FEMS MicrobiologyLetters 95 ( 19921241-246 ~'~ 1992 Federation of European MicrobiologicalSocieties 0378-1097/92/$115.1~) Published by Elsevier

FEMSLE 051106

Membrane-protein phosphorylation in the Bacillus subtilis cell cycle Naomi Sandier and Alex Keynan Department of Biological C'hcmistry. The Alexander Silberman hrstitt,te of Lil't" Sciences, The Hebrew Unit'ersity of Jerusalem. Jerusalem. Israel

Received 5 February 1992 Revision received 14 May 1992 Accepted 21 May 1992 Key words: Bacillus; Membrane; Phosphorylation: Cell cycle 1. S U M M A R Y Sphingosine, an inhibitor of Ca-'+-dependent protein kinases in eukaryotic cells, inhibited initiation of D N A replication in Bacillus subtilis at a concentration of 10 p.M, without inhibiting elongation. The tumor promoter 12-tetradecanoyl 13phorbol acetate, (TPA), an activator of protein kinase C in eukaryotic cells, partially counteracted the inhibition of initiation by sphingosine. Phosphorylation of polypeptides was observed in vivo at initiation of D N A replication in B. subtilis. Sphingosine, TPA, and vancomycin affected this protein phosphorylation.

2. I N T R O D U C T I O N Phosphorylation and dephosphorylation of proteins have recently been found to be basic Correspondence to: N. Sandier, Department of Biological

Chemistry, The Alexander Silberman Institute of Life Sciences. The Hebrew Universityof Jerusalem, Jerusalem 919114. Israel.

regulatory mechanisms in eukaryotic cells, and protein kinases reportedly trigger both the initiation of D N A replication and mitosis [2,8,10]. Protein phosphorylation has also been observed in bacteria. Phosphoproteins have been implicated in citrate metabolism in Escherichia colt [i] and in D N A replication in response to heat shock in this organism [17]. Sporulation-dependent phosphorylation of peptides was found in Bacillus subtilis [5,1 I] and in B. thuringiensis var. k u r s t a k i [15]. The known requirements for initiation of D N A replication at the origin of the B. subtilis chromosome include protein synthesis [6], an R N A synthesis step dependent on R N A polymerase [7], and, at least under certain conditions, ongoing synthesis of the cell wall [13]. Before initiation can occur in B. subtilis, D N A in the neighborhood of the replication •rigin must become attached to the cytoplasmic membrane [16]. Binding of D N A to a specific mentbraae subfraction has been found to be periodic, occurring prior to initiation of D N A replication in the B. subtilis cell cycle, and is prevented by inhibitors of protein or cell-wall synthesis including vancomycin [131.

242 Ylae present report describes several expcriments that were done to test the hypothesis that protein kinases also play a role in the control of bacterial replication, and specifically in initiation of D N A replication in B. subtilis. We looked for the occurrence of protein phosphorylation preceding and during initiation of D N A replication, and also for possible effects of a eukaryotic protein kinase C activator and inhibitor on such phosphorylation, and on initiation of D N A replication. It was observed that specific polypeptides were phosphorylated in vivo prior to and during induction of D N A replication in B. subtilis, and that the phosphorylation and also initiation of replication were affected by sphingosine, an inhibitor of eukaryotic protein kinase C and Ca -'+, calmodulin-dependent protein kinase [3,4]. The tumor promoter and activator of eukaryotic protein kinase C 12-tetradecanoyl 13-phorbol acetate (TPA) [9] also affected protein phosphorylation and the initiation of D N A replication.

3. M A T E R I A L S A N D M E T H O D S

3.1. Organism and growth conditions B. subtilis 168 trpC was used in all experiments. Synchronous growth and initiation of D N A replication were induced by transfer of stationary-phase ceils to fresh medium. The diluted cultures were incubated at 37°C.

the cells was measured immediately, and again after 120 min at 37°C. in this system, a doubling of the counts represents the completion of one round of D N A replication, while a flat curve appearing in contrast to a steep rise in the control implies prevention of initiation of a round of replication. Only the first one or two rounds of replication were observed in these experiments, due to the rapid loss of synchrony in the system by 45-60 min after dilution of a stationary-phase culture.

3.3. Deternzblation of protein phosphorylation For observation of protein phosphorylation in vivo, cells were grown overnight in casein amino acid medium containing un[abelled inorganic phosphate [14], after which the culture was filtered and the cells were resuspended in approximately 10 times the original volume of the medium containing 5% of the original concentration of phosphate and with the addition of carrier-free [3-' P]-Pi (5-h~/xCi m l - I). At 5-min intervals after dilution, 5-ml aliquots of the culture were filtered and suspended in a buffered solution containing sucrose and chloramphenicol. The samples were incubated with lysozyme (2 mg ml-~) at 37°C to form protoplasts, as described previously [13]. The protoplasts were ruptured by strong vortexing and the particulate (membrane) fraction was obtained by centrifugation for 5 min in a microcentrifuge. The particulate fraction was washed once with 1.0 ml buffer solution.

3.Z Measuremolt of bdtiation o f DNA replication For measurement of the number of chromosomes in a cell culture which had initiated replication, residual D N A synthesis was determined while further initiation was prevented by inhibition of protein synthesis. This method is based on the fact that protein synthesis is required for initiation of D N A replication but not for elongation [6]. The determination was done by transferring portions of the cultures to test tubes containing labelled thymidine (10 tzCi [3H]-thymidine ml -I or 1.4 or 2.8/.tCi [14C]thymidine ml - l ) and unlabelled uridine (250 p.g ml-~), as well as 100 /zg chloramphenicol m l - ~ (Sigma) to inhibit protein synthesis. The radioactivity associated with

4. R E S U L T S A N D DISCUSSION

4.1. Lf]'ect o f .~phbtgosble and TPA on #utiation of replication T P A at concentrations from 100 nM to 16/xM generally stimulated initiation of D N A replication, but the effect was slight (data not shown). Since intact cells presumably already contain the factors required for initiation and are provided with strict controls to prevent excess replication, it was considered worthwhile to assess the effect of sphingosine, which is a T P A antagonist in eukaryotic systems. It was found that sphingosine inhibited initiation of D N A replication at a con-

243 in T a b l e I. In t h e p r e s e n c e o f s p h i n g o s i n e , i n i t i a tion of DNA replication was prevented without affecting binding of DNA to the cell membrane (data not shown).

centration which did not inhibit chromosome elongation (Fig. la). TPA was able to counteract the inhibition of DNA replication by sphingosine t o s o m e e x t e n t , a s m a y b e s e e n in F i g . l b , c a n d

,

b

,

,

i

i

A.

13

I

I

I

I

v

al

b 2c

D 6O

i •

//

×

77 `/

E

o

50

u

IO'

n- 4 o v

30 0 - -

I

I0 Time

210 Time

(rain)

I

i

I

I

I

50

410

(min)

]

5O

0 ×

E 2O o.. i LO

,~ 25 5~ 4'o ~ 6'o Time

( m,n )

Fig. 1. Effect of sphingosine and TPA on initiation of DNA replication in B..subtili,s. A. Effect of sphingosine. D sphingosine in DMSO was added to a B. subtilis culture grown in brain-heart-infusion broth (Difco) at the time of dilution. At the indicated times after dilution aliquots were taken for the measurement of initiation of DNA replication. DMSO alone at the maximum final concentrations used had no effect on initiation. Open and filled symbols represent the results of two separate experiments, v . control ( . . . . ): ix. 2 # M sphingosine; c~, lit .o.M sphingosine: (5. 511 # M sphingosine. B, C. Effect of TPA and sphingosine. B. Initiation of DNA replication in a control culture was compared with initiation in cultures grown in the presence of sphingosine (411 #M), TPA (8 #M), vancomycin (3 # g ml - i) or TPA (S # M) and sphingosine i40 #M). v . controh ©, vancomycin: A. sphingosine: e, TPA: Lx, TPA and sphingosine. C, In an experiment similar to that represented in B. cultures contained sphingosine (20 #M). TPA (8 #M), or both sphingosinc (211# M ) and T P A (8 #M).

244 Table I Growth of freshlydilutedstationary.-phaseculturesin the presenceand absenceof TPA and sphingosinc OD~) immediately folh)wing dilution 0.79

TPA (.aM)

Sphingosine (21) .aM)

()D~,~,) after incubation at 37°C Ih 3h

-

2.3

8.4

1.6

2.4 2.5 2.4

9.4 8.2 8.8

8.1) 16 -

÷

1.11

4. I

1.6

+

I.I

s.()

+

1.3

+

0.9

5.8 6.9 4.5

lb

4.Z Protein plu~7~ltotylation dttring hzitiation of replication Phosphorylation of proteins in vivo was observed immediately on induction of growth in both the membrane and cytoplasmic cell fractions, although only in the membrane fraction were changes in the intensity of specific phosphorylated peptide bands observed in response to the presence of the various agents tested. The phosphorylation of membrane proteins on induction of growth by dilution of stationary-phase cultures is shown in Fig. 2a. After 5 min of incubation phosphorylated polypeptide bands were clearly seen in the 15-45 kDa range and also in higher molecular mass regions. An overall inhibitory effect on the appearance of phosphorylated membrane polypeptides was obvious when 40 /xM sphingosine was present in the cultures. Low concentrations of sphingosine (10 or 20 t.tM) induced the appearance of several phosphorylated membrane polypeptides in the range of 15-45 kDa in cells initiating DNA replication, (Fig. 2c, d). Addition of TPA, either by itself or together with sphingosine, to cells initiating growth in the presence of [a-'P]-Pi stimulated the appearance of phosphorylated polypeptides, especially in the range of 15-45 kDa, the same molecular-mass region in which phosphorylation was stimulated by low concentrations of sphingosine (Fig. 2a). Vancomycin, an inhibitor of cell-wall synthesis in Gram-positive organisms and of membrane

binding of DNA and initiation of DNA replication in B. subtilis cultures [12,13] inhibited the appearance of phosphorylated membrane proteins, and appeared specifically to inhibit phosphorylation of two peptides of approximately 3540 kDa whose phosphorylation was stimulated by 10 or 20/,,M sphingosine (Fig. 2d, lanes C and V, 20 min). it should be noted that both sphingosine and TPA affected the distribution of protein between the particulate and the cytoplasmic fractions of the cell during growth induction, though not in such a way as to account for the altered amounts of phosphorylated membrane proteins (Fig. 3). It thus seems that rapid changes occur in the phosphorylation of specific proteins of the membrane fraction of B. subtilis cells during initiation of chromosome replication, and that these changes are affected differently by vancomycin, an inhibitor of initiation which prevents binding to the membrane, and by sphingosine, which was also found to inhibit initiation of DNA replication in B. sttbtilis without, apparently, affecting membrane binding. Also, concentrations of sphingosine which inhibited only initiation of replication had a different effect on the appearance of phosphorylated proteins in the cell membrane than a concentration which also inhibited elongation. The conclusion that cell cycle controls similar to those recently described in eukaryotic systems exist in Bacillus would require the identification

245 of specific phosphorylated proteins playing a role in i n i t i a t i o n o f D N A r e p l i c a t i o n . H o w e v e r , t h e occurrence of protein phosphorylation at the time of initiation of DNA replication and the prcvi-

T T S T S C V S TS

A. MW

CCV

ously unreported effects of an activator (TPA) and an inhibitor (sphingosinc) of eukaryotic protein kinasc on this phosphorylation and on the initiation of DNA replication raise the possibility

B.

T T C C V ST S C V S T S

MW E

!~

946743302014/L~JI

Jl

0

5'

I0'

C. MW kDa

0

5'

I0'

D.

- + - + - + -+ l ! l ~

S C V

CVSCVSCV

S

MW CDo

94 67 -- 43

II

, 5'

ii

I0'

II

15'

20'

I --

30

--

20

--14

4t 5'

i

i

L

I O'

15'

20'

Fig. 2. Protein phosphorylation. A, Effect of sphingosinc, TPA, and vancomycin. The pattern of phosphorylated proteins in the membrane fraction of ¢onlrol cells was compared with that of cells diluted in [~2P]-Pi medium containing, where indicated, vancomycin (3 p,g ml -z) (V), sphingosine (40 p,M) (S), TPA (5 p,g ml i), IT), or TPA and sphingosinc (T, S). B. CL~)massic blue stained gel of A. C. Membrane proteins as in A. l0 p,M sphingosine was present in the culture where indicated (+). D. An experiment similar to those of A and C, Cultures contained vancomycin (3 ,u.g ml n) IV). or sphingosine (2(1 p,M) (S).

246

REFERENCES

7'.4¸

2.0

~

1.6

1.2 ~8

¢_ 0.8

0"°4O

I0 20 Time ( min )

50

Fig. 3. Protein concentration of membrane extracts. Aliquots of samples taken from diluted stationary phase cultures for determination of protein phosphorylation were frozen immediately. Subsequently. protein concentration was measured using the Bradford protein assay reagent of Bio-Rad Laboratories, Munich. v . Control: ,~. 41) # M sphingosine: o. 8 # M TPA; A 40 # M sphingosine plus 8/.tM TPA.

that protein phosphorylation may play a role in control of the cell cycle in B. subtilis.

ACKNOWLEDGEMENT We wish to acknowledge expert technical assistance by Mrs. Anita Kol.

[1] Garnak, M. and Reeves. H.C. (1978) Science 203, 111 I 1112. [2] Gautier, J., Matsukawa, T., Nurse. P. and Mailer, J. (1989) Nature 339, 626-629. [3] liannun, Y.A.. I~omis. C.R. Merill, A.H., Jr. and Bell, R.M. (1986)J. Biol. Chem. 261, 12604-12609. [4] Jefferson, A.B. and Schulman. H. (1988)J. Biol. Chem. 263, 15241-15244. [5] Kohler, E. and Antranikian. G. (1989) FEMs Microbiol. Lett. 57. 87-92. [6] Lark, K.G., Repko. T. and Hoffrmm, E.J. (1903) Biochim. Biophys. Acta 76, 9-24. [7] Laurent, S.J. (1973) J. Bacteriol. 116, 141-145. [8] Murray, A.W. and Kirschner. M.W. (1989) Science 246. 614-621. [9] Nishizuka, Y. (1984) Nature 308, 693-698. [10] Pardee, A.B. (1989) Science 246, 603-608. [11] Perego, M., Cole. S.P., Burkuly, S.D.. Trach. K. and Hoch. J. (1989) J. Bacteriol. 171, 6187-6196. [12] Sandier, N. and Keynan. A. (1981) J. Bacteriol. 148. 443-449. [13] Sandier. N. and Keynan. A. 0988) J. Gen. Micmbiol. 134. 1155- 1163. [14] Sterlini. J.M. and Mandelslam, J. (1969) Biochem. J. 113. 29-37. [15] Watson, G.M.F. and Mann, N.H. 0988) J. Gen. Microbiol. 134, 2559-2565. [16] Winston, S. and Sueoka. N. (1980) Proc. Natl. Acad. Sci. USA 77, 2834-2838. [17] Zylicz, M., Lehowitz. J.H.. McCaken, R. and Georgopoulos. C. (1983) Prec. Natl. Acad. Sci. USA 8(1, 6431-6435.