~1 [NSTITUTPASTEUR/ELSEVIER Paris 1991
Res. MicrobioL 1991, 142, 137-140
A calcium.binding protein that may be required for the initiation of chromosome replication in Escherichia coli E . C . G u z m a n n), R , H . P r i t c h a r d ~2~a n d A. Jim6nez-Szinchez I" m Departamento de Bioquimica y Biologia Molecular y Gendtiea, Universidad de Extremadura, 06080 Badajoz (Spain), and ¢2)Department o f Genetics, University o f Leicester [ E l 7 R H (UK)
SUMMARY Starvation for isoleucine but not for other amino acids in an iJv- strain or the addition of vanne in an tlv + strain inhibits initiation of chromosome and mlnlohromosome replication in stringent (Rel ÷ ) Escherichia coil, but il does not inhibit repllcation in relaxed {ratA) mutants (Guzman et at, 1988). From these results, we con¢ludod that, ( 1 ) o r f C initiation of replication is inhibited by ppGpp, and (2) isoleucine is not needed for the protein synthesis required at initiation. These results led us to find an isoleucine-free protein whose de novo synthesis is the sole protein synthesis requirement for oriC Initiation. We also present evidence that this protein may be a calcium-binding protein located at 73 rain in the genetic map, Key-words : Replication, Initiation, Calcium; oriC ; Escherichia coil
INTRODUCTION Initiation o f c h r o m o s o m e replication requires RNA and protein synthesis, and inhibition of the synthesis of either of these macromolecnles has long been used to inhibit the initiation step w i t h o u t a f f e c t i n g elongatiGn (McMacken et aL, 1987). RNA synthesis can be specifically inhibited by the use of several drugs, but the inhibition of protein synthesis by amin o acid deprivation also affects R N A synthesis through the stringent response (Cashel and Ru d d , 1987). Thus, inhibition of the initiation of chromosome replication by amino acid starvation could be the consequence of the
inhibition of a stringently regulated RNA or protein synthesis required for this step. In this work, we show that in vivo initiation of chromosome and minichromosome replication in E. coil is inhibited by ppGpp (Guzm/m et al.. 1988l, and that de novo synthesis of an isnleucine-free protein, a probable calcium-binding protein, is the sole protein synthesis requirement for DNA initiation. MATERIALS AND METHODS Bacterial strains and plasmids Bacterial strains used in this work were E. coil LE234, K12
F- metB argH ilv thi; FC200 is LE234 l i v e ; BP225 is LE234 relA ; BP226 is FC200 relA. Plasmid pSY3i7 is a minichronzosome containing a 5.6 kb oriC Eco R.I fragment from E. coli and a 7.9-kh gene for kanamycine resistance ; it was received from Kornberg's laboratory (Kaguni el al., 1982). Growth conditions
Bacteria were grown in M9 minimal medium with appropriate supplements. Protein synthesis was inhibited by chloramphenieol ( 2 0 0 t ~ g / m l ) or by removing the required amino acid by filtration or by adding
t38
E.C. GUZMAN ET AL.
'-'aline (500 i~g/ml)
to
ilv'
strains.
Measurement of D N A p r o t e i n synthesis
and
Chromosomal DNA synthesis was measured by acidprecipitable ~H-thyntidine (Amersham) at a concentration of I ng/ml (37 kBq/,ag} accumulation in the presence of uridine (1.5 raM) to avoid degradation of labelled nucleoside. Minichromosome synthesis was determined by the accumulated ~H-thymidine in the corresponding band isolated t h r o u g h a 0,7% agarose eh:ctrophoresis. Protein synthesized in the absence of an amino acid was determined by 5 min of amino acid s t a r v a t i o n f o llo w ed by 5-rain labelling with 3Hmetbionine (Amersham} in the absence of the amino acid. Aliquots of the culture were lysed and run into 12% PAGE. Bands were viewed by autofluorography,
RESULTS Starvation for isoleucine in E. coli by removing this required amino acid to an ilv- strain, or by a d d i n g valine to an ilv" strain, does not inhibit initiation of chromosome replication in a relaxed RelA- strain, but starva;ion for isoleucine in a stringent strain or starvation for other amino acid or chloramphenicol addition in either strain does (fig. 1). T o show that this replication in the absence of isoleucine and the absence of the stringent response initiates at oriC, we measured replication of minichromosome pSY317 under the saran conditions to find the same results (fig. 2). These results led us to conclude that initiation of replication at oriC is inhibited by p p G p p and that isoleucine is not needed for the
protein(s) synthesis required at initiation. These results suggest that de novo synthesis of an isoleucine-free protein is the sole protein synthesis requirement for D N A initiation and that this protein can be made in the absence
~1/ .~ -- 2
RolA+
of isoleue!ne only in a relaxed strain. To show whether there is some protein synthesis in the absence o f i s o l e u c i n e , an exponentially growing culture was starved for isoleuclne for
~,
RolA-
,, II
~
~t .x
•
t 1 5
~
_~ '~ '~ ~ "1
o~r~°" O
wo omoo Io~ ° °
!
i
I
I
I
I
1
2
3 Time
0 "I (hours)
2
3
F i g . I. Relative amount of chromosomal DNA synthesized in Rel + strains LE234 and FC200 (left panel) and gel- strains BP22-c and BP226 (right panel) without any treatment (A), or after addition of chloramphenieol (11), addition of valine (k), starvation for isoleucine (Q), starvation for valine (O), or starvation for arginine ([~1,
RelA+
~_-- 3
,',
RelA-
"
~| &e
&e
A
~" 2 t... ~. .~ ¢¢ ~ 1
2
/,. A 0
0
• kc, O •
1
o
2
3
O
1
2
3
Fig, 2. Relative amount of pSY3[7 minichromosomal DNA synthesized in R-:I" strains LE234 and FC200 (left panel) and Rel- strains 8P225 and BP226 (right panel). Symbols as in figure 1.
CALCIUM-BINDING PROTEIN IN E. COLI CHROMOSOME REPLICATION
5 min followed by 5 rain in the absence of this amino acid and in the presence of 3Hmethionine. After this treatment, cell lysates were run in 12070 P A G E and proteins synthesized during the labelling period were viewed by autofiuorography (fig. 3). We found a single band of a protein of about II kDa synthesized under isoleucine starvation only in the RelA- mutant. We carried out a computer search in the E M B L Data Library for a protein with no isoleucine in its sequence and identified an open reading frame at rain 73 o f the E. coil genetic map (Olins and Nomura, 1981) which, if translated, would give a polypeptide of 11.6 kDa with n o isoleucin¢ a n d with the sequence for a stringently regulated promoter 98 nucleotides upstream. An a iditional computer search for sequence homologies with other pro- and eukaryotic proteins showed that this polypeptide had four potential calcium binding domains in highly hydr ophobic-hydrophilic transition regions of the polypeptide sequence (fig. 4). The low M W of this polypeptido ( l l . 6 kDa), its high glutamate and aspartate coment (21°/0 o f its amino acid composition) and low iseelectri¢ point (4.4) give it a strong resemblance to eukaryotic ¢almodulin (Klee et aL, 1980). U s i n g a synthetic oligonuclcotide, we have been able to localize this sequence by D N A hybridization in fragment 3F8 of Kohara's physical map (Kohara et at., 1987) which will be used for cloning experiments.
DISCUSSION Our data show that isoleucine starvation of (or valine addition to) relaxed ( R e l ) strains of E. coli permits continued initiation o f chromosome replication. As
1
2
3
4
5
6
139 7
66-
45-36-29-24-20--
14--
Fig. 3. Exponentially growing cultures of Rel + (1-,3) and Rel- strains (4-6) were starved for arginine (I and 4) or for isoleucine to ilv strains (2 and 51, starved for isoleucine by valine addition to ilv strains (3 and 61 or not treated (7). After 5-rain, c~:[;ares were pulse-labelled for 5 min with 3H-methionine and crude lysates were run in a 1207oPAGE. After dried, gel was autofluorographied.
hydroph i I i¢ ,41-3
I
I
. . . . . . . . . . . . . . . . .
-2
1
10
20
30
40
50
~0
70
80
90
100
amino acid position Fig, 4. Hoop-Woods hydrophilicity plot of Pin sequence. Segments show the positions of the four potential calcium binding domains.
lap this resnh can only be observed in a RelA strain, we conclude that initiation of chromosome repiicalion is inhibited by tile stringent response. Either the inhibition by ppGpp and tile non-requirclnent for isoleucine are o r i C - d e p e n d e n t , since a minichromosnnte iucluding the minimal oriC behaves as the bacterial chromosome. T h e only protein which accumulates in significant amounts under isoleueinc deprivation has MW of nearly I1 kDa and its synthesis is p p G p p repressed, since it was only present when a relaxed strain was used. We tentatively call this protein Pin for "initiation prot e i n " . A data library search showed an open reading frame at position 73 minutes of the E. coli chromosome m a p (Olins and Nomura, 1981) that, if translated, will code for an isoleucinefree protein with a MW of I 1.6 kDa and with a ppGpp-repressed promoter. We have not shown that the protein which accumulates in isoleucine.starved cultures is specifled by this reading frame, nor that the protein hand we see is the protein which we postulate must be synthesized de novo to permit continued initiation during isolcucin¢ starvation. The fact that the postulated protein appears to be a calcium-binding protein makes this possibility particularly attractive. This will raise a putative role of calcium in the regulation of chromosome initiation and extends to bacteria the presumed role of calcium in
E.C. G U Z M A N E T AL. replication of E. coil chromosomal origin is bidirectional Nature (Lond.), 296, 623-627. Klee, C.B., Crouch, T.H. & Richman. P.G. (1980L Calmodulin. Ann. Rev. Biochem., 49, 489-515, Kohara, Y., Akiyama, K. & Isono, K. 0987), The physical map of the whole E, coil chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell, 50, 495-508, McMacken, R., Silver, L. & Gemgopoulos, C. (1987), DNA replication, in "Escherichia coil and Salmonella typhimurium. Cellular and Molecular BiolAcknowledgements ogy" (J.L. Ingraham, K.B. Low, B. Magasanik, M. We thank Y. gohara for the gene Schaeehter & H.E. Umbarger) bank, A. Kornhtrg for minichromosome (pp. 564-612). American SocipSV3i7, J.M. Diaz for olignnucl¢otide ety for Microbiology, Washingand C. Guti~rrez for helpful discussions. ton, D.C. This work was supported by grant Norris, V. 0989), A calcium flux at PB8%097S from MEC. Programa the termination of replication General d~ ICYT, Spain. triggers cell division in Escheriehia coll. Cell Calcium, I0. 511-517. Norris. V., Seror. S.J., Casaregola. References S. & Holland, LB. (1988), A single calcium flux triggers Cashel. M. & Rudd, K.E. (1987), chromosome replication, segleThe stringent response, in gation and septation in bac"Escherichia colt and teria: a model. J. 7~eor. Bivl., Salmonella typhimurium. Cel134, 341-350. lular and Molecular Biology" Olios, P.O. & Nomura. M. (1981), (J.L. [ngraham, K.B. Low, B. Regulation of the Sl0 Magasanik, M. Schaechter & ribosomal protein operon in E. H .E. Umbarger) (pp. coil: nucleotide sequence at the 1410-143g). American Society start of the operon. Cell, 26, for Microbiology, Washington, 205-211. D.C. Rasmussen, C.D. & Means, A.R. Guzmltn, E.C., Carrillo, F.J. & (1989), Calmodulin is required Jim~nez-Sanchez, A. (1988l for cell-cycle progression durDifferential inhibition of the ing Gl and mitosis. EMBOJ., initiation of DNA replication in 8, 73-82. stringent and relaxed strains of Singh, J. & Chanerjee, S. (1988), Escherichia coil. Genetics Re&, Cell-cycle-dependent variation Sl. 173-177. of calmodulin in tetrahymena. Kaguni, J.M., Fuller, R.S. & KornCytobios, 55, 95-103. berg, A. (1982). Enzymatic
the regulation of the eukaryotic cell cycle (Singh and Chetterjee, 1988; Rasmussen and Means, 1989). This conclusion emphasizes the notion of calcium as a key element in regulating different bacterial cell cycle events recently proposed by Norris et al. (Norris el at., 1988; Norris, 1989). Its verification will reveal a calmodulin-lik¢ protein whose de novo synthesis may he the sole protein synthesis required for oriC replication.
Res. MicrobioL 1991, 142, 137-140
A calcium.binding protein that may be required for the initiation of chromosome replication in Escherichia coli E . C . G u z m a n n), R , H . P r i t c h a r d ~2~a n d A. Jim6nez-Szinchez I" m Departamento de Bioquimica y Biologia Molecular y Gendtiea, Universidad de Extremadura, 06080 Badajoz (Spain), and ¢2)Department o f Genetics, University o f Leicester [ E l 7 R H (UK)
SUMMARY Starvation for isoleucine but not for other amino acids in an iJv- strain or the addition of vanne in an tlv + strain inhibits initiation of chromosome and mlnlohromosome replication in stringent (Rel ÷ ) Escherichia coil, but il does not inhibit repllcation in relaxed {ratA) mutants (Guzman et at, 1988). From these results, we con¢ludod that, ( 1 ) o r f C initiation of replication is inhibited by ppGpp, and (2) isoleucine is not needed for the protein synthesis required at initiation. These results led us to find an isoleucine-free protein whose de novo synthesis is the sole protein synthesis requirement for oriC Initiation. We also present evidence that this protein may be a calcium-binding protein located at 73 rain in the genetic map, Key-words : Replication, Initiation, Calcium; oriC ; Escherichia coil
INTRODUCTION Initiation o f c h r o m o s o m e replication requires RNA and protein synthesis, and inhibition of the synthesis of either of these macromolecnles has long been used to inhibit the initiation step w i t h o u t a f f e c t i n g elongatiGn (McMacken et aL, 1987). RNA synthesis can be specifically inhibited by the use of several drugs, but the inhibition of protein synthesis by amin o acid deprivation also affects R N A synthesis through the stringent response (Cashel and Ru d d , 1987). Thus, inhibition of the initiation of chromosome replication by amino acid starvation could be the consequence of the
inhibition of a stringently regulated RNA or protein synthesis required for this step. In this work, we show that in vivo initiation of chromosome and minichromosome replication in E. coil is inhibited by ppGpp (Guzm/m et al.. 1988l, and that de novo synthesis of an isnleucine-free protein, a probable calcium-binding protein, is the sole protein synthesis requirement for DNA initiation. MATERIALS AND METHODS Bacterial strains and plasmids Bacterial strains used in this work were E. coil LE234, K12
F- metB argH ilv thi; FC200 is LE234 l i v e ; BP225 is LE234 relA ; BP226 is FC200 relA. Plasmid pSY3i7 is a minichronzosome containing a 5.6 kb oriC Eco R.I fragment from E. coli and a 7.9-kh gene for kanamycine resistance ; it was received from Kornberg's laboratory (Kaguni el al., 1982). Growth conditions
Bacteria were grown in M9 minimal medium with appropriate supplements. Protein synthesis was inhibited by chloramphenieol ( 2 0 0 t ~ g / m l ) or by removing the required amino acid by filtration or by adding
t38
E.C. GUZMAN ET AL.
'-'aline (500 i~g/ml)
to
ilv'
strains.
Measurement of D N A p r o t e i n synthesis
and
Chromosomal DNA synthesis was measured by acidprecipitable ~H-thyntidine (Amersham) at a concentration of I ng/ml (37 kBq/,ag} accumulation in the presence of uridine (1.5 raM) to avoid degradation of labelled nucleoside. Minichromosome synthesis was determined by the accumulated ~H-thymidine in the corresponding band isolated t h r o u g h a 0,7% agarose eh:ctrophoresis. Protein synthesized in the absence of an amino acid was determined by 5 min of amino acid s t a r v a t i o n f o llo w ed by 5-rain labelling with 3Hmetbionine (Amersham} in the absence of the amino acid. Aliquots of the culture were lysed and run into 12% PAGE. Bands were viewed by autofluorography,
RESULTS Starvation for isoleucine in E. coli by removing this required amino acid to an ilv- strain, or by a d d i n g valine to an ilv" strain, does not inhibit initiation of chromosome replication in a relaxed RelA- strain, but starva;ion for isoleucine in a stringent strain or starvation for other amino acid or chloramphenicol addition in either strain does (fig. 1). T o show that this replication in the absence of isoleucine and the absence of the stringent response initiates at oriC, we measured replication of minichromosome pSY317 under the saran conditions to find the same results (fig. 2). These results led us to conclude that initiation of replication at oriC is inhibited by p p G p p and that isoleucine is not needed for the
protein(s) synthesis required at initiation. These results suggest that de novo synthesis of an isoleucine-free protein is the sole protein synthesis requirement for D N A initiation and that this protein can be made in the absence
~1/ .~ -- 2
RolA+
of isoleue!ne only in a relaxed strain. To show whether there is some protein synthesis in the absence o f i s o l e u c i n e , an exponentially growing culture was starved for isoleuclne for
~,
RolA-
,, II
~
~t .x
•
t 1 5
~
_~ '~ '~ ~ "1
o~r~°" O
wo omoo Io~ ° °
!
i
I
I
I
I
1
2
3 Time
0 "I (hours)
2
3
F i g . I. Relative amount of chromosomal DNA synthesized in Rel + strains LE234 and FC200 (left panel) and gel- strains BP22-c and BP226 (right panel) without any treatment (A), or after addition of chloramphenieol (11), addition of valine (k), starvation for isoleucine (Q), starvation for valine (O), or starvation for arginine ([~1,
RelA+
~_-- 3
,',
RelA-
"
~| &e
&e
A
~" 2 t... ~. .~ ¢¢ ~ 1
2
/,. A 0
0
• kc, O •
1
o
2
3
O
1
2
3
Fig, 2. Relative amount of pSY3[7 minichromosomal DNA synthesized in R-:I" strains LE234 and FC200 (left panel) and Rel- strains 8P225 and BP226 (right panel). Symbols as in figure 1.
CALCIUM-BINDING PROTEIN IN E. COLI CHROMOSOME REPLICATION
5 min followed by 5 rain in the absence of this amino acid and in the presence of 3Hmethionine. After this treatment, cell lysates were run in 12070 P A G E and proteins synthesized during the labelling period were viewed by autofiuorography (fig. 3). We found a single band of a protein of about II kDa synthesized under isoleucine starvation only in the RelA- mutant. We carried out a computer search in the E M B L Data Library for a protein with no isoleucine in its sequence and identified an open reading frame at rain 73 o f the E. coil genetic map (Olins and Nomura, 1981) which, if translated, would give a polypeptide of 11.6 kDa with n o isoleucin¢ a n d with the sequence for a stringently regulated promoter 98 nucleotides upstream. An a iditional computer search for sequence homologies with other pro- and eukaryotic proteins showed that this polypeptide had four potential calcium binding domains in highly hydr ophobic-hydrophilic transition regions of the polypeptide sequence (fig. 4). The low M W of this polypeptido ( l l . 6 kDa), its high glutamate and aspartate coment (21°/0 o f its amino acid composition) and low iseelectri¢ point (4.4) give it a strong resemblance to eukaryotic ¢almodulin (Klee et aL, 1980). U s i n g a synthetic oligonuclcotide, we have been able to localize this sequence by D N A hybridization in fragment 3F8 of Kohara's physical map (Kohara et at., 1987) which will be used for cloning experiments.
DISCUSSION Our data show that isoleucine starvation of (or valine addition to) relaxed ( R e l ) strains of E. coli permits continued initiation o f chromosome replication. As
1
2
3
4
5
6
139 7
66-
45-36-29-24-20--
14--
Fig. 3. Exponentially growing cultures of Rel + (1-,3) and Rel- strains (4-6) were starved for arginine (I and 4) or for isoleucine to ilv strains (2 and 51, starved for isoleucine by valine addition to ilv strains (3 and 61 or not treated (7). After 5-rain, c~:[;ares were pulse-labelled for 5 min with 3H-methionine and crude lysates were run in a 1207oPAGE. After dried, gel was autofluorographied.
hydroph i I i¢ ,41-3
I
I
. . . . . . . . . . . . . . . . .
-2
1
10
20
30
40
50
~0
70
80
90
100
amino acid position Fig, 4. Hoop-Woods hydrophilicity plot of Pin sequence. Segments show the positions of the four potential calcium binding domains.
lap this resnh can only be observed in a RelA strain, we conclude that initiation of chromosome repiicalion is inhibited by tile stringent response. Either the inhibition by ppGpp and tile non-requirclnent for isoleucine are o r i C - d e p e n d e n t , since a minichromosnnte iucluding the minimal oriC behaves as the bacterial chromosome. T h e only protein which accumulates in significant amounts under isoleueinc deprivation has MW of nearly I1 kDa and its synthesis is p p G p p repressed, since it was only present when a relaxed strain was used. We tentatively call this protein Pin for "initiation prot e i n " . A data library search showed an open reading frame at position 73 minutes of the E. coli chromosome m a p (Olins and Nomura, 1981) that, if translated, will code for an isoleucinefree protein with a MW of I 1.6 kDa and with a ppGpp-repressed promoter. We have not shown that the protein which accumulates in isoleucine.starved cultures is specifled by this reading frame, nor that the protein hand we see is the protein which we postulate must be synthesized de novo to permit continued initiation during isolcucin¢ starvation. The fact that the postulated protein appears to be a calcium-binding protein makes this possibility particularly attractive. This will raise a putative role of calcium in the regulation of chromosome initiation and extends to bacteria the presumed role of calcium in
E.C. G U Z M A N E T AL. replication of E. coil chromosomal origin is bidirectional Nature (Lond.), 296, 623-627. Klee, C.B., Crouch, T.H. & Richman. P.G. (1980L Calmodulin. Ann. Rev. Biochem., 49, 489-515, Kohara, Y., Akiyama, K. & Isono, K. 0987), The physical map of the whole E, coil chromosome: application of a new strategy for rapid analysis and sorting of a large genomic library. Cell, 50, 495-508, McMacken, R., Silver, L. & Gemgopoulos, C. (1987), DNA replication, in "Escherichia coil and Salmonella typhimurium. Cellular and Molecular BiolAcknowledgements ogy" (J.L. Ingraham, K.B. Low, B. Magasanik, M. We thank Y. gohara for the gene Schaeehter & H.E. Umbarger) bank, A. Kornhtrg for minichromosome (pp. 564-612). American SocipSV3i7, J.M. Diaz for olignnucl¢otide ety for Microbiology, Washingand C. Guti~rrez for helpful discussions. ton, D.C. This work was supported by grant Norris, V. 0989), A calcium flux at PB8%097S from MEC. Programa the termination of replication General d~ ICYT, Spain. triggers cell division in Escheriehia coll. Cell Calcium, I0. 511-517. Norris. V., Seror. S.J., Casaregola. References S. & Holland, LB. (1988), A single calcium flux triggers Cashel. M. & Rudd, K.E. (1987), chromosome replication, segleThe stringent response, in gation and septation in bac"Escherichia colt and teria: a model. J. 7~eor. Bivl., Salmonella typhimurium. Cel134, 341-350. lular and Molecular Biology" Olios, P.O. & Nomura. M. (1981), (J.L. [ngraham, K.B. Low, B. Regulation of the Sl0 Magasanik, M. Schaechter & ribosomal protein operon in E. H .E. Umbarger) (pp. coil: nucleotide sequence at the 1410-143g). American Society start of the operon. Cell, 26, for Microbiology, Washington, 205-211. D.C. Rasmussen, C.D. & Means, A.R. Guzmltn, E.C., Carrillo, F.J. & (1989), Calmodulin is required Jim~nez-Sanchez, A. (1988l for cell-cycle progression durDifferential inhibition of the ing Gl and mitosis. EMBOJ., initiation of DNA replication in 8, 73-82. stringent and relaxed strains of Singh, J. & Chanerjee, S. (1988), Escherichia coil. Genetics Re&, Cell-cycle-dependent variation Sl. 173-177. of calmodulin in tetrahymena. Kaguni, J.M., Fuller, R.S. & KornCytobios, 55, 95-103. berg, A. (1982). Enzymatic
the regulation of the eukaryotic cell cycle (Singh and Chetterjee, 1988; Rasmussen and Means, 1989). This conclusion emphasizes the notion of calcium as a key element in regulating different bacterial cell cycle events recently proposed by Norris et al. (Norris el at., 1988; Norris, 1989). Its verification will reveal a calmodulin-lik¢ protein whose de novo synthesis may he the sole protein synthesis required for oriC replication.
