Department of Microbial Cell Biology, Graduate school of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0101, Japan1
Author for correspondence: Naotake Ogasawara. Tel: +81 743 72 5430. Fax: +81 743 72 5439. e-mail: nogasawa{at}bs.aist-nara.ac.jp
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ABSTRACT |
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Keywords: bacterial GTPase, molecular switch protein, bacterial growth, regulation of initiation of chromosome replication
b The primer sequences used for PCR in this study are shown as supplementary data on Microbiology Online (http://mic.sgmjournals.org).
a Present address: Faculty of Science, Saitama University, 255 Ohkubo, Saitama, Saitama 338-8570, Japan.
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INTRODUCTION |
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Among bacterial GTP-binding proteins of the Era/Obg family, the E. coli Era and the B. subtilis Obg proteins have been well studied, but their precise roles are still poorly defined. The Era protein has been shown to be essential for cell growth in E. coli, Salmonella typhimurium and Streptococcus mutans (March et al., 1988 ; Takiff et al., 1989
; Gollop & March, 1991
; Anderson et al., 1996
; Sato et al., 1998
). Era binds to 16S rRNA and contains an RNA-binding KH domain at the C terminus (Meier et al., 1999
; Sayed et al., 1999
). In E. coli, depletion of Era at 27 °C has been shown to cause cell filamentation (Gollop & March, 1991
) and a mutation in the GTP-binding domain was found to suppress temperature-sensitive chromosome partitioning mutations, suggesting that Era is a cell-cycle checkpoint regulator (Britton et al., 1997
, 1998
; Britton & Lupski, 1997
). The B. subtilis and Streptomyces spp. Obg proteins are essential for vegetative growth and initiation of sporulation (Trach & Hoch, 1989
; Okamoto et al., 1997
; Okamoto & Ochi, 1998
; Vidwans et al., 1995
; Welsh et al., 1994
). Furthermore, B. subtilis Obg has been found to be essential for stress activation of transcription factor
B and has been shown to cofractionate with ribosomes, together with regulators of
B activity (Scott & Haldenwang, 1999
; Scott et al., 2000
). The existence of Obg is not restricted to bacteria that undergo cellular differentiation. The Caulobacter crescentus Obg homologue (CgtA) has been shown to be indispensable for growth (Maddock et al., 1997
). Very recently, the E. coli homologue YhbZ (renamed ObgE) has been reported to be an essential gene involved in chromosome partitioning (Kobayashi et al., 2001
). In addition to Era and Obg, a GTP-binding protein encoded by the engA gene of Neisseria gonorrhoeae has been suggested to be essential for growth (Mehr et al., 2000
). B. subtilis YsxC is also an essential GTP-binding protein (Arigoni et al., 1998
; Pragai & Harwood, 2000
) and the depletion of its homologue (YihA) in E. coli caused defective cell division (Dassain et al., 1999
). Thus, experimental results are accumulating to suggest that many GTP-binding proteins of the Era/Obg family are essential for bacterial growth, often related to cell cycle progression such as chromosome replication and partitioning, and cell division and cellular differentiation.
When the complete genome sequence of B. subtilis was surveyed for genes encoding GTP-binding proteins of the Era/Obg family, we identified in addition to obg and ysxC, homologues of the E. coli era and N. gonorrhoeae engA genes, as well as five additional family members. As a first step in elucidating a functional network of those nine GTP-binding proteins, we found that six of them are essential for B. subtilis viability. In vitro studies demonstrated that the six essential proteins specifically bind GTP and GDP. Furthermore, we analysed the effect of depletion of these proteins in the B. subtilis cells on cell morphology and chromosome replication, and found that Bex (a homologue of E. coli Era) and YqeH appeared to participate in the regulation of initiation of chromosome replication.
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METHODS |
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Plasmids pTM403 and pTM409, used to replace the yphC or yqeH genes by the tetracycline resistance gene, were constructed as follows. The upstream and downstream regions of yphC or yqeH were amplified, digested at the BamHI/XhoI sites or XhoI/HindIII sites introduced in the primers, ligated using the XhoI sites, and inserted between the HindIII and BamHI sites of pBR322. Then, an XhoI fragment containing the tet gene derived from pBEST307 (Itaya, 1992 ) was inserted into the XhoI site of the resultant plasmids in the same direction as the yphC or yqeH gene.
Purification of the GTP-binding proteins.
To express the GTP-binding proteins with a histidine tag (His6) at the C terminus, the coding sequences of Obg, Bex, YphC, YsxC, YlqF and YqeH (from the initiation codon to the C-terminal amino acid codon, except the termination codon) were amplified by PCR and cloned into the pET28b or pET29b plasmid (Novagen) to obtain pTM282, pTM291, pTM293, pTM294, pTM298 and pTM299, respectively. E. coli BL21(DE3)pLysS derivatives (Novagen) containing each of the plasmids were grown at 30 °C in 500 ml LB medium and the his-tagged GTP-binding proteins were purified according to the pET system protocol (Novagen). When the culture reached OD600 0·6, IPTG was added to a final concentration of 1 mM. The cells were grown 3 h more and then harvested by centrifugation. Collected cells were washed with binding buffer (0·5 M NaCl, 5 mM imidazole, 20 mM Tris/HCl, pH 7·9) and resuspended in 20 ml of the same buffer. The cells were broken by sonication on ice and the lysate was centrifuged at 39000 g for 30 min at 4 °C. The supernatant fraction was applied to a Ni2+-NTA resin column (Novagen), and the column was washed with 30 ml binding buffer followed by 10 ml washing buffer (0·5 M NaCl, 20 mM imidazole, 20 mM Tris/HCl, pH 7·9). The GTP-binding protein in the column was eluted with 10 ml binding buffer containing 60 mM imidazole. The resin-bound YphCHis6 in the Ni-column was cleaved with 2 U thrombin protease in cleavage buffer (20 mM Tris/HCl, pH 8·0, 150 mM KCl, 2·5 mM CaCl2), incubated at 4 °C overnight. Purified proteins were stored at -80 °C after addition of 10% (v/v) glycerol.
GTP-binding assay.
GTP-binding activity was analysed essentially following the procedure of Lin et al. (1999) . Purified BexHis6, YlqFHis6, YphC, YsxCHis6 or YqeH protein (50 pmol) was added to 1·3 pmol [4 µCi (1·5x105 Bq)] of [
-32P]GTP [3000 Ci (1·1x1014 Bq) mmol-1; NEN Life Science] in 20 µl GTP-binding buffer (50 mM Tris/HCl, pH 8·0, 50 mM KCl, 2 mM DTT, 5 mM MgCl2, 10%, v/v, glycerol) (Sullivan et al., 2000
) and incubated on ice for 10 min. The bound radioactive labelled GTP was cross-linked to the proteins by UV light (254 nm, 1 J cm-2). Excess unbound [
-32P]GTP was eliminated with a Microcon centrifugal filter (Millipore). For reactions with competing nucleotides, 40 µM nonradioactive nucleotide (GTP, GDP, GMP, ATP, UTP or CTP) was mixed with [
-32P]GTP (with a ratio about 600:1) prior to protein addition. Radiolabelled proteinGTP complexes were separated by SDS-PAGE. After electrophoresis, the gel was dried and exposed to an imaging plate for 1 h and signals were detected by BAS2500 (Fuji Film).
Immunoblotting.
Rabbit polyclonal antibodies raised against purified Bex, Obg, YphC, YsxC, YlqF and YqeHHis6 proteins were obtained from Takara Shuzo. Preparation of cell lysates from exponentially growing B. subtilis cells and separation of proteins were carried out as described previously (Hassan et al., 1997 ). Proteins were blotted on a Hybond-P PVDF membrane (Amersham Pharmacia), and the membrane was incubated with rabbit polyclonal antibodies. After the membrane was treated with a second antibody (goat anti-rabbit IgGhorseradish peroxidase conjugate), signals were detected by the ECL-Plus enhanced chemiluminescence system (Amersham Pharmacia).
Fluorescence microscopy.
Cell morphology and nucleoid distribution were examined as described under fluorescence microscopy after DAPI (4',6-diamino-2-phenyl indole) staining (Hassan et al., 1997 ).
Flow cytometry.
Chloramphenicol was added at a concentration of 200 µg ml-1 to exponentially growing cells, which were then incubated for 5 h to ensure both inhibition of new rounds of replication initiation and completion of ongoing replication. The cells were fixed with ethanol and treated as described previously (Løbner-Olesen et al., 1989 ), and the number of replication origins per cell was measured with a Bryte HS flow cytometer (Bio-Rad).
Measurement of DNA/protein ratio.
Nucleic acid and protein fractions were extracted from cells by Schneiders method (Herbert et al., 1971 ) with some modifications as described elsewhere (Kadoya et al., 2002
). Briefly, cells were suspended in 0·6 M perchloric acid and heated at 70 °C for 15 min. The soluble fraction was recovered by centrifugation and used as the nucleic acid fraction. The remaining insoluble materials were resuspended in 1 M sodium hydroxide and heated at 95 °C, followed by centrifugation. The soluble fraction was used as the protein fraction. DNA and protein concentrations in these fractions were determined by colorimetric methods described by Burton (1956)
and the Lowry method
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RESULTS |
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To begin examination of Era/Obg family members in the B. subtilis genome, we searched all ORFs of B. subtilis by PSI-BLAST (Altschul et al., 1997 ) using the amino acid sequence of the GTP-binding domain of E. coli Era as a probe. We identified twelve related ORFs, including those encoding ribosomal function proteins IF2, LepA and YlaG. Further analysis using the twelve proteins identified as probes detected EF-Tu and EF-G. The predicted amino acid sequences of the detected GTP-binding domains were aligned by CLUSTALW (Thompson et al., 1994
) and their phylogenetic tree was constructed using TreeView (Fig. 2a
). The resultant tree indicated that the five ribosomal function proteins and the remaining nine form distinct subfamilies, leading us to conclude that the B. subtilis genome contains nine GTP-binding proteins that are structurally related to Era and Obg. The amino acid sequences of the GTP-binding domain of these nine proteins are shown in Fig. 2b
and the location of the domain in each protein is shown schematically in Fig. 2c
. The YphC protein contains two GTP-binding domains. Three motifs (G1, G3 and G4) were well conserved, and a conserved threonine was identified in G2 among the ten GTP-binding domains (Fig. 2b
), while amino acid sequences outside of the GTP-binding domains were unique to each protein.
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yphC and yqeH are the first genes of operons containing two and seven genes, respectively (Fig. 3h and i
). Therefore the artificial control of their expression by integration of pMUTINT3 should cause a polar effect on the expression of downstream genes. To avoid this, we first cloned the complete yphC and yqeH genes downstream of the spac promoter in pDLT3 or pAPNC213 and integrated them into the amyE or aprE loci, respectively, through transformation of wild-type cells with linearized plasmid DNA and selection for chloramphenicol or spectinomycin resistance (Fig. 3h
and i
). Then, each original gene was replaced by the tet gene and the expression of downstream genes were maintained by the tet promoter to obtain IPTG-dependent mutants. This work showed that Bex, YphC, YqeH and YlqG are essential in B. subtilis.
These essential B. subtilis GTP-binding proteins bind specifically to GTP and GDP in vitro
Specific GTP/GDP binding and intrinsic GTPase activities have been demonstrated for B. subtilis Obg, C. crescentus CgtA, and Thermotoga maritima TrmE in vitro (Welsh et al., 1994 ; Lin et al., 1999
; Yamanaka et al., 2000
), so we next analysed the GTP/GDP binding activities for Bex, YlqF, YphC, YqeH and YsxC proteins. Test proteins were fused to a histidine tag (His6), expressed in E. coli and purified to homogeneity (Fig. 4a
). Since B. subtilis cells with His-tagged replacement genes grew normally except in the case of yphC (data not shown), we used purified His-tagged proteins for the GTP-binding assays except for the case of YphC, in which thrombin digestion was used to remove the His-tag before the GTP-binding assay (Fig. 4a
).
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The effect of depletion of these essential GTP-binding proteins on cell morphology and chromosome replication
Next we used B. subtilis cells to analyse the effect of depletion of these essential GTP-binding proteins on cell morphology and chromosome replication, to begin understanding whether they are involved in regulating cell cycle progression. IPTG-dependent mutants were cultivated in antibiotic medium 3 with 100 µM IPTG to OD600 0·4, and cells were washed and diluted in the same medium without IPTG. Immunoblotting indicated that the amount of each tested protein decreased to less than 10% of control levels after approximately four generations in the absence of IPTG, which is the point at which the growth rate of the mutant cells started to decrease. Microscopic examination of Bex- and YqeH-depleted cells cultivated for nine generations without IPTG revealed that cells became 1·5 to 2 times longer compared to wild-type, and nucleoid distribution dispersed (Fig. 5b and g
). On the other hand, cell length increased more than threefold and cell shape was abnormally curved in Obg-, YphC-, YsxC- and YlqF-depleted cells. Furthermore, nucleoid condensation was observed in these cells (Fig. 5c
, d
, e
and f
). No further changes in the morphology of cells and nucleoids were observed after a longer period of depletion.
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DISCUSSION |
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Three of the identified Era/Obg family members were found to be dispensable for normal growth in B. subtilis: ThdF, YnbA, and YyaF. The homologue of ThdF in E. coli (TrmE) was reported to be involved in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine (found in the wobble position of some tRNAs) and dispensable in certain genetic backgrounds (Cabedo et al., 1999 ). The E. coli counterpart of ynbA (called hflX) encodes a regulatory subunit of the CII repressor of lambda phage. However, there is no data to suggest that B. subtilis ynbA might relate to phage function. Although dispensable in B. subtilis, yyaF is conserved in all micro-organisms including archaea and yeast (COG database in NCBI). Furthermore, the YyaF homologue has been identified in the genomes of higher organisms. Therefore this protein could also play an important role in cell growth. Although phenotypes of the thdF, ynbA and yyaF disruptants were not studied in this work, their preliminary characterization by the Japanese and European consortia for functional analysis of the B. subtilis genome (Ogasawara, 2001
) is available in BSORF (http://bacillus.genome.ad.jp/) and Micado (http://locus.jouy.inra.fr/cgi-bin/genmic/madbase/progs /madbase.operl) databases.
Based on the observed phenotypes of cells in which each protein was depleted, the six essential GTP-binding proteins were classified into two groups. Depletion of Bex or YqeH induced an excess initiation of DNA replication, suggesting that these proteins negatively control initiation of chromosome replication. Both positive and negative regulators of replication initiation have been reported previously in E. coli. An overproduction of the replication initiator protein DnaA resulted in overinitiation (Atlung & Hansen, 1993 ). Sequestration of newly duplicated hemimethylated origins into the cell membrane by the SeqA protein (Lu et al., 1994
; Onogi et al., 1999
) and inactivation of the DnaA protein by the DnaN and Hda proteins (Katayama et al., 1998
; Kato & Katayama, 2001
) are known negative regulatory mechanisms for suppression of initiation. Recently, we reported that DnaA levels in the B. subtilis cell could also act as a positive regulatory system for the initiation of chromosome replication (Ogura et al., 2001
). However, no negative regulatory system controlling chromosome replication has been previously identified in B. subtilis. Thus the finding that Bex and YqeH negatively regulate replication initiation is an important step towards understanding the regulatory network of chromosome replication in B. subtilis. In E. coli, a mutation in era (homologue of B. subtilis bex) was reported to suppress several temperature-sensitive lethal alleles of genes involved in chromosome partitioning. On the other hand, the mutation did not suppress any of the cell division and DNA replication initiation mutations (Britton et al., 1998
). The suppression of the defect in chromosome partitioning by the era mutant might be due to overinitiation of replication. The genes encoding the Bex and YqeH homologues are located side by side in T. maritima. Powers & Walter (1995)
demonstrated that the GTPases FtsY and Ffh interact directly and regulate their GTPase activities reciprocally in E. coli. Such direct coupling between Bex and YqeH in B. subtilis will be an interesting model for future study.
The depletion of Obg, YphC, YsxC, and YlqF resulted in cell elongation, abnormal cell curvature and nucleoid condensation without apparent change in the DNA histogram upon flow cytometry analysis. Although we did not fully examine the various roles of these proteins in a direct manner, our observations are compatible with the previous observation that depletion of the Obg homologue (ObgE) or YsxC homologue (YihA) in E. coli caused defects in cell division (Kobayashi et al., 2001 ; Dassain et al., 1999
). In B. subtilis, it has been proposed that Obg is involved in the initiation of chromosome replication (Kok et al., 1994
). However, our results do not support this hypothesis, necessitating further work in this area.
The complete genome sequences of diverse organisms have revealed the existence of more unknown genes than had been anticipated. Our work revealed that many GTP-binding proteins of the Era/Obg family play essential but unknown functions in the growth of B. subtilis. Our results together with previously published reports indicate that many Era/Obg family members are conserved and play important roles in different facets of bacterial function. Thus, as we look towards the future, the GTP-binding proteins of the Era/Obg family will be an important protein family whose members require further study to elucidate their precise roles in bacterial growth and health.
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ACKNOWLEDGEMENTS |
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Received 18 April 2002;
revised 21 June 2002;
accepted 2 July 2002.