper balance of putres-cine to spermidine is necessary for division (67).A mutant of E. coli (MA-135), conditionallydeficient in putrescine synthesis, forms fila-ments in rich medium, but not in minimalmedium. When putrescine levels are decreasedfurther, growth stops (149). The level of polya-mines depends on amino acid metabolism. Itseems possible, therefore, that a block in pro-tein synthesis makes amino acids increasinglyavailable for polyamine synthesis. This, in turn,may stimulate division and account for thephenotype of E. coli ts 52.

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v,.,y:> K. ., A,>t

00- "nt,1f,,-0-,,

. J.S-

.,J l

I.'4 ]:.s s

a

OL

FIG. 5 Grampositive cells: altered shape with normal division in B. subtilis mutant tag-1. Thistemperature-sensitive mutant was grown for 3 h at 45 C. OL indicates the dense and particulate outer layer ofwall. Note the lack of inner layer and periplasm. The distance between arrowheads indicates the width of themembrane. Magnifications: high power, x49,000; low power, insert, x10,500. Both bars designate 1gm;reproduced from reference 118 by permission of N. H. Mendelson and the publisher, Springer- Verlag,Berlin-Heidelberg-New York.

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When a S. typhimurium mutant constitutivein the histidine operon is placed in a mediumcontaining a high concentration of glucose, itforms filaments (100). Another S. typhimuriummutant, divA (ts), forms filaments at the re-strictive temperature in glucose, but not insuccinate-containing medium (24). Cyclic aden-osine 5'-monophosphate causes division ofthose filaments. Azide stimulates division in E.coli ts 52, but azide and iodoacetamide togetherblock it (152).Purines and pyrimidines. E. coli tif- forms

filaments at 40 C. Division takes place in thepresence of added guanine, cytidine, hedacine(an inhibitor of adenylic acid synthesis), andpantoyl lactone. The protection is reversed byadenine. Mutant and wild-type cells exhibit thesame changes in nucleotide pools at restrictiveand permissive temperature, but the mutantmay respond differently to these changes. Thekey small molecule effector appears to be asubstituted furan moiety (80, 81, 124).

CONCLUSIONSWe conclude that bacterial cell division is a

complex series of events which can be measuredin different ways, and that its causal dependen-cies are diverse, interrelated, and variable.Division may be measured by the functionalseparation of sister cytoplasms, by the comple-tion of the septum, or by separation of the sistercells. There are indications that sister cyto-plasms become functionally separated only atthe time of completion of the septum. Little isknown about the timing of septum formationduring the cell cycle. Cell separation in B.subtilis apparently requires the same time,regardless of the rate of growth. Studies withmutants of gram-negative bacteria indicatethat the cell separation step is under geneticcontrol and related to the relative timing of thesynthesis of various envelope components of the"septum."There is reason to believe that gram-negative

rods, like gram-positives, divide by formation ofa morphologically distinct septum and not, ascommonly held, by gradual constriction of thecell envelopes. The details of the mode ofdivision and its timing must be known for theproper definition of mutant phenotypes.

Specificity of division controls. In manybacteria, division is extremely sensitive to awide range of growth inhibitors and gene muta-tions. Low doses of inhibitors, insufficient toslow growth, will preferentially stop division.This makes it difficult to define the degree ofspecificity with which physiological manipula-

FIG. 6. Gram-negative cells: altered shape withnormal division. Electron micrograph of thin sectionof cells of the rodA mutant of E. coli grown in L-brothat 37 C. Marker bar represents 0.2 Am; reproducedfrom reference 92 by permission of H. Matsuzawa andthe publisher, The American Societ~y for Microbiol-ogy.

tions or gene mutations are involved in theregulation of division.When conditional mutants are used to probe

the causal connections of a cell cycle, it isusually assumed that the target of a restrictivecondition is the sensitized product of a mutatedgene. In several cases, such as E. coli Ion-, B.lichenformis mutants, and probably E. coli tif-,this is not so. Thus, while the target for ultravio-let light in E. coli ion- is DNA, the mutant isnot defective in DNA synthesis or its regulation,but in the response of the cell to unbalanceddecreases in DNA synthesis.

Diversity of division controls. Division de-pends on many cellular activities, among whichwe can list the synthesis of macromolecules, themetabolism of small molecules, and certainproperties of the cell envelope. More specifi-cally, division is related to several aspects ofDNA metabolism: replication, repair, and per-haps recombination functions, and the levels ofprecursor pools. Division also requires the syn-thesis of proteins made at or near the comple-tion of rounds of DNA replication, as well asproteins normally made during the round ofreplication but dissociable from it. Division ap-

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parently depends on properties of the cell en-velopes that contribute to the rigidity of thewall and probably to the determination of theproper shape of the cell. Although murein playsan important role in determining and maintain-ing the shape of the cell, recent evidence in-dicates that special envelope proteins may alsobe involved in this process. Despite uncer-tainties, the biochemistry of envelopes andchromosomes is more thoroughly understoodthan the myriad of other factors upon which di-vision depends. It must be emphasized that atpresent there is no biochemical means to assaythe initiation of division or to study septumsynthesis separately from the synthesis of pe-ripheral wall or membrane.Interrelationships of division controls.

Factors related to division are related to eachother. Thus, shape determination, permeabili-ty, and envelope composition are related toDNA metabolism-possibly by the same effec-tors that link DNA synthesis to division. In nocase is there a molecular explanation for theserelationships.

Variability of division controls. While someof the basic assumptions in the use of inhibitorsor mutants may be too simplistic for the com-plexity of the system being investigated, wemay conclude that there is not a unique andobligatory set of causal connections in the cellcycle. When normal relationships are inter-rupted they may be bypassed or they may bemodified to produce artifacts.Bypasses and artifacts have complicated

studies of viral systems where the genes and thegene products can be defined. In bacterialdivision where such knowledge is not availableand where a black box approach must be used,the possibility of such bypasses and artifactsrenders the study even more intractable. Thereare several indications that normal causal rela-tionships in cell division can indeed be by-passed. Thus, although division normally de-pends on DNA synthesis, this requirement islost in some mutants. Sometimes the newpathway may require additional mutations forits expression. Division also appears to requirenormal cell shape and structure of the wall, butcertain mutants with altered shape and/or wallstructure can divide. The main event may bethat, at restrictive conditions, there are pos-sibilities for artifacts, and that what is observedunder such conditions is not relevant to the"normal" cycle.We would like to end by stating that in cell

division, as in other cellular phenomena, whatcan be learned from conditional mutants isrestricted to what can be learned about their

phenotype. Unfortunately, present knowledgeof the morphological and biochemical eventsleading to cell division does not permit thedetailed characterization even of the mutantsthat are available. A recent method for orderingthe sequence of complex functions by using analternation of different restrictive treatmentshas recently been developed for the study of thebacteriophage P22 (70). It is being used in theanalysis of division mutants of yeast and maywell be useful in studies with bacteria. The useof such genetic tools, along with further bio-chemical studies of cell functions likely to berelated to division, remains still the most hope-ful of the available approaches.The complexity in regulation of cell division

in bacteria may be attributed in part to theirrelative structural simplicity. Bacteria have notevolved the complex machinery that ensuresequipartition of the genome in higher cells. Itseems likely that they utilize structures andregulatory mechanisms that may be used alsofor other cell functions. Therefore, it is notsurprising that there is not a unique cell cycle,but rather that there is a "normal" sequence ofevents with alternate sequences, should thenormal one be interrupted. Speaking of celldivision in higher cells, Mazia (94) stated in1960 that "complex" does not necessarily mean"hard to understand." It should mean, simply,"composed of many parts and of definite rela-tionships between the parts." In 1974, thisstatement is no less true, but, in the case ofbacterial cell division, its optimism may betempered by recognizing that the structuralsimplicity of bacteria is deceptive.

ACKNOWLEDGMENTSWe are grateful to B. Beck, D. Boyd, W. D.

Donachie, E. A. Grula, R. James, 0. Maal0e, M. H.Malamy, L. A. McNicol, N. H. Mendelson, S. Nor-mark, J. T. Park, H. J. Rogers, R. Rowbury, B. M.Shapiro, G. Shockman, S. Torti, and A. Wright fortheir helpful comments. They are not responsible forwhat we say because we did not always take theiradvice, a fact we may live to regret.

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Control of Cell Division Bacteria · directly involved in cell division). Thepresence orabsenceofa morphologically distinct septum may be a real difference between species and mayreflect