Toporegulation of bacterial division according to the nucleoid occlusion model

Res. Microhiol.

INSTITUTPASrEUR/ELSEVIEIt Paris 1991

[991, 142, 309-320

Toporegulation of bacterial division according Io the nucleoid occlusion model C . L . W o l d r i n g h ('), E. M u l d e r , P . G , H u l s and N. Vischer

Department o f Molecular Cell Biology, Section of Molecular Cytology, Plantage Mnidergracht 14, 1018 TV Amsterdam

SUMMARY

A model for the toporegulation of division in Escherichia coli is presented in which call constriction is initiated by the combined action of a biochemical and a structural event. It is proposed that the biochemical event of termination of DNA replication causes a transient change in the pool of deoxyrlbonucleofides, which serves as a localized trigger that is converted to a diffusible, cytoplasmic activator of peptidoglycen synthesis. The second event involves the segregation of the nucleoids. Evidence is presented that the nucleoid suppresses the activity of pepti. dogtycan synthesis in its vicinity, it is proposed that active transcription/translation around the nucleolds produCes a strong but short-range inhibitor which prohibits division (nucleoid occlusion). The combined effects of the locally produced termination-activator :~ndof the diminished occlusion as a result of nucleold segregation, guarantee that division Is normally placed between the separated nucleoids. The model can explain the pattern of division-recovery of filaments, the maiortty of which constrict at sites which produce polar daughter ceils containing two nun|acids. In addition, the model offers an explanation for the occurrence of minicells under a variety of conditions. Key-words : Nucleoid occlusion, Toporegulation, Cell division; fts mutant, Filaments, Minicells, E. coil, Model.

INTRODUCTION In spite of extensive analyses o f wild type a n d m u t a n t Escherichia coli cells, the m e c h a n i s m of c o o r d i n a t i o n between D N A replication and cell division has remained elusive. One of the reasons may be that the mechanism is complicated because it has not only to regulate the timing but also the

(=) Correspondingauthor.

positioning of the division event. In this respect, the mechanism can be expected to be universal, because the same timing and p o s i t i o n i n g is required in eukaryotie cells. It should be emphasized, however, that e~'en when comparing E. coli with a lower eukaryote like the yeast Schizosaccharomyces pombe, the division processes are executed

on a completely different scale. Considering the size of the slructures involved (see fig. 1), the constriction site in E. coil should rather be compared with the yeast spindle pole body than with its septation machinery. Because of this size difference, the hypothetical signalling system in E. coli c o u l d occur directly between the metabolic or conformational state of the DNA

310 lind its recciviug envelope. Ill the ellkat)olic cell, such a gigltal hits I0 be tralisdaced Io inlcrlltedial¢ Mrgctures, poMponcd for mitosis and als~ amplified. These hitter i~ioce~L-~, have but'l) nnravclled wi0| much success ill tile past years (Hartwcll and Welnert, 1989). Ilowever, the primary signal, which ilt eakaryotes regnlilies Ibe accnnlnlation Of, for instance, the MPF-aclivating cyclin (Murray and Kirschner, 1989), has remained as elusive as in the bacterial eel1 cycle.

Reversed signal Iransduetion In bacteria, extensive signaltransfer reactions occur when cells adapt to a change in their physical or chemical environmeal, Such environmenlal signals arc sensed by membrane proteins (kinases) and through the transfer of phosphorylgroups, transmitted to cytoplasmic response-proteins, which may cause a change ill tile expression of target genes ISIoek et a(., 1989). Tile response, however, can also be direct, i.e. nol dependent on gene expression, as in the case of chemotaxis {fig. 2A). Conversely, a discontinuity or subtle change in the "imernal environment" of tile growing cell (see D'Ari and Bouloc, 1990), simply caused by its enlargemeat, may trigger an inverse signal transduction pathway from the cyloplasm or nucleoplasm towards the cell envelope (fig. 2B). In the nucleoid occlusion model (Mulder and Woldringh, 19891; Woldringh er al., 1990), it is proposed that the abrupt stop of DNA synthesizing replication forks at termination and the subsequent separauon of the daughter nucleoids are internal signals to which membrane proteins respond by The synthesis of a division site. The biochemical DAP = diaminopclic add

f'.l.. H ' O I DR/NGH ET /IL. Schlzosaecharom¥ce$

pombe

Escherichia colJ

Fig, I, Comparison of the sizes in their living {hydrated) state of two rod-shaped cells. In Ihe Iowe~ eukaryote, Schizosaecharomyces pombe, the duplicated spindle pole body embedded in the nuclear envelope is indicated by the t'~o black dots. In the dividing E. coil cell, the annular constriction site base been thickened to emphasize that its size is similar to that of the spindle pole body.

A. ADAPTATION

B. CELL CYCLE

Environmental change

Response

1

t

Response

Protoplasmic growth

F-

Fig. 2. Flow of information in adaptation (A) and in the cell cycle (B). In adaptation (A), the flow of information from the external evironment to an internal response can occur either directly, as in the case of ehemotaxis, or indirectly via gene expression, as in most other cases (osmoregulation, nitrogen and phosphate regulation). In the cell cycle (B), a reversed flow of information from the metabolic and structural state of the DNA to the plasma membrane is assumed, analogous Io the direct signal transduetion in chemo~axis. I

IPTG = isopropyl lhiogalactosidc.

NUCLEOID

OCCLUSION

discontinuity in DNA svnthesis may repres&l a signals mainly involved in the timing of the division event. The spatial discontinuity caused by nucleoid separation may repreSent a positional rignal. In the steady state of growth. the immediate wcce?.sion of termination and DNA segregation, characteristic for the bacterial cell cycle, will ~uarantee that division takes place between the separated nucleoids. When, under unbalanced growth conditions. the two signals cannot mmbine their action and strengthen each other, aberrant divisions will occur (see below).

AND

OF CELL

D,WW,N

A nucleoid

B

2 0

0

e

a= .c

(Z)

tndirect evidence that transcriptionltranslation activity suppresses pepddoglycan symhesis comes From the following observations. 1) In growing cells, it wiii never occur that a constriction is initiated at the site of

NORMALIZED

occlusion

termination

(

The principle assumption in the nucleoid occlusion model is that the metabolic and confoymational state of the DNA informs the cell surface how to grow and where to divide. This requires a continuous Flow of information from the nucleaid towards the peptidoglycan synthesizing enzymes in the plasma membrane. The model p~oposcs that the information is derived from the transcription/rranslation activity occurring in the vicinity of the nucleoid and that this is sensed as an inhibitinn but short-range effect on pephdbglycan synthesis. In figure 3. the wcureace of this hypothetical inhibitor has been Schematically depicted together with the termination signal (see below). It should be noted here that the abovementioned spatial signal caused segregation nucleoid by represents, in our model, a decrease in the short-range occlusion effect.

311

Steadystate:

Y The oeelusion mle of the nnckoid

TOPOREGULATlON

CELL LENGTH

C.L, tVOLDRINGH E T A L.

312 the nllC[eoid. NO aclivcly growing i n u l a a l s have yet been described which show such all aberration, not even the one isolated by Hiraga (1990). 2) A c t i v a t i o n o f t r a n s c r i p tion/Iranslation as taking place after a nutri|ional pulse (Kepes and D'Ari, 1987) results in u postponement of division and. after a shifiup, in a transient drop in tile percentage of constricling ceils (from 12 to 6 % during 40 rain afler a shift of E. coli Br; Woldringh and Zarilsky, unpublished observations). 3) Slowing down or inhibition of transcription/translation allows for Ih¢ progression of initiated constrictions resulting in residual division. In addition, we can now observe cells in the population with a constriction at the site of the nnclcoid. Such continuous nucleoids in constricting cells have been observed in slow growing B/r cells (e.g. fig. 4 in Woldrlngh e: al,," 1977), in cells treated with chloramphenicol (unpnhlished observations) or starved for amino acids (Grossman el aL, 1982) and in cells stored at lOW temperature (Van Bagel et a/., submitted). These observations could be taken as evidence against nucleoid occlusion. In o u r i n t e r p r e t a t i o n , however, we assume that in all these cases, DNA synthetis ran to c o m p l e t i o n and that the hypothetical termination signal was sufficient to initiate a constriction in i'~s vicinity because of the diminished nncieoid occlusion effect (¢f. fig. 4A). More direct evidence for an inhibitory effect of the transcription/translation activity around the nucleoid on peptldogLycan synthesis was obtained from the a u t o r a d i o g r a p h i c analysis o f d i a m i n o p i m ¢ l i c acid ( D A P ) incorporation along the cell's length axis, Previously, we have shown (c.f. fig, 4 in Woldringh et al., 1987) that in E. c o l i f t s Z filaments containing well-segregated nucleoids, DAP incorporation is evenly distributed over the length

A. Storvotion .....

*d

3 .

~.,L+ - - - ,

(

Y)

B.SOS in SfiA"

v%

w

Z LLI Z

o

C,)

C. Recovery from SOS

0.5

NORMALIZED CELL LENGTH

1.0

NUCLEOID OCCLUSION AND TOPOREGULATION OF CELL DIVISION induced by some artefact or by the mere lack of cytoplasm which reduces the synthetic capacity: the experiment was repeated with leucine. In contrast to DAP, leucine incorporation showed a peak value around the central nucleoid. These results suggest that, in growing filaments, high protein synthesis activity at the site of the nucleoid coincides with a low activity in peptidoglycan synthesis.

of the filaments. To oblain cells with a clear separation between the nucleoid-containing and nueleoid-free parts, E. t'nli dnaX(tsllysA was allowed to grow into short filaments with a single, centrally located nucleoid. The results, plotted in figure 5, show a lower incorporation of DAP at the site of the nucleoid relative to the nucleoid-free call ends. To see whether the central dip in grain number is not

I. Positioning of constriction sites in relation to segregated nucleoids in DAPl-stained f t s Z filaments recovering from division inhibition at 42 °C for two mass doublings r*~.

Table

Call number scored

Number of nucleoidsper filamentl")

Percentage of cells showing a constriction at positions indicated by Lhe arrows

(~

o~.o

I

t

o

oQ

t

t 21

170

8

8

71

]

44

S

3O

60

10

44

4

7

9S

Qo 3

Constrictions and nucleoids were visualized by ph~e contrast and fluorescence m~eroscopy. C')Data are compiled from three samples taken at 30, 45 and 60 rain after downshift to 28 oC ISee also table ll). ~") The ten constricting filaments wilh 7 and 5 nucleoids all pinched off a polar Lo cell.

Fig. 4. Schematic representmions, as in figure 3, of cells under the condition of starvation (A), of SOS response in a SfiA background (B), and of division recovery after SOS inhibition (CI. In IA) transcripton/translation activity and thus the nucleoid occlusion effect (dashed line) is low ; therefore, the termination activator is sufficient by itself to induce a constriction in its vicinity, without the nucleoids having to segregate apart. In (B), the few continuing constrictions which occur at 42 °C in dnaA or dnaX sJ't/i filaments are positioned scmewhere in the DNA free cell ends. Although it is generally accepted that the resulting DNA-less ceils are of "normal size" (Jaffd et al., 1988), their length distribution is at least twice that of newborn cells. In (C), restoration of DNA synthesis first results in large masses of unsegregated nucleoids. The first terminations induce a high activator concentration leading to constrictions close to the nucleoids.

313

The termination signal Just as a subtle change in ext'~'n~l phosphate concentration or osmolarity revcesents a signal in the process of adaptation (cfl~ fig. 2)~ a subtle change in the pool of deoxyribonueteotides may represent a stimulus for the process of division. Such a change in pool size could result from the abrupt stop of dNTP incorporation at termination. At present, it can only be speculated as to how a transient increase in the dNTP pool could be converted into a diffusible cytoplasmic signal that ultimately regulates the activity of membrane-bound enzymes. Donachie and colleagues have previously suggested (Teather et aL, 1974) the idea of a diffusible factor which was produced in a limited amount, sufficient for one division event. These " q u a n t a " of division capacity could be used up at any of the potential division sites in a cell or filament. It was later suggested that this division factor might be the FtsZ protein (Donachie and Begg, 1990). Characterization of genes near the E. coil terminus (terC; Cr.=':sman et aL, 1989) led to the suggestion that other gene products may also play the role of termination protein. In our proposal, a diffusable effector originates in the nucleoid, is relatively stable and acts evenly at any position in cell or filament. The proposal was based on observations of recovering SOS filaments (Matdec and Woldringh, 1989): In these filaments, constrictions are initiated only when the first reinitiated or restarted rounds of replication can be expected to have terminated. In addition, those constdctioos are positioned close to the still unsegregated nucleoids, resulting in the pinching off of relatively large DNAless cells (fig. 4C). This contrasts the more random positioning of

C.L. IVOLDRINGH E T AL.

3t4 the relatively few constrictions ill I:. cob dnaX, 4fiA or dtlaA46 ¢11iltalltS at 42 °C (fig. 411l,

l)ivisinn recovery in filaments of ft.~Z, pbpB and ftsA mu,*ants A striking p h e n o m e n o n which the iluclcoid ocelnsion model or any o!her model should explain is the pattern of division recovery in E. coil filaments. It has been shown that pbpB filaments conltlining segregated nucleoids recover division by pincbittg off relatively long cells (¢:f. fig. 5 in Taschner et a!., 1988) The length distribution iv figure 6 obtained from electrt3u microscope measarements also ~hows lha= in f t s Z filaments recovering from a shift to 42 °C, the majorit.v of cells constrict at sites corresponding In twice the length of newA~orn cells (2 x L0). htlere~;tingly, lhe coeffb.icut t~( variation o f this di;tribution (26°/0) is large compared to that of newborn ceils ~about 10%), Thai these relatively Ion~ celt~ are also 2×Ltrce![s with resp~l to the DNA ~ep~ication cycle is sustained by the resuhs in table 1, which show ~hat 71% of constrictions in cells containing 8 segregated nucleoids is initiated al a position be~.ween the second and third nucleoid from the pole. As a result, many daughter cells should contain 6 nucleoids. This is confirmed in table 1!, which gives an ir=dlcation of the number of fully segregated nucleoids occurring in frsZ filaments at dilferent t rues "after the downshift to 28 °C. The division and segregation pat~err~ shogun in table II have not been interpreted further. The analysis is complicated by the fact that filaments not oniy divide up, but also continue to grow, as well as to segregate nucleoids. It can be seen, however, tha~ at 45 and 60 min, 30~0 of the filaments contain 6 nucleoids. Similar results were obtained after dowashift of ft~:4

ftsA filaments, they also found a higher percentage of division at positions near the poles. Tile data in table 11 also show that abo¢.r 4% of the f:~Z filaments c o n t a i n 3, 5 a n d 7

apd pbpB filaments, although in those strains the analysis is complicated because of the occurrence of double constrictions in about half of the filaments. In some pbpB cells, 3 to 5 constrictions per filament were found. In the ftsZ strain, doubleconstriction filaments were only rarely observed. Although our observations do not agree with the recovery pattern observed by Tormo and Vicente (1984) in

nueleoids, These can be ascribed to irregularities in nueleoid segregation. In m a n y of the 2XLo-cells the two nucleoids were not fully segregated as in the filament indicated by the arrow in figure 7. Surprisingly,

2o-'1 A O..-

-~.

-20

0

201B -20

. . . . . . 20

"%,..

40

60

80

100

120

100

120

n o r m a l i z e d cell l e n g t h (%)

0

20 40 60 80 n o r m a l i z e d cell l e n g t h (%)

20

-.~ 0

.

-20

0

.

.

.

20

.

.

.

40

.

.

60

.

.

-:

80

100

_

.

120

n o r m a l i z e d cell l e n g t h (%) Fig. $. Silver grain distributions over whole ftsZ (A) and dnaX (B) filaments, growing at 42 °C, puke labelled with tritiated DAP and prepared by agar filtration (for technique, see Woldringh el aL, t987). DnaX filaments were also pulse labelled with tfitiated leucine (C) before preparation for autoradioglaphy.

NUCLEOID OCCLUSION AND TOPOREOULATION OF CELL DIVISION these irregularities were more manifest in pbpB and still more in f t s A cells. For instance, in 37% of 75 pbpB filaments, tl~e 2 x Lc-cell which was being pinched off contained an elongated but not fully segregated nucleoid. We have not been able to detect any system in the occurrence of segregation defects.

irregularities in the displacement and separation of nueleoids, one cannot escape the impression that it is these seemingly random irregularities in segregation (decatenatinn ?) which ultimately determlpe the site o,~ division and which can violate any rule ,Jaff6

Based on the above results (table I and II; figs 6 and 7), our contention is that constrictions are effectuated at a position of 2 × L 0 from the pole because of an additive effect of the terminations at the two a d j a c e n t nueleoids. According to the nuclcoid occiusion model, the inhibition is minimal at this site o f the previous termination, where segregation has been fully accomplished, In addition, it is assumed that the level of unused activator from the previous termination at this site can still be relatively high. These combined effects induce a constriction at 2 x L 0, before a new site is c r e a t e d between the lastterminated nucleoids at 1 × L0. As a result, cells are produced containing 2 and 6 segregated nueleoids, as found in table I. These ideas have been schematically depicted in figure 8A and B. From the observation of m a n y filaments with subtle

315

e! aL, 1990) on the pattern of division recovery. Mioisell fo,m=tion Already in 1974, Donachie and coworkers proposed a model

~0

10

~0]

~ °7,,,7;,='°'L',27~,

~-~o

s ~

~

Lj

Z~LU~FL~

Lo 2Lo

~

CELL LENGTH(pml

Fig. 6. Length distribution of E. coli tysA ftsZ, after cell division inhibition at 42 °C for two mass doub;ings and downsbfft Io 23 °C for 60 min, to allow division recovery. Hatched areas represent constricting ceils. For a better estimation of the length of newborn cells, a separate distribution was measured (N 462) of small cells and of prospective cells being pinched off at the poles of filaments. The average length of this population is 3.4 g~m. The average length of newborn ceils (Lo) in the control population at 28 °C was 1.9 #m (cf. fig. 5 in Tasehner et aL. 1988).

Table 1I. Distribution of nucleoids in E. coF #isZ filaments (see table l) recovering from division ;nhibitioo at 42 ~C for differca! periods. Times after downshift 42° ~ 2 8 *C (min)

Cell number scored

0 30 45 60 90

81 142 184 130 182

1

--3 0 18

Percentage of filaments (constricli~ag and non-const ricting) ~il.h the number of segreg~.~e~5 ~ucie~id~ per filament indicated 2 3 4 5 6 7 8 5 2 20 29 46

--4 4 3

54 44 16 9 10

i 3 4

3 29 31 9

-1 4 1

41 50 24 ~0 5

9

10/16

--1 2 i

-1 1 5 2

“,,,a

JllM

;,>ym

sp!oa(s””

$ Pzm?2,“03 S,,JJ JO (U/,9,, laqul”” me, e ‘“o!r!ppe u, ‘pJn,azqo 2,11.* sp!oa,J”” 1155,X!S q&M auou pw Il,ti!J ,,,!a SIUJL” -e,FJ Ma, e Qla ‘,“e,“e glr!w ix,, “, ‘E”!BIIE S”!J”po,d-,,JJ!“!m ,“aKlJJ!p “! “o!,“‘,!l,c!p p!lm,,“” JO S!Sl,m”! at,, moqs

B,!ru!S m ‘,I,

r JO Y,,“IJ, .l,qs, u,

NUCLEOID OCCLUSION AND TOPOREGUI,A TION OF CELL DIV,'SIOIV

first polar constrictions were initiated after 15 rain of induction. Both after 20 and 60 min, the average miniceU-forming cell was smaller (Lm=2.6,um) than the average centrally-constricting cell (Ld=3.0/~m). Interestingly, 90% of these minicell-forming cells contained an unsegregated nu¢leoid. The average cell in the population segregates its nucleoids at a length of 2.3 ~m (calculated from the percentage o f ceils with two separated n u c l e o i d s a n d a s s u m i n g the population to be in a steady state o f growth)• It can thus be expccied that most polar constrictions have been induced in cells in which termination of D N A replication has taken place but in which, for some unknown reason, segregation has been retarded. Autoradiographie experiments will be performed o n cells pulse-labelled with tritiated thymidine, to verify this expectation. In terms o f the nucleoid occlusion model, minicellformation can be explained by assuming the termination activator to be overproduced or to be m o r stable. Consequently, it reaches a higher concentration at the cell poles, where it can now induce a c o n s t r i c t i o n before nucleoid segregation (cf. fig. 8C). This premature division results in decreased cell volume and this, in turn, could hamper further nucleoid segregation. Segregation defects are readily observed in filaments of minicellforming strains (Jaff~ et al., 1988; Mulder et al., 1990).

Predetermined division sites or coupling ? in the nucleoid occlusion model, we have not adopted the idea that c h r o m o s o m e s are attached to periseptal annuli (Rothfield et aL, 1990) or to any other membrane site, in the sense that such attachments function

317

A. Division inhibition at 42"C ,. _ ~. -p i~- ....

,

, / - " A " - "

....

s

~,1,~ ....

/ - ""~"~,~-

//,/- v~///~,, I."/~"~"~--

r

J

unused

"~'

,y

nctivator

',

~o B. Recoveryafter downshift

•

t,,'.-7~_.',..7-,~..

'-v,~.

,~-,j

I:_

UJ O

C. Minicell torrnotion

0

0.5

1~)

NORMALIZED CELL LENGTH

Fig. 8. Schematic representation of the nueleoid occlusion model applied to division inhibition in fts strains filamenting at 42 °C IA), to filamems recovering division after downshift to 30 ~C (B) and to a miniceU-forming strain (C). The situations i , B and C are similar in the sense that the termination activator reaches a relatively high concentration outside of the terminating nucleoid, before segregatioa has taken place. During the time a triggered division is effectuated, segregation continues, producing at division one cell with two nucleoids and either one with six nucleoids (table I and Ill or a minicell.

318

C.L. IVOLDRINGH

ET AL.

Table I I I , Distribution o f nueleoids in d i f f e r e n t m i n i c e l l - f o r m t n g strains n) as d e t e r m i n e d by fluorescence m i c r o s c o p y o f D A P I - s t a i n e d cells (cf. fig. 7).

Slrain

Cell n u m b e r scored

m # l B O'

318 415 289 443

m i n B I~]

P B 1 1 4 q~) M i n e + (~)

I

2

33 46 12 42

34 31 54 47

P e r c e n t a g e cells o r filaments w i t h t h e n u m b e r o f nueleoids indicated 3 4 5 6 7 8 9 16 8

7 II

2 1

3 2

-~

5 1

1

22

--

3

--

5

4

7

10

12/16

'"In m)ne of Ihese populalions could anueleat© rods be observed (of. Jaff~ el aL, 19881. :'t.MC IDII Crown in glucose minimal medium until sleady slale. ,~,t.MC 1011 grown in glycerol milllli~al medium ulll[l steady slate, '4q)11114 A min/hDB m i n C + D + (De Boer et aL. 19891, grown in glucose minimal medium until steady stale. (~LMUSO0/pDBISC ~,rown in glycerol MOPS-meditlm + 0,5 mM IPTG for 60 mln.

T a b l e iV. C e n t r a l a n d p o l a r c o n s t r i c t i o n s in d i f f e r e n t m i n i c e l l - p r o d u c i n g strains as d e l e r m i n e d by electron m i c r o s c o p y o f a g a r filtered cells.

Strain

G C 7 1 0 6 m i n B ~(31 GCT115 m i n B O) LMC5fJO m i n B ~141 L M C 5 0 0 m i n B t~) L M C S 0 0 m i n B +(~) L M C 5 0 0 m i n B IB L M C 5 O O / p D B 156(6) idem + 60 rain I P T G 161 L M C 5 0 0 / p D B 156 {71 i d e m + 6 0 m i n I P T G I~)

Doubling time , i m e (rain)

A v e r a g e cell l e n g t h It) ~ m )

52 56/48 56 54

1,9 5.2/7.4 2.2 5.0

26 11/13 28 21

76 84 52 52 80 80

2.1 4.7 1.7 2.5 2.3 3.2

18 16 25 21 30 34

central (z) ('70)

Constrictions p o l a r ~21 (o70)

total (~o)

2"5

26 38/41 28 46

-45 -23 6 29

18 61 25 44 36 63

27"}'28

'L)Delermnied from length distributions,not including minice]Is. 1~'Includinghalf of lh£ 7~r~taCe of double-~on~tlictinccalls. (')T~o independent exper~mems in glucose minimal medium. See for the strains: Jaffg et aL, 1988. L')LMCS00 i~ E. c~li MC4100 lysA; the minB mutation was introduced by PI (GC7115) transduclion. Glucose minimal medium. ~Glyc~ol miv.it~ medium. L¢'Glucose min[mai medium; pDBI56 is pMLUlll5 P~;:rainB+. See De Boer et aL) i989. IPTG concentration was {).5 raM. '7)Sam~ strata crown in MOPS + glycerol minimal medium; IPTG conccmration was 0.5 raM.

NUCLEOID OCCLUSION AND TOPOREGULATION OF CELL DIVISION

in D N A segregation. Although specific sites of the chromosome may attach to the membrane, we think that the principle drivh,g forge able to move the nueleoid mass lies in the g : o w i n g cytoplasm and not in the synthesis of specific, newly synthesized proteins (Hiraga et aL, 1990), Also, the distribution mechanism, which determines the pattern of segregation of a particular D N A strand with respect to one cell pole, is suggested to be localized in the cytoplasm (see r i b o s o m e - a s s e m b l y - m e d i a l ed model for segregation in Woldringh and Nanninga, 1985). In addition, we assume that the replicating and segregating DNA, through the hypothetical inhibitor and activator, determines, how the envelope is being synthesized in the vicinity of the nueleoids. This does not contradict the occarence of specific membrane compartments as proposed in the periseptal-annulus model o f Rothfield and coworkers (1990). We challenge, however, the existence of an e n v e l o p e - b ased mechanism which can pre-determlne and position division sites independently from the nucleoid, Our m o d e l thus implies a strict coupling between the additive effects of D N A termination and nncleoid segregation with the initiation of cell constriction, as ~;sentially suggested more than twenty years ago. Recemly, a positive coupling between chromosome replication and cell divi'finn has been negated on the basis o f the behaviour of strains in which the origin o f replication has been replaced by a temperature-sensitive but cellcgele-independent mechanism for initiation, derived from plasmid RI (Bernander and Norsdstr6m, 1990). When, in the future, similar systems may be appli=d in studies which also include cytological screening of nucleoid segregation in populations growing under near-steady-state conditions, we should be able to

make a choice between predetermination and coupling or, in other words, between envelopegenerated and nucleoid-directed division sites,

Acknowledgements

The discussions with N. Nanninga and the ohserva0ons and mua~utcm~nls pgrformed by M. El'Bouhali and J.P.L, Cameron are gratenllly acknowledged. We Ihank J. Leul~cher far mak;ng the drawings.

Re|erences

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