Temporal control of the cell cycle in Caulobacter crescentus: Roles of DNA chain elongation and completion

J. Mol. Biol. (1980) 138, 109-128

Temporal Control of the Cell Cycle in C a u l o b a c t e r crescentus: Roles of D N A Chain Elongation and Completion MARY ANN OSLEY AND AUSTIN NEWTON

Department of Biology Princeton University Princeton, N.J. 08544, U.S.A. (Received 29 August 1979) The functional relationships between cell cycle steps in Caulobacter crescentus have been examined by reciprocal shift experiments (Hereford & Hartwell, 1974; Jarvick & Botstein, 1973) using temperature-sensitive mutations (Osley & Newton, 1977) and drugs that reversibly block specific cell cycle steps. Hydroxyurea was used to inhibit DNA chain elongation (DNAe) and penicillin was used to block the initiation of cell division (DIVI; Terrana & Newton 1976}. These experiments show that the cell cycle of Caulobacter can be organized into at least two dependent pathways. One of these pathways mediates the sequence of DNA events, DNAI (initiation of chain elongation) -~ DNA. -> DNAc (completion of chain elongation), and the other pathway mediates the sequence of division events, DIVi-->])IV. (progressive pinching at the division site)--> CS (cell separation). Shift experiments in synchronous cultures showed that the initiation and completion of the cell division pathways require two successive stages of the DNA synthetic pathway: DIV l steps are dependent on DNAe and CS steps are dependent on DNAr These and earlier results suggest that chromosome replication may act as a cell cycle " d o c k " to time events in the cell division pathway and to control the periodic expression of genes required for formation of the flagellum {Osley et at., 1977) and other developmental structures.

1. I n t r o d u c t i o n The p a t t e r n of asymmetric cell divisionin Caulobacter crescentus has made this aquatic bacterium a useful system for the s t u d y of several problems of cell differentiation. I n contrast to most bacteria, this organism divides to yield two morphologically and functionally distinct progeny which follow different patterns of development (for reviews: Poindexter, 1964; Shapiro, 1976). The parental stalked cell cycle, shown below in Figure 1, is composed of a period of D N A synthesis (S period) and a postsynthetic gap period (G2 period) (Deguen & Newton, 1972a). A division site which first defines the new swarmer cell is formed during S (Terrana & Newton, 1975), and subsequently in late S and G2 a single flagellum, pili, and other structures are assembled at the stalk distal pole (Poindexter, 1964; Shapiro & Maizel, 1973; Lagenaur & Agabian, 1977). After asymmetric cell division, the stalked cell repeats this cycle, while the new, motile swarmer cell enters a presynthetic gap period (G1 period) (Degnen & Newton, 1972a) ; during this period, the swarmer cell loses motility and develops into a stalked cell. 109

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9 1980 Academic Press Inc. (London) Ltd.

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M. A. OSLEY AND A. N E W T O N

Previous studies have shown t h a t the cell cycle of Caulobacter is responsible for controlling m a n y of the characteristic developmental events in these cells. The completion of specific steps in DNA synthesis and cell division is required for flagellum formation (Osley & Newton, 1977; Osley et al., 1977), stalk formation (Osley & Newton, 1977) and surface morphogenesis (unpublished observations) at the swarmer pole. (We refer to these changes as "developmental" events, because they characterize the two different cell types and none of them are cell cycle events, i.e. none of the events are required for cell division (0sley & Newton, 1977).) The best studied of these events is flagellum formation, and recent work has shown t h a t the periodic synthesis of at least two flagellar components, flagellin (Osley et al., 1977) and the hook protein (Sheffrey & Newton, unpubhshed results), is achieved b y coupling gene expression to a late stage of chromosome replication. Since steps required for DNA replication and cell division appear to regulate differentiation in Caulobacter, w e have examined how these steps are temporally controlled and organized. Our approach has been similar to the one developed b y Hartwell and co-workers in their analysis of the yeast cell cycle (Hereford & Hartwell, 1974; Hartwell, 1976). Using reciprocal shifts with temperature-sensitive cell cycle m u t a n t s and drugs which block specific cell cycle steps, we have identified two dependent (cf. Mitchison, 1971; Hereford & Hartwell, 1974) pathways t h a t control the cell cycle in Caulobacter. One of these pathways is made up of steps t h a t are required for D N A replication and the other contains steps t h a t are required for cell division. The results also show t h a t progression through the division p a t h w a y is dependent on two successive steps in DNA synthesis, DNA chain elongation and DNA chain completion. These data have been incorporated into a model in which chromosome replication is considered as a cellular clock responsible for initiating the dependent division p a t h w a y and subsequently for completing the cell cycle.

2. Materials and M e t h o d s (a) Strains

All conditional cell cycle mutants are independent isolates that were derived by u.v. mutagenesis from strain PC1, and, with the exception of strains 1053 and 2116, they have been described in detail elsewhere (Osley & Newton, 1977). Strains 1053 and 2116 were identified in the mutant screen described earlier (Osley & Newton, 1977) and they have a Dna + D i v - phenotype. Since eomplementation studies cannot be carried out in C. crescentus at present, some of the mutants with a common phenotype, e.g. Dna~', may be defective in the same gent. All of the strains studied behave as single site mutants, as characterized by reversion frequencies (10 -6 to 10 -7 ) to normal colony formation and cell division at the restrictive (or non-permissive) temperature. (b) Media and cell growth Strains were grown in M3 minimal salts medium (Poindexter, 1964) with 0.2~/o (w/v) glucose and supplemented with eyst6ine (10 ~g]ml) and Difco Casamino acids (10 ~g]ml). The permissive temperature for growth of cell cycle mutants was 30~ and the restrictive temperature was 37~ Synchronous cells were obtained by the plate-release method previously described (Degnen & Newton, 1972a).

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(c) D N A synthesis Synchronous cells were incubated with [8-3H]guanosine (25 ~Ci/ml; Amersham) and carrier guanosine (2 to 5 ~g/ml; Calbioehem), and incorporation of label into base-stable, acid-insoluble material was measured as described (Osley & Newton, 1978). (d) Reciprocal shift experiments The protocol for reciprocal shift experiments was identical to that described by Hereford & Hartwell (1974), and is outlined in Table 1. Two experiments, each consisting of 2 incubations, were performed with exponential populations of the conditional cell cycle m u t a n t s . I n the first experiment, cells were held at 37~ (restrictive temperature) for 1 generation, a length of time sufficient to complete all steps in the cell cycle in an unperturbed culture (first incubation), to uniformly block cells at the temperaturesensitive step. For some m u t a n t s incubation at 37~ was only for 90 m i n (half a generation) to ensure their viability and rapid reversibility. The cells were then shifted back to 30~ (permissive temperature) in the presence either of hydroxyurea (4-5 mg/ml, Sigma) or penicillin-G (300 units/ml, Calbiochem) (second incubation), and the incubation was continued for 6 to 9 h. Somewhat higher concentrations of penicillin and hydroxyurea were used in the second incubation than in the first incubation (see below) to prevent a n y leakage of division resulting from the higher cell concentrations and longer periods of incubation with the drugs. The reciprocal shift was performed in the second experiment. During the first incubation the culture was grown at 30~ in the presence of either hydroxyurea (3 mg/ml) or penicillin (210 units/ml) for 1 cell generation to block all cells at the appropriate drugsensitive step. The drugs were removed either by addition of penicillinase (Calbiochem: 2.5 • 104 units/ml) or filtration through 0-2 ~m Millipore fi]ters and washing, a n d the culture was immediately shifted to 37~ I n all of these experiments the completion of cell cycle steps was assayed by measuring cell n u m b e r on a Coulter Counter model ZB1. Cell division was used as an assay because it is dependent on the completion of each temperature-sensitive (Osley & Newton, 1977) and inhibitor-sensitive step (Degnen & Newton, 1972b; Terrana & Newton, 1976). Since each condition was normally imposed for one cell generation, successful execution of steps resulted in a doubling of cell n u m b e r (Table 1). W h e n the first condition was imposed for only half a generation the age distribution previously calculated for exponentially growing Caulobaeter cells (Osley & Newton, 1978) was used to estimate the n u m b e r of cells that would divide if this incubation had been for one generation. I t was shown in all experiments b y appropriate controls t h a t the temperaturesensitive or drug-sensitive block was reversible. After the first incubation, a sample of cells was shifted from the restrictive to the permissive conditions, and the resumption of cell division was followed by observing cell morphology a n d determining cell number. The failure of some cultures to fully double in n u m b e r (100~o increase; Tables 2 and 3) results from incomplete recovery of these cells from non-permissive conditions. Previous results have sho~m t h a t rates of R N A and protein synthesis are not significantly affected by t r e a t m e n t with hydroxyurea (Degnen & Newton, 1972b), penicillin (Terraria & Newton, 1976) or by 37~ in temperature-sensitive m u t a n t s (Osley & Newton, 1977).

3. Results T h e sequence of six f u n c t i o n a l a n d morphological l a n d m a r k s identified w i t h chromosome replication a n d cell division i n t h e Caulobacter cell cycle is described i n F i g u r e 1. A n u m b e r of t e m p e r a t u r e - s e n s i t i v e m u t a n t s blocked a t specific p o i n t s i n this sequence has been isolated ( 0 s l e y & Newton, 1977) a n d used to define essential cell cycle steps or gene p r o d u c t s which are required for each of the l a n d m a r k s (Fig. 1). T h e properties of these m u t a n t s suggested t h a t t h e t e m p o r a l sequence of four of t h e events, D N A

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FTG. 1. Steps in the DNA-division cycle of G. crescentus blocked by mutations and inhibitors. Stages in the stalked cell cycle that can be identified by characteristic structures or functions are termed events or landmarks (cf. Hartwell, 1974). The timing of landmarks associated with DNA synthesis (Osley & Newton, 1977,1978) and cell division (Osley & Newton, 1977) is shown as a fraction of the stalked cell cycle; in the experiments described here, and as shown in this Figure, the time for the stalked cell to divide in minimal medium (120 min) is defined as 1-0; the swarmer cell is not depicted, but it would require an additional 60 min to develop into stalked cells and to divide. The events are defined as DNAI, initiation of DNA synthesis; DNA~, DNA chain elongation; DNAc, DNA chain completion; DIVI, initiation of cell division by formation of division site (Tcrrana & Newton, 1975); DIVp, progressive pinching at location of division site; and CS, cell separation. The periods of the cell cycle occupied by DNAe (S period) and DIVp are indicated by the horizontal arrows. The steps that mediate the events are indicated under the verticle arrows. These steps are defined by either temperature-sensitive mutations (indicated here and in the text without the alphabetical prefix, cf. Tables 2 and 3) or by the drugs hydroxyurea (HU) and penicillin (PEN). With the exception of the 1040 temperature-sensitive step (see text), the execution point of the cell cycle steps (last time in the cell cycle a gene product is required; Table 2) coincides with the time of the cell cycle landmarks that they mediate. chain i n i t i a t i o n (DNAI) , D N A chain e l o n g a t i o n (DNAe), progression of division (DIVp), a n d cell separation (CS), was controlled b y their o r g a n i z a t i o n i n t o a d e p e n d e n t p a t h w a y , i n which the execution of the two division events r e q u i r e d prior D N A replication (Osley & Newton, 1977). A t h i r d division event, i n i t i a t i o n of division (DIV1) ( T e r r a n a & Newton, 1975,1976; Fig. 1), was n o t e x a m i n e d i n the earlier s t u d y . T h e a i m of the e x p e r i m e n t s reported below was twofold : first, to d e t e r m i n e how cell cycle events i n Caulobacter (see Fig. 1) are organized b y e x a m i n i n g the f u n c t i o n a l d e p e n d e n c e b e t w e e n pairs of steps required for i n d i v i d u a l D N A a n d division events i n reciprocal shift e x p e r i m e n t s (see section (a), below), a n d second, to d i s t i n g u i s h b e t w e e n two different stages of chromosome replication, DNAe a n d DNAo, i n order to d e t e r m i n e t h e r e q u i r e m e n t s of each of these stages for cell division. (a) Experimental rationale (i) Exponential cultures T h e f u n c t i o n a l relationship b e t w e e n two steps i n a n y t e m p o r a l sequence c a n be described as dependent, independen.t, or interdependent (Hereford & Hartwell, 1974;

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Hartwell, 1976; see Table 1). W h i c h of these relationships applies to two steps, including those t h a t appear to occur concurrently, can be determined using reciprocal shift requirements as described b y Hereford & Hartwell (1974) and J a r v i c k & Botstein (1973). These experiments require t h a t the two steps examined be blocked reversibly and in sequence within the same cell, and t h a t a later step which is dependent on each of the two earlier steps be assayed.

TABLE 1

Protocol and expected results of reciprocal shift experiments Experiment A Incubation 1 Incubation 2

37~ 30~

Possible arrangement of steps

(1) Dependent .~x ~ (2) Dependent _~Yx (3) Interdependent x,Y> (4) Independent x

Experiment B

-- drug (X) + drug (Y)

30~ 37~

+ drug (Y) -- drug (X)

Cell division after incubation 2 Expt A Expt B _ + _ +

+ _ _ +

Y .->

All experiments were performed with temperature-sensitive cell cycle mutants as described in Materials and Methods. X refers to a cell cycle step blocked by a temperature-sensitive mutation and Y is a cell cycle step inhibited by penicillin or hydroxyurea. The first incubation was normally for 1 generation (3 h) (see Materials and Methods), and the extent of cell division was measured 6 to 9 h after the shift to the second incubation condition. The 4 possible dependent arrangements have been discussed in detail by Hereford & Hartwell (1974) and Hartwell (1976). (+), Doubling in cell number; (--), no increase in cell number.

I n our experiments, one step was defined b y a temperature(heat)-sensitive cell cycle m u t a t i o n (Osley & Newton, 1977; Fig. 1), and it could be blocked b y shifting cells from 30 to 37~ The other step was blocked b y one of two drugs: DNAe was blocked b y h y d r o x y u r e a (Degnen & Newton, 1972b; Fig. 1), a n d D I V I was inhibited b y a low concentration of penicillin ( T e r r a n a & Newton, 1976; Fig. 1), Since every cell cycle landmark, with the exception of D N A c, is mediated b y at least one temperaturesensitive step (Fig. 1 ; Tables 2 a n d 3), it was possible to examine the functional relationship of each temperature-sensitive cell cycle step to the hydroxyurea-sensitive step (DNAe) in one set of experiments a n d to the penicillin-sensitive step (DIVI) in a second set of experiments. Successful completion of cell cycle steps was monitored b y following cell division as outlined in Materials a n d Methods. The results o f these experiments discriminate a m o n g four possible relationships between a n y pair of temperature-sensitive a n d drug-sensitive cell cycle steps examined (Table 1). F o r convenience in discussing these experiments we h a v e assumed t h a t a different

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TABLE 2

Dependence relationships between temperature-sensitive and hydroxyurea-sensitive steps Strain

Cell cycle event blocked

Execution pointt

PC2076 PC1042 PC2153 PC2179 PC1053 PC1049 PC2244 PC1029 PC2269 PC2116 PC1040

DNAI DNA e DNAe DNAe DIVI DIVp DIVp CS CS DIV,-DIVp 82 CS

0.00 0.75 0"75 0-75 0.50 1"00 1-00 1.00 1.00 1"00 0"00

~oCells dividing after incubation 2 Expt A:~ Expt B w 0 0 0 0 95 100 99 97 100 102 80

90 0 0 0 15 11 12 0 0 0 90

Dependence relationship _ ~ --~u ~, ~u ta, ~u) ~, ~u1, __~u _ ~ HU . . ~ mr ~ ~u _~ HU _ ~ __~u _ ~ HU

(dependent) (interdependent) (interdependent) (interdependent) (dependent) (dependent) (dependent) (dependent) (dependent) (dependent) (independent)

ts

Reciprocal shift experiments were performed with exponential cells of conditional cell cycle mutants using the protocol outlined in Materials and Methods and Table 1. t Execution points were identified as the latest time in the stalked cell cycle at which mutant cells could be shifted to the restrictive temperature and still divide, and they denote the time of synthesis or function of the cell cycle gene product (Hartwell, 1974). :~Results of 37~ to hydroxyurea shift (see Table 1). wResults of hydroxyurea to 37~ shift (see Table 1). 82See Discussion. ts, temperature-sensitive step; HU, hydroxyurea-sensitive step.

cell cycle s t e p is b l o c k e d in each t e m p e r a t u r e - s e n s i t i v e m u t a n t . Since genetic complem e n t a t i o n c a n n o t be done (see M a t e r i a l s a n d Methods), however, some of t h e m u t a n t s w i t h i d e n t i c a l p h e n o t y p e s m a y be b l o c k e d in t h e s a m e cell cycle step. This p o s s i b i l i t y does n o t affect t h e results a n d m o d e l t h a t a r e p r e s e n t e d here a n d in t h e Discussion. (ii)

Synchronous cultures

A v a r i a t i o n of t h e p r o t o c o l d e s c r i b e d a b o v e is t o c a r r y o u t shift e x p e r i m e n t s w i t h s y n c h r o n o u s cells. B y blocking D N A r e p l i c a t i o n a t different t i m e s d u r i n g t h e S p e r i o d as t h e first condition, i t is possible, for e x a m p l e , to d i s t i n g u i s h D N A e f r o m DNAo a n d , m o r e generally, t o d e t e r m i n e t h e r e q u i r e m e n t o f a n y p h a s e o f D N A r e p l i c a t i o n for a cell division step. (b)

Sequence of the temperature-sensitive and hydroxyurea-sensitive steps

T h e results of r e c i p r o c a l shifts .between h y d r o x y u r e a a n d 37~ a r e t a b u l a t e d in T a b l e 2. T h e m u t a n t s fall i n t o four categories w i t h r e s p e c t to t h e i r d e p e n d e n c e on t h e h y d r o x y u r e a - s e n s i t i v e step. D n a m u t a n t s 2076, 1042, 2153 a n d 2179 failed to d i v i d e w h e n t h e y were first i n c u b a t e d a t t h e r e s t r i c t i v e t e m p e r a t u r e a n d t h e n s h i f t e d to

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TABLE 3

Dependence relationships between temperature-sensitive and penicillin-sensitive steps Strain

PC2076 PC1042 PC2153 PC2179 (HU) PC1053 PC1049 PC2244 PC1029 PC2269 PC2116 PC1040

Cell cycle event blocked DNAI DlffAe DNAe DNA. DNAo DIVI DIVp DIVp CS CS DIVI-DIVpw CS

Execution point 0.00 0.75 0.75 0.75 0.75 0-50 1"00 1"00 1.00 1.00 1.00 0.00

%Cells dividing after incubation 2 Expt A t Expt B:~ 13 11 14 7 17 0 101 83 88 94 30 75

96 96 70 93 80 0 0 0 0 0 4 110

Dependence relationship ~ ---~PEN(dependent) _.~ ~rEN (dependent) ~ ~rsN (dependent) _ ~ ~rEN (dependent) ~u rztr (dependent) ~, ran) (interdependent) ~FEN~ (dependent) ~r~'N~ (dependent) PEN ~ (dependent) PEN ~ (dependent) ~. rBN (interdependent) PEN (independent) t~

Experiments were performed as described for Table 2. Results of 37~ to penicillin shift (see Table 1). :~ Results of penicillin to 37oC shift (see Table 1). wSee Discussion. ts, temperature-sensitive step; PEN, penicillin-sensitive step; HU, hydroxyurea-sensitive step.

hydroxyurea a t 30~ 9(Table 2, expt A). When the reciprocal shift was performed, however, this group could be subdivided further: m u t a n t s 1042, 2153 and 2179 were unable to execute the temperature-sensitive step after a prior incubation in hydroxyurea, while the 2076 strain performed the temperature-sensitive step under the same conditions (Table 2, expt B). These results establish the interdependence of the hydroxyurea-sensitive step with the 1042, 2153 and 2179 temperature-sensitive steps, which mediate D N A e (Osley & Newton, 1978), and the dependence of the hydroxyureasensitive step on the prior execution of the 2076 temperature-sensitive step, which mediates a D N A l event (Osley & Newton, 1978). I n contrast, m u t a n t s 1053, 1049, 2244, 1029, 2269, 2116 and 1040 (which specify cell division steps) were all able to execut6 the hydroxyurea-sensitive step when t h e y had been placed at 37~ during the first incubation (Table 2, expt A and Fig. 2(a)). I n the reciprocal experiment, however, only m u t a n t 1040 could divide at 37~ after prior incubation in hydroxyurea; the remaining six m u t a n t s failed to perform the temperature-sensitive cell cycle steps when DNA, was blocked first (Table 2, expt B and Fig. 2(b)). The significance of the small a m o u n t of division in the hydroxyurea to 37~ shift with strains 1053, 1049 and 2244 and in the 37~ to penicillin shift with the Dnae m u t a n t s is considered below and in the Discussion. The temperature-sensitive step defective in strain 1040 is therefore independent of the hydroxyurea-sensitive step,

M. A. O S L E Y AND A. N E W T O N

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FIG. 2. Functional relationship between hydroxyurea-sensitive step and a temperaturesensitive cell separation step. Exponential cells of strain 1029 (Tables 2 and 3) growing at 30~ were divided into 2 parts. One part (expt A; Table 1) was placed at 37~ for 90 min, and then it was shifted back to 30~ in the absence or presence of hydroxyurea (4"5 mg/ml) Ca). I n this experiment, the cells were held at the restrictive temperature for 90 rain instead of 180 rain to ensure their viability and rapid reversibility. The second portion Cexpt B; Table 1) was placed in hydroxyurea C3mg]ml) at 30~ and incubated for 180 min Cb). The inhibitor was removed by filtration and the cells were incubated either at 30 or 37~ Cell number was determined in all subcultures. Ca) 30~ (O); 37~ (A); 37 to 30~ shift (A); 37 to 30~ + HU shift (El). (b) 30~ (O); 30~ + HU (A); HU to 30~ shift (A); HU to 37~ shift (El).

while the e x e c u t i o n of the t e m p e r a t u r e - s e n s i t i v e steps defined b y the r e m a i n i n g s t r a i n s of this group is dependent on the completion of the h y d r o x y u r e a - s e n s i t i v e step. (c) Identification of an early cell division event (DIVI) W h e n penicillin is a d d e d i n low c o n c e n t r a t i o n s to e x p o n e n t i a l l y growing cells of

Caulobacter, cell division c o n t i n u e s for 50 to 60 m i n u t e s before s t o p p i n g ( T e r r a n a & N e w t o n , 1976; Fig. 3(a)). This period of residual division, together ~4th the observ a t i o n t h a t penicillin filaments have no d e t e c t a b l e division sites, suggested t h a t t h e drug m i g h t i n h i b i t division b y blocking a step r e q u i r e d for D I V i (cf. Fig. 1). Thus, D I V I is the earliest k n o w n division e v e n t i n the Caulobacter cell cycle a n d the first

CAULOBACTER CELL CYCLE

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marker that distinguishes the incipient swarmer cell from the parental stalked cell (Terrana & Newton, 1975; Fig. 1). The temperature-sensitive mutation in strain 1053 also appears to affect an early step in cell division. When cells of this mutant are shifted from 30 to 37~ they continue to divide for 50 to 60 minutes (Fig. 3(b)), and their phenotype after 3 to 6 hours at the |

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F I o . 3. E f f e c t o f penicillin a n d 1053 on cell division. E x p o n e n t i a l cells of p a r e n t a l s t r a i n PC1 (a) a n d 1053 (b) were g r o w n a t 30~ ( O ) . Penicillin (210 u / m l ) w a s a d d e d to PC1 cells a n d i n c u b a t i o n w a s c o n t i n u e d a t 30~ ( A ) . S a m p l e s o f 1053 cells were s h i f t e d to 37~ ( A ) , or penicillin (500 u / m l ) w a s a d d e d a t 30~ ( A ) . Cell n u m b e r w a s followed in all c u l t u r e s .

restrictive temperature is similar to that of penicillin-induced filaments (Terrana & Newton, 1976). These results and the observation t h a t strain 1053 is partially resistant to penicillin at the permissive temperature (2-fold more penicillin is required to inhibit cell division in 1053 than in ~dld-type cells ; Fig. 3(b)) suggest that DIV~ is affected by both the 1053 mutation and penicillin. This conclusion is supported by the reciprocal shift experiments presented in the next section showing that the 1053 temperaturesensitive step and the penicillin-sensitive step are interdependent (Table 3). (d) Sequence of temperature-sensitive and penicillin-sensitive steps The functional relationships of the penicillin-sensitive DIVI step to each of the temperature-sensitive DNA and DIV steps are listed in Table 3. The penicillin-sensitive step is not executed in D n a - strains 2076, 1042, 2153 or 2179 when the cells are first incubated at 37~ to block DNA synthesis (Table 3, expt A). The reciprocal experiment shows that prior incubation of these mutants in penicillin does not block performance of the temperature-sensitive steps (Table 3, expt B). Thus, execution of the penicillinsensitive DIV1 step is dependent on the DNAI and DNA e temperature-sensitive steps. An identical conclusion was reached when hydroxyurea was used to block DNA synthesis in place of temperature-sensitive mutations (Table 3). Examination of Div mutants 1049, 2244, 1029 and 2269 in similar experiments shows that the penicillinsensitive step is executed in these mutants when the temperature-sensitive step is blocked in the first incubation (Table 3, expt A and Fig. 4(a)) ; however, the tempera-

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FIG. 4. Functional relationship between penicillin-sensitive step and a temperature-sensitive DIVp step. Strain 1049 cells growing exponentially at 30~ were divided into 2 parts. One subculture (expt A; Table 1) was held at 37~ for 90 min prior to incubation at 30~ in the presence of penicillin (300 u/ml) Ca). The other subculture (expt B; Table 1) was incubated at 30~ in the presence of penicillin (210 u/ml) for 180 min. Penicillin was then removed by addition of penicillinase and the culture was incubated at 30 or 37~ (b). Cell number was followed in all subcultures. (a) 30~ (C)); 37~ (A); 37 to 30~ shift (A); 37 to 30~ + PEN shift (F-l). (b) 30~ (O); 30~ -F PEN (A); PEN to 30~ -b penicfllinase (PENase) shift (A); PEN to 37~ shift ([3). ture-sensitive D I V steps cannot be performed if cells are first arrested at the penicillinsensitive step (Table 3, expt B and Fig. 4(b)). These results show t h a t the four steps defined by the temperature-sensitive Di% and CS mutations are dependent on the execution of the penicillin-sensitive step. The temperature-sensitive ])IV steps defined by mutants 1040 and 2116 display different categories of dependent relationships to the penicillin-sensitive step than the division steps just considered above. Mutant cells of strain 1040 double in number whether the first incubation is at 37~ or in the presence of penicillin (Table 3, expts A and B). This establishes the independence of the penicillin-sensitive and 1040 tempperature-sensitive steps. The temperature-sensitive step defined by strain 2116 is interdependent with the penicillin-sensitive step (Table 3), but in contrast to strain PC1053, which also displays interdependence between the penicillin-sensitive and temperature-sensitive steps, the 2116 m u t a n t does not have a period of residual cell division at 37~ and it is not resistant to penicillin at 30~ (unpublished observations). As considered in the Discussion, these data suggest t h a t the gene product defined b y this strain m a y be required continuously during division from DIVI through CS.

CA ULOBACTER

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(e) Requirement of DNA chain elongation for i)lVl step8 The observation that DIV~ occurs before the completion of chromosome replication in the cell cycle (Fig. 1) suggested that the dependence of DIV~ steps on DNA synthesis reflects a requirement of DNA~, rather than DNAc (Fig. 1). Using synchronous cultures it was possible to determine the temporal relationship between the 90 minute S

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F~o. 5. Timing of DlgA e requirement for execution of penicillin-sensitiveD I V l step. Synchronous cells of strain 1042, a temperature-sensitive DN-A chain elongation m u t a n t , were incubated a t 30~ A t 100 rain a sample of cells was placed at 37~ for 70 min, and t h e n shifted back to 30~ D~rA ~ a : at 0 rain synchronous swarmer cells were incubated w i t h [aH]guanosine at 30~ (Materials and Methods) (O) ; a subculture was shifted to 37~ at 100 rain (A) and t h e n back to 30~ at 170 rain ( / \ ) . Incorporation of radioactivity into DNA was determined as described in Materials and Methods. D i z ~ o n : cell n u m b e r was determined during the 30~ incubation (C)) and in subculture after it was shifted to 37~ and back to 30~ (Q). Execution of the pencillinsensitive step was determined in b o t h the control ( - - / ~ - - A - - ) and shifted ( - - A - - A - - ) cultures; 0.2 ml portions of cells were periodically removed and incubated a t 30~ in the presence of penicillin (210 u/ml). Final cell number achieved in each subculture was determined a t 360 rain. Verticle lines indicate the midpoints for the times of execution of the PEN-sensitive step ( P E N g) and cell separation (CS) in each culture. (a) DNA. (b) ]Division.

120

M. A. OSLEY AND A. NEWTON

S period (DNA~) and the execution point of the penicillin-sensitive DIV, step. I t is clear from the data shown in Figure 5 that the penicillin-sensitive step is executed during the S period and that it precedes DNA~ by 20 to 30 minutes; thus, this step must be dependent on DNA~. Since the 1053 temperature-sensitive DIV~ step and the penicillin-sensitive step are interdependent (Table 3), DNA~ is presumably also required for the 1053 step. The question of when the requirement of DNA synthesis for DIV~ steps occurs during the first 60 minutes of the S period was also examined in a synchronous culture. DNA synthesis was inhibited for a short period of time before the penicillin-sensitive step, and then the execution point of this step was determined after DNA synthesis had resumed (Fig.5(b)). Mid-way in the S period, approximately 30 minutes before DIVI, DNA synthesis was blocked in a Dna~ m u t a n t for 70 minutes, a length of time sufficient to allow completion of the DIVi step in a 30~ control culture. The penicillinsensitive step was not executed until 40 minutes after the resumption of DNA synthesis, and it was completed 20 to 30 minutes before the end of a round of chromosome replication, as also observed in the control culture (Fig. 5). These data confirm the dependence of this DIV~ step on DNA chain elongation, and they indicate t h a t the penicillin-sensitive step requires chromosome replication occurring very close in time to its execution. (f) D N A chain completion and D N A chain elongation are required for cell division The above results suggest a role of DNA chain elongation in initiating the division pathway by mediating the sequence of events, DNA, --> DIVI --> CS. Previous results in Caulobacter (Degnen & Newton, 1972b) have indicated that cells must normally complete DNA replication to divide. We have re-examined the apparent requirement of DNAc for cell division to determine if it can be distinguished from the requirement of DNA e. Figure 6 shows an experiment in which DNA synthesis was inhibited in a synchronous culture of a temperature-sensitive DNa~-mutant either b y shifting samlJles to 37~ or b y the addition of hydroxyurea or mitomycin-C to samples at the permissive temperature. The final cell number achieved in each of the samples generated a curve identifying the latest time in the cell cycle at which DNA synthesis was required for cell division. In parallel samples the time of execution of the penicillin-sensitive DIV 1 step was also established. The results (Fig. 6) demonstrate that cell division requires DB[A replication after the time it is needed for execution of the penicillin-sensitive step, and that the last time DNA synthesis is required coincides with the completion of a round of chromosome replication (DNAc). The dual requiremnt of DNAe and of DNAr for cell division was confirmed by reciprocal shift experiments. After shifting Dnae-mutants from 37~ to 30~ in the presence of penicillin, approximately 15% of the cells divided in the presence of the drug (Table 3). This increase in cell number can be accounted for b y cells in the population which were between DIVi and DNA~ at the time of the DNA synthetic Iblock (Osley & Newton, 1978). Since these cells had completed chromosome replication required to execute the penicillin-sensitive step, but not DNA~, they would be expected to divide in the presence of the drug when the round of replication was

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FIG. 6. Execution of penicillin-sensitive step and hydroxyurea-sensitive step in synchronous cells. Synchronous cells of strain 1042 were grown at 30~ and cell number was followed to determine the time of cell division ((9). Execution of the penicillin-sensitive ( 0 ) and hydroxyureasensitive (A) steps was determined by periodically removing 0.2 ml samples of cells and incubating t h e m at 30~ in the presence of penicillin (210 u/ml) or hydroxyurea (3 mg/ml). A t 360 min the final cell number achieved in each of these subcultures was measured. DNA synthesis was also inhibited b y the addition of mitomyein-C (4 ug/ml) at 30~ or by shifting cells to 37~ for 360 rain with identical results to those shown for hydroxyurea-treated cells. Vertieall lines indicate the times of execution of the penicillin-sensitive (PEN') and hydroxyurea-sensltive (HU') steps and cell separation (CS). The G1, S, G2 periods are shown at the top of the Figure.

allowed to complete. The remaining cells in the population could not divide in penicillin because they had not completed DNA synthesis needed for DIVi before the initial shift to 37~ (g) Division step mediating CS is dependent on completion

of chromosome replication The dependence of cell division on completion of chromosome replication could be mediated through the execution of temperature-sensitive division steps required for DIVp or CS (Fig. 1). To distinguish between these possibilities synchronous cells of a DIVp or CS temperature-sensitive m u t a n t were allowed to develop at the permissive temperature, and hydroxyurea was added at 135 minutes to block DNA synthesis (Figs 7 and 8) ; a large fraction of the cells in the population had executed the penicillinsensitive DIV~ step at this time, but they had not completed a round of chromosome replication (DNAr Hydroxyurea was left in the culture for 75 minutes, to allow time for execution of the DIVp (Fig. 7) or CS (Fig. 8) temperature-sensitive steps. The drug was then removed from the culture and the cells were shifted to 37~ The pattern o f cell division was followed both in the temperature shifted culture and in a control sub, culture which was left at 30~ after treatment with the inhibitor. Cell cultures were

M. A. O S L E Y A N D A. ~ E W T O N

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Fro. 7. DNAe is required for a DIVp step. Synchronous cells of strain 2244 were grown at 30~ and cell division was determined (O). The execution points of the penicillin-sensitive (@) and hydroxyurea-sensitive (ram) steps in this culture were determined as described in the legend to Fig. 6. At 135 min hydroxyurea (3 mg/ml) was added to a portion of cells and incubation was continued at 30~ until 210 min (Z~). Hydroxyurea was removed by filtration of cells, and the culture was incubated at 30~ (A) or 37~ (A). Cell number was monitored in all subcultures. The proportion of cells at different stages of the cell cycle at the time of hydroxyurea addition arc noted (see test). n o t entirely synchronous, a n d the results of these experiments can be interpreted knowing the age distribution of cells present at 135 minutes when h y d r o x y u r e ~ was added: (a) cells t h a t had completed D N A c ; these "G2 cells" divided in the presence of the inhibitor at 30~ (b) cells between the penicillin-sensitive D I V I step and DNAo ; these cells do n o t divide in the inhibitor since DNAc cannot be performed, b u t t h e y will execute the DIVp or CS steps in the presence of the drug a n d subsequently divide at 37~ if these steps are dependent on the D I V I step and not on D N A c ; a n d (c) cells t h a t have not completed the penicillin-sensitive step; these cells will not divide in h y d r o x y u r e a or at 37~ after the inhibitor has been removed, since b o t h D I V , and CS temperature-sensitive steps are dependent on the execution of the penicillin-sensitive D I V I step (Table 3), The difference observed between the two m u t a n t s is seen in the " b " class of cells: this fraction divided at 37~ after h y d r o x y u r e a was r e m o v e d from the Divp m u t a n t (Fig. 8), b u t n o t after the drug was r e m o v e d from the CS m u t a n t (Fig. 9). The delay in division of the Divp m u t a n t at 37~ was f o u n d consistently and it reflects a failure of this strain to recover immediately from h y d r o x y u r e a inhibition at 37~ (unpublished results). A p p a r e n t l y the temperature-sensitive D I V , step can be executed w i t h o u t prior completion o f chromosome replication, b u t the CS step is

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DNA~ a r e r e q u i r e d for a CS step. T h e e x p e r i m e n t a l protocol d e s c r i b e d in t h e u s e d w i t h s y n c h r o n o u s cells of s t r a i n 1029 (Tables 2 a n d 3). Cell division a t of penicillin-sensitive s t e p ( 9 ; e x e c u t i o n of h y d r o x y u r e a - s e n s i t i v e s t e p ( 9 ) ; + H U ( A ) ; cell division a f t e r H U t o 30~ s h i f t ( / k ) ; cell division a f t e r H U

dependent on DNA c for its execution. We interpret these results to show that the coupling between DNA c and the cell division pathway in Caulobacter is after steps mediating DIVp and before execution of the steps required for CS ; thus, execution of DIVp steps requires only the completion of DIV 1 steps, while CS steps require completion of both DIVI steps (and presumably DIVp steps) and DNAo. 4. Discussion Data from shift experiments presented above, together with previous results (Osley & Newton, 1977,1978; Terrana & Neuron, 1976), have been used to construct the model for temporal control of the stalked cell cycle in Caulobacter (Fig. 9). We propose that steps in the cell cycle are organized into at least two dependent pathways. One of these pathways contains the steps that are required for chromosome replication and other steps that are required for cell division events. A unique feature of the model is the regulation of the division pathway by two separate branches from the DNA synthetic pathway. DIVI is dependent on DNA chain elongation and CS is dependent on the completion of chromosome replication: additional cell cycle steps may be required during the G1 period for the swarmer cell cycle (cf. legend to Fig. 9: Osley & Newton, 1978), but the organization of dependent steps shown in Figure 9

124

M. A. O S L E Y A N D A. N E W T O N

,o,,,,,,, : H

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Fro. 9. Organization of DNA synthesis and cell division pathways in C. crescentus. The model pravoses~that t~ho:t~emporalorder of events in the Ca,ulobacter cell cycle (Fig. 1) is determined by . the functional dependence of both the gene-mediated steps (shown by sequence of arrows) and the cell cycle events themselves (connected by the arrows). Reciprocal shift experiments in exponential cells have established the dependence relationships among the gene-mediated steps (cs Tables 2 and .3). The~posslble role of DNA~ and DNAo as part of the pathway is suggested by shift experiments' in synchronous cultures which implicate these 2 events as requirements for execution of DIVI steps and CS steps,, respectively (see text). There is no direct evidence that DNAI, DIVe, DIVp and CS are also part of the dependent pathways and these events have been placed in braeket~. The organization of the DNA and division pathways shown should apply to stalked cells produced at division and stalked cells which develop from swarmer cells. I t is likely, however, that additional cell cycle steps are required to initiate the cell cycle in newly developed stalked cells during the preceding G1 period (Osley & Newton, 1978). Numbers refer to cell cycle mutations (Tables 2 and. 3) which define temperature-sensitive cell cycle steps; it is not known whether .mutations blocking the same event define different gene-mediated steps (cf. Results). Cell cycle events are defined in the legend to Fig. 1. The model summarizes data presented in this paper and elsewhere (Osley & Newton, 1977,1978). The role of the 2116 step, which acts at DIVI and throughout the D'IVb period, and the 1040 step, which acts at CS, are considered in the text.

a c c o u n t s for t h e o r d e r of cell cycle e v e n t s in b o t h t h e s t a l k e d cell p r o d u c e d a t division a n d t h e ~ a l k e d cell t h a t d e v e l o p s from t h e s w a r m e r cell. S e v e r a l f e a t u r e s o f t h e m o d e l are considered below. (a) D N A pathway R e c i p r o c a l shift e x p e r i m e n t s show t h a t t h e s t e p m e d i a t e d b y t h e 2076 gene p r o d u c t is a p r e r e q u i s i t e for t h e s t a l k e d cell to u n d e r g o D N A r e p h c a t i o n (Table 2). This t e m p e r a t u r e : s e n s i t i v e s t e p can be c o m p l e t e d in t h e presence o f h y d r o x y u r e a (Table 2), a finding c o n s i s t e n t w i t h t h e p r e v i o u s r e p o r t t h a t t h e 2076 gene p r o d u c t is r e q u i r e d for i n i t i a t i o n of D N A s y n t h e s i s (Osley & N e w t o n , 1978). T h r e e o t h e r D n a m u t a n t s were p r e v i o u s l y c h a r a c t e r i z e d as defective in D N A chain e l o n g a t i o n (Osley & N e w t o n , 1978) a n d , as w o u l d be e x p e c t e d , t h e i r t e m p e r a t u r e - s e n s i t i v e s t e p s a r e i n t e r d e p e n d e n t w i t h t h e h y d r o x y u r c a - s e n s i t i v e s t e p (Table 2; Fig. 9). T h e d e p e n d e n t p a t h w a y which m e d i a t e s t h e sequence o f events, D N A l -+ D N A e --> DNAc, is d e m o n s t r a t e d b y reciprocal shift e x p e r i m e n t s a n d D N A i n t e r r u p t i o n experim e n t s in s y n c h r o n o u s cells: t h e h y d r o x y u r c a - s e n s i t i v e D N A e s t e p (and, p r e s u m a b l y , t h e i n t e r d e p e n d e n t t e m p e r a t u r e - s e n s i t i v e D N A . steps) c a n n o t occur in t h e a b s e n c e of t h e t e m p e r a t u r e - s e n s i t i v e D N A I s t e p (Table 2), a n d D N A c c a n n o t t a k e p l a c e w h e n c h r o m o s o m e r e p l i c a t i o n is blocked a t a n y p o i n t in t h e S p e r i o d (Fig. 5 ; Osley & N e w t o n , 1978). (b) 19ivision pathway W e h a v e a n a l y z e d six t e m p e r a t u r e - s e n s i t i v e m u t a t i o n s t h a t define a d e p e n d e n t cell division p a t h w a y (Fig. 9). T h e steps in this p a t h w a y m e d i a t e t h e t h r e e cell division

GAULOBAGTER

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landmarks, D I V , DIVp and CS (Fig. 1). The earliest of these events, DIVI, is characterized by the initial placement of the division site, and it can be blocked either by addition of penicillin to wild-type cells or by growth of mutant 1053 at the restrictive temperature (Fig. 3). The interdependence of the 1053 temperature-sensitive step and the penicillin-sensitive step (Table 3) suggests either that the gene product defined by strain 1053 is the target of the drug or that it interacts with a penicillin-binding protein (cf. Spratt, 1977). Prior completion of the penicillin-sensitive step is required for execution of the temperature-sensitive steps defined by four mutants (1049, 2244, 1029, 2269 ; Table 3 ; Fig. 9). This result is consistent with the morphology of these mutants grown at 37~ : strains 1049 and 2244 (defective in steps required during the DIVp period) become long, variably pinched filaments with multiple division sites, while mutants 1029 and 2269 (defining steps required for CS) produce a uniform population of very pinched filaments also containing multiple division sites (Osley & Newton, 1977). By comparison, filaments blocked in the earlier steps required for DIVI have no detectable division sites (Terrana & Newton, 1976). We propose in Figure 9 that division steps are arranged in a dependent pathway to account for the sequence of events DIV t -> DIVp --> CS. Although DIVI steps are required for execution of both DIVp and CS steps (Table 3), the functional dependence between DIVp and CS steps cannot be examined in reciprocal shift experiments since all of these steps are defined by heat-sensitive mutations. The dependent relationship shown between DIVp and CS (Fig. 9) is supported, however, by evidence that the DIV, steps precede CS steps in time and that DIVp must be completed before CS can occur: (1) the majority of ])IV, filaments at 37~ are not highly pinched, suggesting that the CS event cannot occur when the DIVp period is blocked; (2) protein synthesis is required after execution of DIVp steps, but not after CS steps or during the G2 period (unpublished data). This observation is consistent with the conclusion that CS steps act just prior to cell division, while DIVp steps occur earlier in the cell cycle during the period when protein synthesis is required. All of the division mutations considered above define individual steps on the dependent pathway, and these steps have execution points that coincide with the cell cycle events that they mediate (Tables 2 and 3). Strains 2116 and 1040 are interesting as exceptions to this generalization, and because of their more complicated phenotypes they have not been incorporated into Figure 9. In the case of strain 2116 the gene product defined by the temperature-sensitive mutation appears to act at DIVi and throughout the DIV, period. This conclusion is supported by the observations that (1) the 2116 product is interdependent with the penicillin-sensitive step (Table 3); (2) the fraction of cells dividing in the 37~ --> penicillin shift (30% ; Table 3) is equal to the fraction of cells between DIV1 and CS in an exponential population (Osley & Newton, 1978); and (3) 2116 cells stop dividing immediately at 37~ forming predominately unseptated filaments, which presumably represent those cells before DIV~ at the time of the temperature shift, and a minority of septated filaments, which presumably represent the cells between DIV~ and CS at the time of the temperature shift. Strain 2116 also differs from other Div-, Dna + mutants in its failure to synthesize flagellar proteins at 37~ (Osley & Newton, 1977 ; Osley et al., 1977). This last observation is consistent with the pleiotropic effect of the 2116 gene product during the DIVp

126

M.A. OSLEY AND A. NEWTON

period since it suggests that step(s) coupling DNAc and CS are also affected in this mutant. Strain 1040 is singular among the Caulobacter cell cycle mutants because the execution point and site of action of the 1040 temperature-sensitive step do not coincide in time. The strain forms long, uniformly pinched filaments at 37cC (Osley & Newton, 1977), indicating the requirement of the 1040 gene product for CS, and it divides for approximately 120 minutes at the restrictive temperature, indicating that the execution point of the 1040 mutation is at the beginning of the stalked cell cycle (Tables 2 and 3). A similar phenotype has been observed for a mutant step in bacteriophage P22 development (Jarvick & Botstein, 1973) and for several mutations blocking cytokinesis in Saccharomyces cerevisiae (Hartwell, 1971). The 1040 step could define another division pathway, but we have not identified the step(s) that are required for its execution. Although it is independent of both chromosome replication (Table 2) and the penicillin-sensitive step (Table 3), it could be dependent on the 2076 (DNAI) gene product, which has its execution point at the same time in the cell cycle (Fig. 1). This possibility cannot be tested at this time because both steps are heat-sensitive. (c) Two branches connect the D N A and division pathways Earlier results in Caulobacter (Degnen & Ne~r 1972b) and other bacterial cells (Clark, 1968; Helmstetter & Pierucci, 1968) have suggested that completion of chromosome replication is a requirement for cell division. The execution point of the hydroxyurca-sensitive step supports this conclusion (Fig. 6). The finding that the penicillin-sensitive DIVi step depends on DNA chain elongation identifies a second requirement of DNA synthesis for early cell division steps (Table 3; Fig. 5). The DNAe requirement for initiation of cell division in Caulobacter is particularly interesting because this type of control has not been reported in the cell cycle of other organisms. The reciprocal shift from penicillin to 37~ in Dnae mutants (Table 3) shows that DNA replicated during the penicillin block satisfies the requirement for execution of the DIV1 step. Either the penicillin-sensitive step is dependent on an intervening step which requires DNA~ or, if it is directly dependent on DNA~, this dependence involves replicated, rather than replicating DNA. (A dependent relationship in which replicating, rather than replicated, DNA was required for a division or other cell cycle step would appear as interdependent in the reciprocal shift experiments described here.) The results of a DNA interruption experiment in synchronous cells (Fig. 5) indicate that the requirement of DNA synthesis occurs close in time to the execution of the penicillin-sensitive DIVI step. These data are supported by results of reciprocal shift experiments in exponential cells which showed that 10 to 15% of the cells divided when Dna~ mutants were shifted from 37~ to penicillin (Table 3). If the DNA~ requirement for DIVI steps occurred well before these steps were executed, then substantially more of the population would have divided in penicillin after inhibition of DNA synthesis. None of the experiments, however, can distinguish between the requirement of a certain amount of replicated DNA, i.e. a two-thirds replicated chromosome, and the replication of a chromosomal segment just prior to DIV~ steps in the cell cycle. Evidence that the second branch from the DNA pathway is from DNAo to cell separation steps comes from shift experiments in synchronous cultures (Figs 7 and 8). When DNA synthesis was interrupted before DNAo the DIVp step was completed in

C A U L O B A C T E R CELL CYCLE

127

the presence of hydroxyurea (Fig. 7), but the CS step was not (Fig. 8). Consistent with the conclusion that CS steps are dependent on DNAo, or on a very late stage of chromosome replication, is the observation t h a t CS mutants fail to divide and DIVp mutants show a very small amount of division in reciprocal shifts from hydroxynrea to 37~ (Table 2). (d) D N A synthetic pathway as a cell cycle clock The temporal order of morphological events in cell division is achieved in Caulobacter by the dependence of late steps on the completion of early steps (Fig. 9 ; Table 3). In additiorl to this control, the initiation and completion of the division pathway are regulated by successive stages in the DNA synthetic pathway, and in this sense chromosome replication can be considered as a cell cycle clock (cf., Mitchison, 1971). In our view, the "clock" starts at the initiation of DNA synthesis when the steps required for this event are completed. After chromosome replication is two-thirds 0ompleted, the DNAe requirement for DIVI steps is satisfied and then, at the end of the S period, the DNAo requirement for execution of CS steps is met to allow completion of the cell cycle. Several characteristics of DNA replication in these cells are consistent with its role as a cell cycle clock. Interruption of DNA synthesis early in the S phase prevents initiation of the division pathway, but the normal timing of division events is maintained once DNA synthesis has resumed (Fig. 5). Also, execution of steps in the DNA pathway occurs independently of all division steps t h a t have been examined (Table 2), and preliminary results (unpublished) show that the 2076 DIVi step is dependent on DNAo, indicating t h a t the DNA pathway in the stalked cell is circular. Finally, a more general role of chromosome replication as a timer in the Caulobacter cell cycle is supported by evidence (considered in the next section) that the periodic expression of some developmental genes is controlled by a late step in chromosome replication. When the model for cell cycle control in Caulobacter is compared to those proposed in Escherichia coli (Donachie et al., 1973; Jones & Donachie, 1973) and S. cerevisiae (Hartwell, 1974,1976), the most striking difference is the couple between DNA, and DIVI observed in Caulobacter (Fig. 9). Although independent DNA and division pathways converge prior to cell separation in all three organisms, there is no direct evidence in E. coli and yeast to indicate that ongoing DNA replication acts as a timer to initiate the cell division pathway. In yeast, the initiation of both the division pathway (starting at bud emergence) and the DNA synthetic pathway requires the completion of a common " s t a r t " event in the G1 period (Hartwell, 1974). In E. coli, the morphological sequence of events associated with septation has not been identified (Slater & Schaechter, 1974), but DNA synthesis-arrest experiments indicate that some of the steps t h a t are required for cell division m a y occur early in the cell cycle before the start of chromosome replication (Jones & Donachie, 1973).

(e) Cell cycle and regulation of development C. crescentus appears to have exploited the organization of the cell cycle as a means to separate into two pathways the steps that control swarmer cell and stalked cell differentiation. We have found that the periodic synthesis of flagellin (Osley et al.,

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1977), hook protein (Sheffery & Newton, unpublished results) and expression of phage receptor sites (unpublished results) in stalked cells is controlled by the D N A synthetic pathway. Stalk formation, an event associated with swarmer cell differentiation (Osley & Newton, 1977), and generation of a new cell pole (unpublished results) are coupled to the completion of DIVp steps in the previous cell division cycle. Thus, in general, it appears t h a t the DNA p a t h w a y acts as a " t i m e r " for the periodic expression of the polar structures t h a t characterize the new swarmer cell, while steps on the division p a t h w a y are required for the polar localization of these structures on the dividing cell (unpublished results). The mechanism of cell cycle control proposed for Caulobacter m a y apply more generally to differentiation in other organisms. This is an attractive possibility in systems where the pattern of development is determined b y cell lineage. This work was supported by Public Health Service grants GM25644 and GM22299 from the National Institutes of Health and by the Whitehall Foundation. REFERENCES Clark, D. J. (I 968). Cold Spring Harbor Syrup. Quant. Biol. 33, 823-828. Degnen, S. T. & Newbon, A. (1972a}. J. Mol. Biol. 64, 671-680. Degnen, S. T. & Newton, A. (1972b). J. Bacteriol. 110, 852-856. Donachie, W. D., Jones, N. C. & Teather, R. T. (1973). In Society of General Microbiology Symposium 23, Microbial Differentiation (Ashworth, J. M. & Smith, J. E., eds),

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