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T o M I o ~ , S. & MArSUXASm, M (1978), P u r i f i c a t i o n o f p e n t c d h n - l n s e n s m v e D D - e n d o p e p t i d a s e , a n ew cell wall p e p t t d o g l y e a n - h y d r o l y z i n g e n z y m e m Escherwhsa coh, a n d its in[nbttton by d e o x y r i b o n u c l e i c acids. Broehera. btophys. Res. C o m m u n . , 84, 978-984. TORMO, A . , MARTII~£Z-SALAS,E & VICENTE, M . (1980), I n v o l v e m e n t o f t h e f t s A g e n e p r o d u c t m late s t ag e o f t h e Eschertchia c o b cell cycle. J. Bact., 141, 806-813. WA c m, 1vl , Doi, M . , OKAVA, Y. & MATS0H^Sm, M. (1989), N e w rare genes, #lreC and tareD, responsible f o r f o r r n a t t o n o f t h e r o d s h a p e o f the Eschertchta colt cell. J. Bact., 171, 6511-6516_ WACHI, M , D o l , M . , TAMAKI, S., PARK, W . , NAKAMMA-IIJIMA,S '~" MATSUHASHI, M . (1987), M u t a n t , s o l a t i o n a n d m o l e c u l a r c l o n i n g o f m r e genes, w h i ch d e t e r m i n e cell s h a p e , senslttvity t o m e e i l h n a m , a n d a m o u n t o f p e n i c d l i n - b m d m g p r o t e i n s in l ~ c h e n c h r a colt. J. Bact., 169, 4935-4940. WACHI, M . & MATSLIHASHi,M . (1989), N e g a t i v e c o n t r o l o f cell d i v i s i o n by mreB, a g e n e t h a t f u n c t i o n s m d e t e r m i n i n g the r o d bhape o f Eschertch~a colt cells. J. Bact., 171, 3123-3127. Yi. Q . - M . & Ltrrr~NHA0S, J. (1985), T h e aauc!eotide s e q u e n c e o f the essentml cell d i v i s i o n gene f t s Z . Gene, 36, 241-247. ZlJDE~VELD, C . A . L . , BLAAUWEr%T . D . , EDELMAbl, A . , SPRATT, B . G . & NANbIINGA,N . (1990), P e n i c d h n - b m d m g p r o t e i n 1B o f E ~ c h e r t c h l a c o h is a s s o c i a t e d with a 38 K m o l e c u l a r weight p r o t e i n . J. Bact., 172 (in press).

The work performed in the authors" laboratories was supported, m part, by Grants m Aid for SckentlflC Research and Grants I n Aid for Sctenttflc Research on Priority Areas from the Mmtstry of Education, Science and Culture o f Japan.

POLAR

CAP FORMATION

DURING

IN ESCHERICHIA N . N a n n i n g a (*), F . B . W i e n t j e s , B . L . M .

CELL DIVISION

COLI de J o n g e a n d C . L . W o l d r i n g h

D e p a r t m e n t o f Molecular Cell Btology. Sectaon o f Molecular Cytology. University o f A m s t e r d a m , Plantage g u i d e r g r a c h t 14. 1018 T V A m s t e r d a m

lntroduetion. W h e n r e a d i n g t h e classic " T h e Cell m Development and Heredity" by E . B . W i l s o n (1925), o n e b e c o m e s i m p r e s s e d b y t h e i n f l u e n c e t h e cell c o n c e p t h a s p l a y e d in s h a p i n g t h o u g h t s o n d e v e l o p m e n t a l b i o l o g y , T bh ae s ~ e r p e t u a t i o n o f cells w a s s e e n as a propert y o f life, t h e c h e m i c a l n a t u r e o f hereditary material not being known.

(*) Corresponding author

T h u s , in line w i t h a q u o t a t i o n f r o m Samuel Butler" "'It has, I believe, been o f t e n r e m a r k e d t h a t a h e n is o n l y a n egg's way of making another egg'" ( W i l s o n , 1925, p. 12), W i l s o n s t a t e d that: "thus regarded, the individual app e a r s as a n e v a n e s c e n t b y - p r o d u c t ; it is b u t a n incident - - a l m o s t , w e m i g h t say, art a c c i d e n t " ( W i l s o n , 1925). I t is p r o b a b l y c o r r e c t n o w a d a y s t o t r a n s l a t e ceil by DNA and ask the question whether

104

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t h e cell is a n e v a n e s c e n t b y - p r o d u c t serving the perpetuation of genomes (for r e v i e w , cf. N a n n m g a a n d W o l d r i n g h , 1985). T h e b i o l o g i c a l s i g n i f i c a n c e o f cell d i v m i o n is t h e r e f o r e i n t i m a t e l y l i n k e d with DNA rephcatlon and nuclcold segregation. In fact, the physical presence of the nucleoid at the prospect i v e n o r m a l site o f d i v i e i o n p r e v e n t s c o n s t r i c t i o n ( W o l d r i n g h e t aL, 1 9 7 7 ; H e l m s t e t t e r e t aL, 1979). Whereas t h i s a s p e c t is f u r t h e r discussed by several contributors e l s e w h e r e m t h i s v o l u m e , h e r e w e will try to analyse the dlwsion process mainly at t h e level o f t h e c e l l u l a r e n v e l o p e (cf. a l s o N a n n i n g a , 1989). D i f f e r e n t i a t i o n w i t h i n t h e cell e n v e l o p e . Is t h e site o f division p r e d e t e r m i n e d ? O n e c a n e n v m a g e t w o possibilities. Firstly, o n e c a n a s s u m e t h a t t h e cell envelope, already at an early stage of t h e d i v i s i o n cycle, is c h e m i c a l l y a n d structurally differentiated. Triggenng of the predetermined divimon system could be under temporal and topological control. Secondly, one can assume that t h e r e is n o d i f f e r e n c e in c h e m i c a l a n d structural composition of lateral wall a n d t h e p r o s p e c t i v e d i v i s i o n site. T h e l o c a l c h o i c e o f a p a r t i c u l a r set o f e n z y m e a c t i v i u e s n e e d e d f o r cell e l o n g a tion or division would depend on the presence of particular temporal and topological signals. P r e d e t e r m i n a t i o n is i n d i c a t e d b y t h e following observations. Treatment of g r o w i n g cells w i t h a m p i c i l h n a t h i g h c o n c e n t r a t i o n (500 l z g / m l ) l e a d s t o t h e expulsion of spheres that occur p r e d o m i n a n t l y a t t h e cell c e n t r e , l o n g b e f o r e t h e d i v i s i o n site b e c o m e s visible ( S t a u g a a r d e t al., 1976). I n o t h e r w o r d s , a c e n t r a l a r e a o f t h e cell is h i g h l y s e n sitive t o i n t e r f e r e n c e w i t h m u r e i n biosynthesis. Another argument can be derived from the location of plasmolysis b a y s in d i v i d i n g cells. I n t h i s c a s e , b a y s o c c u r p r e d o m i n a n t l y at t h e p r o s p e c t i v e site o f d i v i s i o n o f t h e d a u g h t e r cells ( C o o k e t al., 1987; of. R o t h f i e l d e t al., 1990) P r e d e t e r m i n a t i o n o n t h e

envelope level implies a different chemical makeup of the envelope along t h e l e n g t h a x i s o f t h e cell, e i t h e r d i s c o n tinuous or more gradient-like. Probably the only candidates with a potentially defined location are the proteins FtsZ (specifically i n v o l v e d in t h e i n i t i a t i o n o f d l v i s a o n ; W a l k e r e t al., 1975; B e g g a n d D o n a c h i e , 1985), p e n i c i l l i n - b i n d i n g p r o t e i n 2 ( P B P 2 , s p e c i f i c a l l y i n v o l v e d in cell e l o n g a t i o n ; S p r a t t , 1 9 7 5 ) a n d P B P 3 (specifically i n v o l v e d in d i v i s i o n ; S p r a t t , 1975, B o t t a a n d P a r k , 1981). T o t h e best of our knowledge, no topographical labelhng studies have been p u b l i s h e d w i n c h give d i r e c t r e f o r m a t i o n o n t h e i r l o c a t i o n in t h e cell. T h e existence o f P B P i n v o l v e d in cell elongation and division points to two d i f f e r e n t f u n c t i o n a l s y s t e m s [ S c h w a r z e~ aL, 1 9 6 9 ; S a t t a e t a L , 1981). I f t h e s e systems do not have a specific location in t h e cell e n v e l o p e , i.e. w i t h r e s p e c t t o l a t e r a l w a l l a n d site o f d i v i s i o n ( n o p r e d e t e r m i n a t i o n o n t h e e n v e l o p e level), t h e y still c o u l d receive specific t e m p o r a l topological signals. The nature of such s i g n a l s is n o t k n o w n , b u t t h e y a r e l i k e ly t o b e in s o m e w a y r e l a t e d t o D N A r e p l i c a t i o n a n d s e g r e g a t i o n (see b e l o w a n - a l s o W o l d r i n g h et a L , 1990). Also, biophysically oriented models d o n o t strictly r e q u i r e p r e d e t e r m i n a t i o n . For instance, according to the variable T (the analogue of surface tension) model of the surface stress hypothesis, d i v i s i o n o c c u r s t h r o u g h a d e c r e a s e in the surface tension at the prospective site o f d i v i s i o n ( K o c h a n d B u r d e t t , 1984). L i k e w i s e , n o p r e d e t e r m i n a t i o n o n t h e e n v e l o p e level is n e c e s s a r y in the flmte zonal growth model (Koch, 1990). In a geometric model based on e l o n g a t i n g c y l i n d r i c a l l y s h a p e d cells o f constant diameter, local surface extens i o n is r e q m r e d at t h e d i v i s i o n site t o restore the original surface-to-volume r a t i o a t b i r t h ( W o l d r i n g h et aL, 1985; W o l d n n g h e t ~I., 1987 ; see b e l o w ) . T h i s descriptive model could also funetton without predetermination, and rather, r e q m r e s m e t a b o l i c r e g u l a t i o n (cf. D ' A r i e t aL, 1990) b e c a u s e o f t h e i m p o r t a n c e of maintaining the surface-to-volume ratio within certain limits.

CELL

SHAPE

AND

DIVISION

T h e t o p o l o g i c a l switch in p e p t i d o g l y e a n syn*hesis. P e p t i d o g l y c a n s y n t h e s t s is u s u a l l y studied by mcorporation of a radioactive p~ e c u r s o ) s u c h a s m e s o - 3 H - d i a m i nopimellc acid (SH-Dap). When the s y n t h e s i s r a t e in t h e cell c y c l e is t o b e determined, two principally different m e t h o d s c a n b e u s e d : 1) T h e cells c a n be s y n c h r o n i z e d a n d t h e s y n t h e s i s r a t e in t h e s y n c h r o n o u s l y g r o w i n g cells c a n be determined b y pulse-labelling. This m e t h o d has the d m a d v a n t a g e o f possible m e t a b o h c disturbances by the sync h r o n i z a t i o n m e t h o d . 2) E x p o n e n t i a l l y g r o w i n g cells a r e p u l s e - l a b e l l e d , a n d a f t e r w a r d s the d i s t r t b u t m n o f the label d u r i n g d i f f e r e n t s t a g e s o f t h e ceil c y c l e is a n a l y s e d . I n thin a p p r o a c h , cells c a n be c l a s s i f i e d a c c o r d m g t o size b y a u t o r a d i o g r a p h y ( R y t e r e t al., 1973; S c h w a r z e t a l . , 1975 ; K o p p e s e t a L , 1978; Verwer a n d N a n n i n g a , 1980; W o l d r i n g h e t a L , 1087 ; W i e n t j e s a n d

L~

u~

Z

e~

IN

E, COLI

105

N a n n m g a , 1989) o r selected o n t h e basis o f a g e b y m e m b r a n e e l u t i o n . I n t h e latter c a s e , 3 H - D a p i n c o r p o r a t i o n w a s measured biochemically (Cooper, 1988). I n c o n t r a s t w i t h m e m b r ~ t , e elut i o n , a u t o r a d i o g r a p h y a l s o F" ,ides topographical information. We used s y n c h r o n i z e d cells as well a s e l e c t r o n microscopic (EM) autoradiography to assess t h e r a t e a n d t o p o g r a p h y o f p e p t ~ d o g l y c a n s y n t h e s i s in E s c h e n c h t a coli, with particular emphasis on the constnction process (Woldringh et aL, 1987; W i e n t j e s a n d N a n n i n g a , 1989; de J o n g e e t al., 1989). E . c o i l M C 4 1 0 0 lysA was s y n c h r o n i z e d by selecting s m a l l cells b y c e n t r i f u g a l e l u t r i a t i o n ( F i g d o r e t al., 1981 ; W i e n t j e s a n d N a n n i n g a , 1989). A l t h o u g h this m e t h o d s e e m s t o d i s t u r b b a l a n c e d g r o w t h less tl~an g r a d i e n t c e n t r i f u g a t i o n ( C r e a n o r a n d M i t c h i s o n , 1979), R is d i f f i c u l t t o ¢.xclude m e t a b o h c d ~ s t u r b a n c e s c o m pletely. T h e r e f o r e , we u s e d as a c o n t r o l asynchronous cultures which had been

Ld

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so

D

O

~

10 LENGTH

"1"

20 30 O F S A C C U L L I S { Izrn |

4"0

~

~0

F I e 1. - - Coral :trison o f theoretmal ( - - ) a n d experimental f - - - ) rates o f s u r f a c e synthesis m M C 4 1 0 0 lysA ftsZ m u t a n t at permtamve temperature, p l o t t e d as a f u n c t s o n o j cell length. The theoretical lines calculated for the present conditions (cf. W o l d r m g h et aL, 1987) predict a si~ghtly larger increase m the rate o f surface synthes~s with cell length m a n shown by the regression lines obtained from the observed g] ain number per individual sacculus (not shown). The regression coefficmnt and hnear correlation coefficient for saccuh that were merely elongating were 2.89 and 0.19, and for constricting saccuh, 3.59 and 0 62, respectively. The theoreUeal and experimental hnes were normah~ed to the cell [ength'correspo" nding to the start o f constriction (O). The two cell figures represent cells that have doubled the volume, Vo, o f a newborn cell Hatched areas reflect the surface synthesized during the constrictton pertod. The black area indicates the extra surface necessary to make polar caps. (From Woldringh et aL, 1987)

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p u t t h r o u g h the e I u t n a t i o n p r o c e d u r e e x c e p t f o r the i s o l a t i o n o f small cells ( C r e a n o r a n d M i t c h i s o n , 1982 ; VVlentjes a n d Na_qninga, 1989). Since the r a t i o o f pulse-labelling d a t a f r o m t h e s y n c h r o n o u s a n d a s ~ a c h r o n o u s cultures did n o t s h o w oscillations, we c o n c l u d e d t h a t the s y n t h e s i s r a t e o f p e p t l d o g l y c a n inc r e a s e d in a m o r e o r less e x p o n e n t i a l w a y d u r i n g the cell cycle ( W i e n t j e s a n d N a n n i n g a , 1989). T h i s r e s e m b l e s the results o f C o o p e r (1988). In any synchronous culture, there will a l w a y s be s o m e o v , ' - l a p b e t w e e n cells m d i f f e r e n t stages o f ~he cell cycle, a n d this will m a k e it d i f f i c u l t t o d e t e c t d e v i a t i o n s f r o m c x p o n e n t i a l i t y . A different approach was therefore used to s t u d y in m o r e detail possible c h a n g e s in rate and t o p o g r a p h y o f peptidogiycan s y n t h e s i s in c o n s t r i c t i n g cells. S t e a d y state c u l t u r e s o f M C 4 1 0 0 l y s A as well as its cell d i v i s i o n m u t a n t f t s Z w e r e pulse-labelled with 3 H - D a p a n d p r o cessed for EM-autoradiography ( W o l d r i n g h e t al., 1987 ; W i e n t j e s a n d N a n n i n g a , 1989). C o n s t r i c t i n g a n d n o n - c o n s t r i c t i n g cel]s were classified

s e p a r a t e l y . I n g e n e r a l , l a r g e r cells c o n t a i n e d m o r e silver grains (fig. 1), w h i c h a g a i n i n d i c a t e s t h a t cells i n c r e a s e their rate o f peptidoglycan synthesis during the life cycle. A l t h o u g h there was a large v a r i a t i o n m t h e n u m b e r o f sliver g r a i n s p e r cell, it w a s o b s e r v e d t h a t c o n s t r i c t ing cells h a d i n c o r p o r a t e d in a statistically significant way more 3 H - D a p t h a n n o n - c o n s t r i c t i n g cells o f t h e s a m e l e n g t h class (fig. 1, see W i e n t j e s a n d N a n n i n g a , 1989). Analysis o f the distribution o f the g r a i n s o v e r t h e wild t y p e a n d m u t a n t cells s h o w e d t h a t the e n h a n c e m e n t o f Dap incorporation during constriction was coupled with a strongly increased i n c o r p o r a t i o n a t t h e c o n s t r i c t i o n site, w h e r e a s , the i n c o r p o r a t i o n in t h e lateral walls d e c r e a s e d s i m u l t a n e o u s l y (fig. 2~0 O u r results w i t h wild t y p e a n d f t s Z m u t a n t s s h o w that a topological switch o c c u r s in s u r f a c e s y n t h e s i s a t t h e o n s e t of constrictiov in E. coil ceils ( W o l d r i n g h et a L , 1987 ; W i e n t j e s a n d N a r . n i n g a , 1989). A s a c o n s e q u e n c e o f this switch, l a t e r a l - w a l l p e p t i d o g l y c a n synthesis d r o p s b y a p p r o x a m a t e l y 40 % .

till

O

0

05

1.0

N O R M A L I Z E D CELL LENGTH

FIG 2. - - Co,nparlson o f the topography o f ~H-Dap mcorporatron m constricting (--J and n o n - c o n s t r w t m g (---) cells o f F,. coil MC4JO0 lysA Labelhng conditions were as described by Wientjes and N a n a m g a (1989). Gram distributions over the cells are plotted as gram densities (number of grams per micrometer o f cell length) vs. normalized cell length. For deeply constricting cells, 399 grains were counted on 23 ceils and average cell dimensions were 2.64 by 0 75 ~m. Cells are positioned with their longer half to the left. For non-constricting cells, 867 grains were counted on 94 cells and the ~vcrage cell dimensions were I 72 by 0.75 tim (From Wlerttjes altd Nanmnga, 1989).

CELL

SHAPE

AND

T h i s f i g u r e is b a s e d o n a c o m p a r i s o n o f t h e a v e r a g e n u m b e r o f s~lver g r a i n s / t a m ceil l e n g t h in t h e l a t e r a l w a i l s o f n o n c o n s t r i c t i n g a n d c o n s t r i c t i n g cells, r e s p e c t i v e l ~ (W~entjes a n d N a n n i n g a , 1989). F o r t h e e x p e r i m e n t s h o w n m f i g u r e 2, t h e s e n u m b e r s w e r e 5.4 a n d 3.1 g r a i n s p e r ~ m , r e s p e c t i v e l y . T h e abruptness of the switch might be a m a t t e r o f d i s c u s s i o n (of. C o o p e r , 1988, a n d C o o p e r , 1990). A s a r g u e d h y Cooper, the transition from lateral g r o w t h t o g r o w t h o f t h e d i v i s i o n site is s m o o t h . T h e a b r u p t n e s s o b s e r v e d in autoradiographic experiments would be c a u s e d b y a p o p u l a t i o n o f d i v i d i n g ceils with a width larger than non-dividing cells o f t h e s a m e l e n g t h . T h i s calls f o r a c c u r a t 0 m e a s u r e m e n t s o f cell w i d t h during the division process. The m e a s u r e m e n t s d o n e s o f a r b y u s inuicate t h a t d i v i d i n g cells a r e n o t w i d e r t h a n n o n - d i v i d i n g cells ; t h u s , C o o p e r ' s s u g g e s t i o n will p r o b a b l y n o t b e c o n f i r m e d by experimental data. One might s p e c u l a t e t h a t a n a b r u p t switch ~s related to the speed by which nucleoids separate (see b e l o w ) .

T h e s w i t c h a n d cell g e o m e t r y . P r e v i o u s l y , it h a s b e e n s h o w n h o w geometric considerations can elucidate what to expect with respect to surface synthesis during division (Woldrmgh et al., 1985). Two situations were presented to illustrate what might happ e n d u r i n g cell g r o w t h i f t h e s u r f a c e (A) to v o l u m e (V) r a t i o o f n e w b o r n cells h a s to be restored to the original value after divasion. I n o n e c a s e , t h e A / V r a t i o rem a i n s c o n s t a n t d u r i n g cell e l o n g a t i o n . T h i s c a n b e a c h i e v e d if cells d e c r e a s e their m e a n width during elongation a n d i n c r e a s e their w i d t h d u r i n g c o n s t r i c t i o n . E . c o l i B / r ceils g r o w n in m i n i m a l m e d i u m a p p r o x i m a t e t h i s s~tuation ( T r u e b a a n d W o l d r m g h , 1980). I n t h e case of the K12 stra;n MC4100 lysA, m e a n cell w i d t h s e e m s t o c h a n g e less d u r i n g t h e cell cycle ( o u r u n p u b h s h e d o b s e r v a t i o n s ; h o w e v e r , see b e l o w ) . Here the A/V ratio probabIy decreases during elongation and thus the A/V r a t i o o f t h e n e w b o r n cells h a s t o m crease during the d~wsion process

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( W o l d r i n g h et al., 1985). It is n o t k n o w n w h e t h e r t h e t w o possibilities e x c l u d e each other. Presumably, intermediate s t t u a t i o n s o c c u r . N e v e r t h e l e s s , cell e l o n g a t i o n w~th co,~stant w i d t h s e e m s to fit w i t h t h e e x p e r i m e n t s with E. co,'i K 1 2 M C 4 1 0 0 l y s A (fig. 1). In f i g u r e 1 it c a n be appreciated how extra surface accumulates during polar cap formation ( b l a c k a r e a in t h e hatclaed p o l a r c a p s ) . A s m e n t i o n e d b e f o r e , thin hag b e e n c o n firmed by autoradlography of Dap p u l s e - l a b e l l e d cell~ ( W o l d r i n g h el a L , 1987 ; W i e n t j e s a n d N a n n i n g a , 1989).

Topographical differentiation qhe p o l a r c a p s .

within

A p r o b l e m m a u t o r a d i o g r a p h y is t h a t e l e c t r o n s m a y t r a v e l s o m e ~,J~tanee b e f o r e h i t t i n g a n A g B r ery,~.=.', a n d silver g r a m s a r e t h e r e f o r e u s ' l a l l y lateral!y displaced from the place of the o r i g i n a l d m i n t e g r a t i o n . T h i s limits t h e accuracy with wluch radioaettve decay can be localized. The resolution of EM-autoradiography o f 3 H - l a b e l in b a c t e r i a l cell w a l l s h a s b e e n e s t i m a t e d t o b e a b o u t 0.2 Izm ( K o c h et a L , 1982). The peak of 3H-Dap incorporation f o u n d in c o n s t r t c t i n g cells c o u l d t h u s b e c a u s e d b y a relatively n a r r o w z o n e o f inc o r p o r a t i o n . W~- s h o w e d t h a t t h i s a c t u a t l y is t h e c a s e ( W ~ e n t j e s a n d N a n n i n g a ~ 1989) b y a c o m p a r i s o n o f 3 H - D e p :c-q~f,or~tion~ in cells w i t h slight, ,nedluc/~ a n d d e e p c o n s t r i c l a o n s (fig. 3). T h r o u g h o u t t h e c o n s t r i c t i o n process, the incorporation pattern did not change. The constant width at the b a s e o f t h e p e a k (0.4 Ixm) i n d i c a t e s t h a t the surface synthesis at the c-qstriction site w a s a t a n a r r o w z o n e f r o m t h e beginning of constriction and remmned that way during constriction. The fact that the two nascent polar caps of deeply c o n s t r i c t i n g cells w e r e l a r g e r t h a n t h e width of the incorporation ~eak also p o i n t s in t h a t direetLon. M o r e o v e r , t h e acttwty remained high throughout the constriction process, considering the constant height of the Dap incorporat i o n p e a k at t h e cell c e n t r e d u r i n g p r o -gressive stages of constriction. We therefore concluded that the enhanced p e p t ~ d o g l y c a n s y n t h e s i s in t h e c o n s t r i c -

108

4 t'~ F O R U M I N M I C R O B I O L O G Y is s t r u c t u r a l l y f i x e d c l o s e t o i t s s u b s t r a t e : a u r e i n ' " ( S c h w a r z et aL, 1969; see a l s o Hoffman et aL, 1972). All these observations at the leading edge seem compatible with a finite zonal growth model (cf. Koch, 1990).

t t o n a r e a is n o t e v e n l y d i s t r i b u t e d o v e r t i l l s a r e a b u t is m a i n l y c o n f i n e d t o t h e leading edge of the invaginating cell envelope (Wientjes and Nanninga, 1989). This central area might ultrastructurally be reminiscent of the membra~e-murein attachment site at the leading edge of the invagmating cell envelope s e e n ira t h i n s e c t i o n s o f plasmolysed 3almonella typhtmurium ( M a c A h s t e r e t a l , 1987). T h e s h a r p c u t visible in the center of saccuh derived from penicilhn-treated dividing cells a l s o s u g g e s t s 4~ 0 ].[ e n y.y~ne s y s t e m w h i c h

Muropeptide composition and mode insertion.

of

Since the switch in peptidoglyc=_.n synthesis probably involves a change in e n z y n ~ e a c t i v i t i e s , it ts c o n c e i v a b l e t h a t

" ' ~ - - _ _ ~

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G=l140 [ =~ t.3~m

20

n,-

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C

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1

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2

CELL LENGIH (pro)

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FIG+ 3. - - S d v e r grctm dtstrtbulton o v e r E. c o h M C 4 1 0 0 l y s A cells tn progressJve stages o f constr~ctLon (cells with s h g h t (A). m e d i u m (B), a n d deep (C) constrictions). G r a m d t s t r z b u t t o n s a r e p l o t t e d as g r a m d e n s i t i e s vs. cell length. C e l l s are p o s i t i o n e d w i t h t h e l o n g ~ cell h a l f to the left D r a w i n g s o f t h e cells o f d i f f e r e n t l e n g t h classes a r e s h o w n a b o v e the d i s t r l b u t l o n s N u m b e r o f cells (N) a n d g r a m s ( G ) , as well as a v e r a g e length o f t h e cells (L), a r e given ( F r o m W m n t j e s a n d N a n n m g a , 1989).

CELL

SHAPE

AND

DIVISION

IN E. COLI

109

d e c r e a s e in d i s a c c h a r i d ¢ t e t r a p e p t i d e (the main monomenc compound) d u r i n g c o n s t n c t m n . T h i s i n c r e a s e in radmactiwty m Tet-Tet during constrict i o n h a s b e e n f o u n d b e f o r e ( O H j h o e k et aL, 1982) a n d it w a s t h e n c o n c l u d e d t h a t the constriction process involved the formation of higher cross-linked peptidoglycan. We now found, however, t h a t it c a n b e e x p l a i n e d b y a n i n c r e a s e in t h e a c c e p t o r - d o n o r r a d i o a c t i v i t y r a t i o ( A D R R ) (de J o n g e et al., 1989). T h e ADRR was determined by dansylation of the Tet-Tct compound isolated by H P L C ( G l a u n e r a n d S c h w a r z , 1987), a n d a p p e a r e d t o rise in p r o p o r t i o n t o t h e p e r c e n t a g e o f c o n s t r i c t i n g cells in t h e c u l t u r e (fig. 4). T h i s i n d i c a t e d t o u s t h a t o n l y o n e d i s a c c h a r i d e u n i t in t h e d i m e r is r a d i o a c t i v e during elongetion, w h e r e a s d u r i n g c o n s t r i c t i o n , b o t h disacc h a r i d e u n i t s a r e latmUed, r e s u l t i n g in a n i n c r e a s e d a m o u n t o f r a d i o a c t i v i t y in this c o m p o u n d in t h e l a t t e r c a s e . T h e r e a l a m o u n t o f T e t - T e t in t h e d i f f e r e n t samples could then be calculated. For this c a l c u l a t i o n , n o t o n l y t h e p e r c e n t a g e r a d i o a c t i v i t y in T e t - T c t , a n d t h e A D R R h a v e to b e k n o w n , b u t also t h e c o n t r i b u t i o n o f non-Iabellcgt p r e c u r s o r t o t h e

the chemical composition of the pept i d o g l y c a n c h a n g e s in t h e c o u r s e o f t h e cell cycle. T h i s q u e s t i o n w a s a d d r e s s e " b y de j o n g e e t aL (1989) b y H P v "~ (high-performance hquid chromatography) analysis of peptidoglycan synthesized during elongation and c o n s t r i c t i o n , respectively. S y n c h r o n i z e d cultures of MC4100 were pulse-labelled w i t h a H - D a p at d i f f e r e n t t i m e s in t h e cell cycle, t h e p e p t i d o g l y c a n was isolated, digested with muramidase and analysed by HPLC according to published procedures (Glauner, PhD. thesis, U n i v e r s i t y o f T u b i n g e n , F R G , 1986; D r i e h u i s a n d W o u t e r s , 1987; G l a u n e r e t a L , 1988). T h e s a m e set o f muropeptides was found during elongation and constriction with some minor c h a n g e s in t h e i r r e l a t i v e a m o u n t s . O v e r a l l p a r a m e t e r s s u c h as c r o s s - l i n k a g e ( D a p - A l a as well as D a p - D a p ) , l i p o p r o tein content and chain length ( l , 6 - a n h y d r o m u r a m y l r e s i d u e s ) a l s o remained constant. The only difference that was consistently observed was an a p p a r e n t i n c r e a s e in t h e a m o u n t o f inc o r p o r a t e d 3 H - D a D in b i s - d i s a c c h a r i d e tetra-tetra (Tet-Tet, the main dimenc compound) and a corresponding

PhDRR in Tet-Tet O~ 04 03

o

02

01 tO

FIG. 4. f

20

40 60 ~, c o n s t r l c l e d c e i l s

80

Correlation o f the A D R R in b~s-dtsacchartde tetratetra c o m p o u n d (Tet-TeO and the percentage o f constricting cells m E coil MC4100 lysA.

Values were obtained frown synchronously growing cells (closed circles) or f r o m an exponentially growing culture (open c~rcles). (From de Jonge et aL, 1989).

110

4 th F O R U M

IN MICROBIOLOGY

peptidoglycan synthesis. E n d o g e n o u ~ s y n t h e s i s o f D a p in M C 4 1 0 0 w a s r e s p o n s i b l e f o r a b o u t 50 % o f t h e i n c o r porated Dap.under the conditions used m o u r e x p e r i m e n t s . T h e r e s u l t o f this c a l c u l a t i o n , g.iven m the append~_x o f o u r r e c e n t m u r e m c o m p o s i t i o n p a p e r (de J o n g e e t a L , 1989), s h o w e d t h a t t h e cross-hnk percentage based on the amount of the predominant dimer was a p p r o x i m a t e l y c o n s t a n t d u r i n g t h e cell cycle. Another consequence of the variable A D R R is t h e d i f f e r e n t m o d e o f p e p t ~ d o g l y c a n c},ain i n s e r t i o n d u r i n g e l o n g a t i o n a n d ,constriction. It is h i g h l y p r o b a b l e that d o n o r s are only derived from newly inserted peptidoglycan, since m a t u r e p e p t i d o g l y c a n h a r d I y c o n t a m s p e n t a p e p t ~ d e side c h a i n s (de P e d r o a n d S c h w a r z , 1981; G l a u n e r , P h D . thesis, U m v e r s l t y o f T u b i n g e n , F R O , 1986). T h e r e f o r e , as p o i n t e d o u t b y others (Burnmn and Park, 1984; C o o p e r e t a l , 1988; K o c h , 1988), a l o w ADRR indicates insertion of pept J O o g l y c a n as single s t r a n d s , w h e r e a s higher ADER would mean doubles t r a n d e d ( c f . B u r m a n a n d P a r k , 1984)

ELONGATION

4/

or even multi-stranded i n c o r p o r a t i o n . E x t l a p o l a _ i o n o f o u r A D R R v a l u e s to 0 a n d 100 % c o n s t r i c t i n g cells in f i g u r e 4, lead t o A D R R o f 0 . 1 0 a n d 0.65~ respectively (de J o n g e e t a L , 1989). Presumably, the observation o f B u r m a n a n d P a r k (1984), w h o r e p o r t e d a n ADRR in e x p o n e n t i a l l y growing c u l t u r e s o f 0 . 2 5 , h a s t o be i n t e r p r e t e d as a m i x e d r e s u l t o f s i n g l e - s t r a n d e d ins e r t i o n in e l o n g a t i n g cells a n d m u l t i - ( o r fast s i n g l e ) - s t r a n d e d i n s e r t i o n a t t h e c o n s t r i c t i o n site m d i v i d i n g cells ( c f . K o c h , 1 9 8 8 ; see al~o H c i l t j e a n d (31auner, 1990}. T h e h i g h A D R R in c o n s t r i c t i n g cells s u p p o r t s t h e c o n c e p t o f a leading edge i.e. a highly c o n c e n t r a t e d w a y o f p e p t i d o g l y c a n synthesis at t h e r e w a r d g r o w m g e d g e o f t h e inv a g i n a t i n g cell e n v e l o p e . T h e n e w l y ins e r t e d d i s a c c h a t i d e u n i t s at t h e l e a d i n g e d g e a r e u s e d v e r y q u i c k l y as a c e e p t o r s in a c r o s s - l i n k a g e , e i t h e r b y s i m u l t a n e o u s i n s e r t i o n o f several g l y c a n c h a m s (multi-stranded insertion), or by immediate cross-linkage of new strands to s t r a n d s i n c o r F o r a t e d j u s t b e f o r e . This has b e e n s c h e m a t i c a l l y d e p i c t e d in f i g u r e 5.

; CONS~TE©N, /

,-,

DIRECTION OF GROWTH

FIo 5 - - A4odel f o r g r o w t h o f t h e p e p t t d o g l y c a n lave t h e cell cycle,

o f E. coil In t h e d z f f e r e n t s t a g e s o f

During elongation, newly synthesized peptidoglycan (thick dashed hne) ts inserted diffusely as single strands in the existing layer (thick unbroken hnes). During dlvlmon, newly synthesized peptidoglycan is mserted at the constriction site, sequential smgle-stranded or multistranded at the leading edge. Cross-hnkmg might be eatalysed by different PBP (A, V , 0 ) .

CELL

SHAPE

AND

E n z y m a t i c d i f f e r e n t i a t i o n in p o l a r c a p formation. Since t h e t o p o l o g i c a l switch involves a strong activation of peptidoglycan s y n t h e s i s m t h e cell c e n t r e a n d a d e c r e a s e m t h e lateral wall, it is c o n eeIvable t h a t t h e s w i t c h p r o c e e d s via activation/deactivation of enzymes. This m i g h t o c c u r b y local stimulation o f a general peptidoglycan synthesizang enz y m e at a p a r t i c u l a r site. A l t e r n a t i v e l y , specific e n z y m e s n u g h t b e i n v o l v e d f o r either elongating or constricting synt h e t i c activities. O r , o n e m a y a s s u m e t h a t t h e r e is a g e n e r a l p e p t i d o g l y e a n s y n t h e s i z i n g a c t i v i t y o n w h i c h specific e n z y r n a t ' e activities a r e s u p e r i m p o s e d . F r o m studies w i t h m u t a t e d penicillinbinding proteins (PBP) and with specific [3-1actam a n t i b i o t i c s it is well k n o w n t h a t P B P h a v e specific r o l e s In peptzdoglyean metaoolism (Spratt, 1 9 7 5 ; M a t s u h a s h i et al., 1981; M a t s u h a s h i et aL, 1990). M u t a n t s w i t h a t e m p e r a t u r e - l a b i l e P B P 3 (pbpB, f t s l o r sep) a r e u n a b l e t o d i v i d e at t h e restrictive t e m p e r a ' u r e ( S p r a t t , 1977; D o n a c h i e a n d B e g g , 1990). A l s o , a n tibiotics w h i c h b i n d e x c l u s i v e l y to PBP3, such a s c e p h a l e ~in a n d f u r a z l o e i l l i n , p r e v e n t cell d i v ~ t o n a n d c a u s e t h e cells t o g r o w as f i l a m e n t s . PBP3 was also reported to have pept i d o g l y c a n - s y n t h e s t z i n g activities (both transglycosylase and transpeptidase) in vitro ( I s h m o a n d M a t s u h a s h i , 1981). B o t t a a n d P a r k (1981) o b t a i n e d evidence using such mutants and antibiotics that PBP3 is a c t i v e as peptidoglycan-synt hesizing enzyme d u n n g c o n s t r i c t i o n o n l y . W e c o n f n reed a n d e x t e n d e d this o b s e r v a t i o n b y inhibition of 3H-Dap incorporation by c e p h a l e x i n a n d f u r a z l o c d h n at d i f f e r e n t stages in t h e cell c y c l e I n e x p o n e n t i a l l y g r o w i n g M C 4 1 0 0 l.vsA cultures, D a p m corporatior~ was inhibited by f u r a z l o c i l l i n b y a b o u t 35 °70 (fag. 6 A ) . T h e s a m e result w a s o b t a i n e d w i t h cephalexin (not shown). We might consider this to be t h e c o n t r i b u t i o n o f P B P 3 to general p e p d d o g l y c a n synthesis. However, m s y n c h r o n o u s cultures, the inhibition d e p e n d e d on the stage o f the cell cycle, t.e. n o m e a s u r a b l e i n h i b i t i o n d u r i n g e l o n g a t i o n , a n d a s t r o n g ~r.hib~-

DIVISION

IN E. COLI

t,on (50-60 % ) (fig, 6B),

during

111 constriction

T h e s e results s u g g e s t t h a t the activit y o f P B P 3 is switched o n at a p a r t i c u l a r t i m e m t h e cell cycle. W e h a v e s h o w n b e f o r e t h a t P B P 3 is c o n t i n u o u s l y pres e n t d u r i n g the cell cycle ( W i e n t j e s et al., 1983)o O t h e r e x p e r i m e n t s r e v e a l e d t h a t P B P 3 ts n o t i n v o l v e d in t h e initiat i o n o f c o n s t r i c t i o n b u t m e r e l y in t h e c o n t i n u a t i o n o f it. S u c h i n d i c a t i o n s came from genetic and morphologqcal studies on different ce!l-diviston m u t a n t s ( W a l k e r et ~1., 1975 ; B c g g a n d D o n a c h i e , 1985; T a s c h n e r et aL, 1988). It w a s s h o w n t h a t m u t a t i o n s in t h e f t s Z g e n e r e s u l t e d in f i l a m e n t s w i t h o u t visible c o n s t r i c t i o n s , w h e r e a s f t s A , risE, f t s Q a n d p b p B m u t a n t s did initiate t h e constricuon process Begg and D o n a c h i e ~1985) conclL=led t h a t the diff e r e n t gene p r o d u c t s m i g h t act in seq u e n c e w i t h F t s Z a c t i n g first, f o l l o w e d b y F t s Q a n d P B P 3 , a n d F t s A be,.ng req u i r e d f o r t h e latest stages in c o n s t r i c t i o n . T a s c h n e r et aL (1988), a f t e r a detailed morphological study, concluded an order FtsZ-FtsQ-FtsA.PBP3. T o p o g r a p h i c a l .~tudaes h a v e also s h o w n t h a t a clear-cut d i s t i n c t i o n c a n be made between imtlation and continuation of constriction. Autoradiography offtsZ filaments that had incorporated 3H-Dap showed a random distribution o f silver g r a m s with n o s~gn o f a n inc r e a s e d i n c o r p o r a t i o n at a specific place o n t h e e n v e l o p e ( W o l d r i n g h et aL, 1987) B y c o n t r a s t , f i l a m e n t s o f t h e pbpB mutant, whether reduced by a t e m p e r a t u r e shift o r b y furazlocillin, did show initiated constrictions with localized i n c r e a s e d i n c o r p o r a t i o n ( W i e n t j o s a n d N a n n i n g a , 1989). T h e s a m e result was o b t a i n e d w i t h f t s 4 g r o w i n g at t h e restrictive t e m p e r a t u r e ( W i e n t j e s a n d N a n n l n g a , u n p u b l i s h e d results). Is t h e a c t i v i t y o I P B P 3 a f f e c t e d b y d i v i s i o n g e n e p r o d u c t s s u c h as F t s A , F t s Q a n d F t s Z ? T o s t u d y a p o s s i b l e int e r a c t i o n o f P B P 3 with t h e g e n e p r o ducts of ftsA, f t s Q and ftsZ, Dap i n c o r p o r a t i o n w a s m e a s u r e d at t h e perm~ssive a n d n o n - p e r m i s s i v e t e m p e r a t u r e as well as in the presence o f furazlocillin (Wtentles and N a n m n g a , manuscript m

1 12

4 th F O R U M

IN MICROBIOLOGY

preparation). As mentmned above, the tn w v o p e p t l d o g l y c a n - s y n t h c s i z m g activity of PBP3 can be measured ind i r e c t l y , I.e. a s i n h i b i t i o n o f 3 I - I - D a p i n c o r p o r a t x o n b y furazlocillin (fig. 6 A ) o r b y c e p h a l e x i n ( n o t s h o w n ) . Tl~e speeifJcity t o w a r d s P B P 3 was s h o w n by performing the same experiment with the pbpB mutant of Me4100 lysA (fig. 7 A ) . 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 , no inhibition of Dap incorporation was observed, whereas at the permissive temperature, the Duo incorporation was inhibited to some extent. This inhibition was less than in the wild type, presumably because of a shght ~mpmrment of PBP3 already at the permissive t e m p e r a t u r e . A l s o in t h e f t s A , f t s Q a n d f t s Z mutants, no inhibition of Dap incorporation was observed at the restrict i v e t e m p e r a t u r e ( s h o w n i n f i g . 7B f o r f l s A ) , i n d i c a t i n g t h a t in t h e s e c a s e s PBP3 was not active. We conclude that P B P 3 n o t o n l y n e e d s i n t a c t F t s A f o r Rs a c t i v i t y ( T o r m o e t at,., 1986), b u t a l s o FtsQ and FtsZ (unpublished observatmns). The envA gene product has been p o s t u l a t e d t o a c t in t h e f i n a l s t a g e o f c o n s t r i c t i o n , t.e. m c e l l s e p a r a t m n (Wolf-Watz and Normark, 1976). Whether EnvA interacts with PBP3 or a n o t h e r p e n i c i l h n - b i n d i n g p r o t e i n ts n o t known. Another protein (MreB) which m i g h t b e i n v o l v e d in t h e r e g u l a t i o n o f the actlvmes of PBP has been described b y W a e h ~ et al. (1987). I n p a r t i c u l a r because mreB gene expression seemed

to be related to pbpB expression (Wachi anrd M a t s u h a s h i , 1989), it w a s s u g g e s t e d that the MreB protein functions t o g e t h e r w i t h F t s A a s r e g u l a t o r s in cell d i v i s i o n a n d c e l l e l o n g a t i o n ( D o i et a l , 1988 ; M a t s u h a s h i e t al., 1990). T h e s e proteins, which have homology with s o m e p r o t e i n k m a s e s m a y very well s e r v e as s w i t c h e s i n t h e E. colt cell d i v i s i o n m a c h i n e r y (cf. a l s o D o n a c h i e a n d B e g g , k990). The question remains whether there a r e still m o r e p r o t e i n s i n v o l v e d i n constrictmn. Inhibition of PBPIA/IB b y s p e c i f i c a n t i b i o t i c s s u c h as c e f s u l o d m l e a d s t o lysis o f t h e cells, a n d xt w a s s u g gested t h a t these e n z y m e s are m a i n l y inv o l v e d in p e p t i d o g l y c a n s y n t h e s i s d u r i n g e l o n g a t i o n ( S p r a t t , 1975). O n t h e o t h e r h a n d , ;.n ceils s t a r t i n g r e g r o w t h o u t o f the stationary phase, PBP1A/IB seem t o b e i n v o l v e d in cell p r o l i f e r a t i o n ( d e i a R o s a e t aL, 1985). A l t h o u g h t h e l a t ter results were not obtained with exponentmlly growing cultures, our results also implicate PBPIA/IB in the constriction process. We found that a u t o l y s i s sites i n d u c e d b y e e f s u l o d i n a n d w s u a l i z e d as vesicles o n t h e cell e n v e l o p e w e r e l o c a t e d in t h e cell c e n t r e in d i v i d i n g cells a s w e l l as i n r e l a t i v e l y l o n g u n d i v i d i n g cells ( W i v n t j e s a n d N a n n i n g a , u n p u b h s h e d r e s u l t s ) . H o w e v e r , so f a r , we have not found a clue to which antibiotics specifically inhibit the mureinsynthesmmg activity needed for initiation o f c o n s t r i c t i o n (see below).

FIG 6 - - [nhtbttJon o f 3l-l-Dap i~corporation by furazloeillitt 3 H - D a p was a d d e d to cultures o f M C 4 1 0 0 / y s A (10 gtgCl/ml) a n d at subsequent ~lmes, 50-~1 ahquots were removed and added to 3 ml o f 10 °?0 Ice-cold T C A . The incorporated radioactivity was measured by filtration over 0 2-tzm mlltipore f l~ers and counting m a liquid scmnllat~on counter. T h e r a d l o a c t ] w t y is piotted against 2 ~,' L O -- 1 to facilitate d e t e r m i n a t i o n o f steady-state m c o r p o r a t m n ( W i e n q e s et a l , 1985). Rate o f pept~doglycan synthes~s ts p r o p o r t i o n a l to the slope o f the line A) Steady-state chlture o f MC4100 lysA m minimal citrate m e d m m wtth glucose, 28°C (O) and 37°C ([3). I n h i b i t i o n by furazlocillia (2 fxg/ml) was 37 070 at 28°C (©), as well as at 37°C (x) The same result was o b t a i n e d with cephalexin (10 g g / m l ) . B) A synchronous culture (obtained by selecting small cells by centrifugal elutnauon) was grown at 28°C ( m a s s - d o u b h n g t~me ca 70 ram) At different times, subcultures were incubated with 3 H - D a p m the presence (x) or absence ( 0 ) o f furazlocillin.

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3H-Dap incorporaUon was measured as in figure 6. A) The p b p B m u t a n t was grown at 28oC with (C)) and without ( 0 ) and at 37°C with (x) and without (El) furazlocillin (2 ttg/m[). B) T h e f t s A m u t a n t (same symbols).

A jump-switch m e c h a n i s m ? I n this s e c t i o n , w e will d i s c u s s whether the presumed abruptness of the switch can be related to another abrupt e v e n t . F u r t h e r m o r e , w e will i n d i c a t e possibilities for the kind of PBP3independent peptidoglycan synthesis needed for the imtiation of constriction. Some years ago, Sargent (Sargent, 1974) p r e s e n t e d w h a t c a m e t o b e k n o w n as t h e j u m p i n g n u c l e o i d ( a l s o q u o t e d b y H o l l a n d e t a l . , 1990). T h e b a s i c (hght microscopic) observation was t h a t , in B a c i l l u s s u b t i h s , n o s t r u c t u r e o f intermediate length was observed between one nucleoid body and two segregated nucleoid bodies. It might, however, be argued that the fixationstaining technique which had been employed could have distorted int e r m e d i a t e s t r u c t u r e s in s u c h a w a y t h a t either separate or compact bodies resulted. Woldringh (PhD. theszs, Untversity of Amsterdam, 1974)

independently measured the length of t h e n u c l e o i d in e l e c t r o n r m c r o s c o p i c t h i n sections of non-dzviding and dividing cells. I n g l y c e r o l - g r o w n E. c o i l cells w i t h elonga te d rod-like nucleozds, the length of the nucleoid (nucleoplasm) increased in step w i t h l e n g t h e x t e n s i o n . I n g l u c o s e g r o w n cells ( c o n v o l u t e d n u c l e o i d s ) , t h i s could not be reliably measured. In d i v i d i n g cells, n o i n d i c a t i o n f o r a j u m p in nucleoid segregation was noticed. However, the sample was small and the technique employed (electron microsc o p i c serial sectzony p r e v e n t e d l a r g e scale sampling. Electron microscopic observations with similar results have been made by Burdett and co-workers o n B. s u b t i h s 0Burdett e t a l . , 1986). T h e electron microscopic data do not necessarily contradict a jumping mode o t n u c l e o i d s e g r e g a t i o n . T h e a n a l y s e s in both cases (Woldringh, PhD. thesis, University of Amsterdam, 1974; B u r d e t t e t a L , 1986) h a v e b e e n b a s e d o n length measurements of nucleoid

CELL

SHAPE

AND

bodies. Presumably the nucleoid shape has to be taken into account as well. Jumping to new positions in the cell could occur with the main part o f the nucleoid body with some trailing behind o f the terminating part. More specific data are, to the best o f our knowledge, not yet available. In summary, then, nucieoid jumping remains a possibility, but clearly needs considerably more corroboration. We refer to these older observations because we detected a coincidence of nucleoid segregation and a change in cell shape. Recently, we found that premature division in EDTA-treated Tris-grown cells was not only accompanied by a change in cell shape (cells becoming longer and thinner; Nanninga et al., 1985), but also premature nucleoid segregation occurred (Wientjes and Nanninga, manuscript in l~reparation). P r o m p t e d by the attentlon that Cooper paid to cell width (Cooper, 1990), we again looked at cell diameter o f dividing and non-dividing ceils. Though the fluctuations were large, we gained the impression that dividing cells were very slightly thinner than nondividing cells. For this reason, we wondered whether what we observe in EDTA-treated cells concerning nucleoid segregation also occurs under normal c i r c u m s t a n c e s . T h a t is, n u c l e o i d segregation would be accompanied by a change in celt shape, though in a less exaggerated way than under artificial cir~tmstances in the presence o f EDTA. T h e p o s t u l a t e d n u c l e o i d segregation/cell shape change mechanism might be mediated b y contractile proteins (cf. Holland e t aL, 1990), mechanocnzymes a n d / o r an overall weakening o f the cell envelope. Such weakening o f the cell envelope might be reflected in the reduction o f P a p incorporation in the lateral wall o f dividing cells (fig. 2). This idea is not formally in conflict with the nucleoid-occlusion model (cf. Woldringh et al., 1990); it only attempts to rel~tte seemingly abrupt phenomena.

DIVISION

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115

The next question to consider is whether a clue can b¢ found with respect to the pepttdoglycan-synthesizingactivity at initiation o f division. PBP3 does not appear to be involved (see above). So one should probably look for penicillin-insensitive enzymes. Is the postulated LD-transpeptidase, which could make Dap-Dap crossbridgcs, a likely c a n d i d a t e ? In synchronized cultures no significant increase in this type o f cross-bridge was observed in constricting cells (de Jonge et aL, 1989). However, since the very initial synthesis is only part o f polar cap formation, a local enrichment o f Dap-Dap bridges might be difficult to demonstrate, It should be noted that Dap-Dap bridges become especially prominent in the sturdy, stationary phase cell wails (cf. H61tje and "31auner, 1990 and references thereiny. One can imagine that peptidoglycan synthesized in the very beginning o f constriction also has to be extra-strong. Another candidate to consider might be the novel gtycan polymerase described by H a r a and S u z u k i (1984), o r p e r h a p s , t h e peptidoglycan-synthesizing activity o f the m u r H gene product (Dai and Jshiguro, 1988). In all these cases, a direct or indirect interaction with FtsZ is to be expected. This cali~ for more knowledge about the interaction o f various division gene products and other proteins with peptidogiycan synthesizing and hydrolysing enzymes (cf. Matsuhashi et al., 1990, and Donachie and Begg, 1990). We already mentioned the possible relationship o f PBP3 with f t s A , Q and Z gene products (fig. 7). Recently, we detected, with antibodies against PBP1B (MW 90 kDa), that this protein occurs in a highmolecular weight complex (140 kDa) under soft lysis conditions (Zijderveld et al., ~ vmanuscript in preparation). The main ta~k will be to identify various protein complexes and their composing parts.

116

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References.

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fins work was supported by the Foundation for Fundamental Blo~oglcalResearch (BION), which is subs~dized by the Netherlands Organization for Scient~hcResearch (NWO) We lhank Mrs. E. Lulz-Langezaal and Mr J D Leutscher for their help m pieparmg the text and figures, resp~ctive|y