The Vibrio harveyi GTPase CgtAV Is Essential and Is Associated with the 50S Ribosomal Subunit
http://www.100md.com
《细菌学杂志》
Department of Molecular, Cellular and Developmental Biology, University of Michigan, 830 North University, Ann Arbor, Michigan 48109,Department of Molecular Biology, University of Gdansk, Kadki 24, 80-822 Gdansk, Poland
ABSTRACT
It was previously reported that unlike the other obg/cgtA GTPases, the Vibrio harveyi cgtAV is not essential. Here we show that cgtAV was not disrupted in these studies and is, in fact, essential for viability. Depletion of CgtAV did not result in cell elongation. CgtAV is associated with the large ribosomal particle. In light of our results, we predict that the V. harveyi CgtAV protein plays a similar essential role to that seen for Obg/CgtA proteins in other bacteria.
TEXT
All living organisms express one or more Obg/CgtA GTPase. Elucidating the function of these GTPases has been complicated by somewhat contradictory phenotypes associated with specific mutants in different model systems. Some of the confusion stems from the dual function of these proteins (ribosome assembly and stress response) in at least some organisms, as well as from potential species-specific differences that may result in different phenotypes observed for orthologous obg/cgtA mutants. Perhaps the most surprising of these inconsistent reports is that of the nonessential nature of the Vibrio harveyi cgtAV gene (5) and its pleiotropic phenotypes (32, 34, 42), observations at odds with what has been reported for other obg/cgtA mutants. Here we show that, in contrast to these studies, the V. harveyi cgtAV gene is essential. Furthermore, we demonstrate that the trimethoprim resistance associated with a cgtAV clone was conferred by the linked folA gene. We also show that CgtAV is associated with the 50S ribosomal particle. From these studies, we predict that the role of CgtAV is similar to that of other bacterial Obg/CgtA proteins.
Bacterial strains, plasmids, and growth conditions. The strains and plasmids used in the present study are listed in Table 1. Escherichia coli cells were grown at 37°C (unless otherwise indicated) in Luria-Bertani media (LB; 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl/liter) or LB agar (1.5% agar) containing antibiotics, as required (gentamicin, 30 μg/ml; kanamycin, 30 μg/ml; chloramphenicol, 10 μg/ml; trimethoprim, 100 μg/ml; ampicillin, 100 μg/ml). V. harveyi strains were derived from BB7 (2) and maintained at 30°C on BOSS medium (10 g of Bacto Peptone, 3 g of beef extract, 30 g of NaCl, and 1 ml of 100% glycerol/liter) or BOSS agar (1.5% agar) supplemented with antibiotics (gentamicin, 30 μg/ml; kanamycin, 50 μg/ml).
The V. harveyi cgtAV gene is essential. We were perplexed by the reported viability of the V. harveyi cgtAV transposon insertion mutant BB7X (5) for several reasons. First, in all other organisms examined, obg/cgtA is an essential gene (1, 16, 22, 25, 37, 40). Second, given the sequence similarity among the Obg/CgtA proteins, we would predict that CgtA proteins play similar roles in all bacteria, and yet the viable nature and reported phenotypes of the V. harveyi cgtAV insertional mutant (5, 32, 34, 42) were different from those reported for other obg/cgtA mutant strains (7, 16, 17, 22, 26, 38). Therefore, we reinvestigated the consequences of CgtAV depletion in V. harveyi. Plasmids used in the present study are listed in Table 1 and were introduced into V. harveyi by conjugation with E. coli S17.1. Cloning and plasmid amplification were performed in E. coli DH5.
Initially, we constructed a chromosomal integrant in which expression of cgtAV was controlled by the PBAD promoter. This strain grew in the presence but not the absence of arabinose (data not shown), indicating that either cgtAV or a downstream gene was essential for viability. To rule out possible contributions from downstream genes, we disrupted the chromosomal cgtAV gene by integration of an internal cgtAV fragment and complemented with a plasmid harboring cgtAV (PCR amplified with the primers 5'-TAATCCACGCTAGCAGTAGTCGGAG and 5'-GGCTTAATGACTGCAGTAGCGATTA) controlled by the PBAD promoter (PBAD-cgtAV; BB7AS31) (Table 1). A control strain, BB7AS26, which expressed PBAD-cgtAV in wild-type BB7 cells, was characterized in parallel. BB7AS31 cells grew in both liquid culture (Fig. 1A) and on plates (Fig. 1C) supplemented with 0.01% arabinose. Under these conditions, the cellular level of CgtAV in BB7AS31 and BB7AS26 was similar and was two- to fivefold more than that of wild-type V. harveyi (Fig. 1B). In the absence of arabinose, however, BB7AS31 cells showed a reduction in cell growth in liquid medium (Fig. 1A) and a reduction in cell viability (Fig. 1C). Furthermore, in the absence of CgtAV expression, no colonies formed on plates (Fig. 1C). We conclude that, in contrast to prior reports (5, 32, 34, 42), cgtAV is an essential gene.
In E. coli, overexpression of CgtAE results in elongated cells with aberrant chromosomal segregation (9, 16). Furthermore, overexpression of Obg in S. griseus impairs differentiation (25). To determine whether overexpression of CgtAV resulted in a growth phenotype, we induced expression of the episomal copy of cgtAV in wild-type cells (strain BB7AS26) with various amounts of arabinose. In the absence of arabinose, growth of BB7AS26 cells was identical to that of the wild-type controls. Up to 10-fold induction of CgtAV protein (0.1% arabinose) had no significant effect on cell growth in liquid medium or on plates (data not shown). Thus, a modest overexpression of CgtAV is not deleterious to V. harveyi. In contrast, in the absence of arabinose the levels of CgtAV in BB7AS31 were significantly reduced after 1.5 h and CgtAV was not detectable after 4.5 h (Fig. 1B). In addition, we note a reduction in the level of CgtAV in the control strain, BB7AS26, as cells entered stationary phase (Fig. 1B). A reduction in the levels of CgtA protein upon entry into stationary phase has been previously been reported for Streptomyces coelicolor (26) and E. coli (16).
The Obg/CgtA proteins have been implicated in cell cycle and/or DNA replication control in a number of bacteria. In E. coli, cgtAE temperature-sensitive mutant cells grown to stationary phase and shifted to the nonpermissive temperature become filamentous and have defects in chromosome partitioning (16). A transposon fusion mutant of CgtAE displays defects in the coordinate timing of DNA replication initiation (12). In the developmental bacterium Caulobacter crescentus, cgtAC(ts) mutants arrest prior to the onset to DNA replication (7). In V. harveyi, however, we did not observe a change in cell morphology, as judged by light microscopy, or DNA partitioning, as judged by DAPI (4',6'-diamidino-2-phenylindole) staining, upon depletion of CgtAV (data not shown). Thus, depletion of CgtAV does not result in either a cell elongation or DNA partitioning phenotype. Therefore, it is likely that a role in cell division and/or DNA replication is not a core function for all Obg/CgtA proteins. One possible explanation is that cell division and DNA replication defects are species-specific downstream consequences of obg/cgtA depletion. In addition, discrepancies between the phenotypes of different cgtA mutants may be due to the nature of the lesion (temperature sensitive, depletion or protein fusion) and/or the status of the cells during analysis (stationary versus exponentially growing cells).
The previously reported cgtA::Tn5TpMCS mutant, BB7X, encodes a wild-type cgtAV gene. There has been a series of studies describing the phenotype of BB7X (5, 32, 34, 42), a strain reported to harbor a cgtA::Tn5TprMCS allele. The plasmid pAC1, encoding at least part of the cgtAV gene, was obtained by digestion of BB7X DNA with EcoRI, ligation to pUC19, and selection for both trimethoprim and ampicillin resistance (5). The expectation was that trimethoprim-resistant transformants would harbor both the Tn5 transposon (encoding dhfrII, the dihydrofolate reductase gene [21]) and flanking chromosomal DNA. Using a primer complementary to the IS50 element of Tn5TprMCS (Tn, 5'-TTCAGGACGCTACTTGTGTA-3'), the sequence of the N-terminal 99 amino acids of cgtAV was obtained, and it was concluded that BB7X contained a cgtAV::Tn5TprMCS insertion mutation (5).
In contrast to this conclusion, we demonstrate through several lines of evidence that BB7X encodes a wild-type cgtAV gene. First, immunoblot analysis of cell extracts from BB7 and BB7X with anti-CgtA antibodies resulted in the detection of a protein band that migrates at ca. 48 kDa, 5 kDa larger than the expected size of CgtAV. A slightly slower migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been previously noted for the C. crescentus (19) and E. coli (39) CgtA proteins. This immunoreactive band migrates at the same position as purified CgtAV protein (data not shown). Second, we designed a primer to a conserved region of rpmA (5'-GAGGATCCATCATCGTTCGTCAACG), the gene predicted to be upstream of cgtAV based on conservation of gene organization in most bacteria, including Vibrio species. PCR amplification of BB7 and BB7X chromosomal DNA with this primer and a primer downstream of cgtAV (5'-ATGGATCCGCAAATCACATCGTCT) produced a 1.6-kb PCR product regardless of the source of chromosomal DNA. Sequence analysis of the PCR products shows that the DNA is identical and encodes a full-length cgtAV gene (data not shown). Third, we sequenced the entire 5-kb EcoRI DNA fragment of pAC1 on both strands. This DNA insert did not contain the Tn5TprMCS transposon (Fig. 2A). Finally, examination of the cgtAV sequence reveals that nucleotides 315 to 326 are 83% complementary to the last 12 bases of the Tn primer used to sequence the N-terminal region of pAC1 (5). We propose that the complementarity between the Tn primer and this region of cgtAV was sufficient to generate the DNA sequence reported previously (5). We conclude, therefore, that BB7X encodes a wild-type cgtAV gene and that publications referring to the characterization of the V. harveyi BB7X mutant (5, 32, 34, 42) do not describe a bona fide cgtAV mutant. The true nature of the Tn5TprMCS insertion in BB7X was not explored further.
Expression of the V. harveyi folA gene in E. coli results in trimethoprim resistance. Because pAC1 did not contain a Tn5TpMCS transposon, we investigated the nature of the trimethoprim resistance conferred by the pAC1 plasmid. Introduction of pAC1, but not the high-copy vector pUC18 or the low-copy plasmid pGD103, conferred trimethoprim resistance to E. coli DH5 cells (Fig. 2B). Trimethoprim resistance was not due to expression of the V. harveyi cgtAV gene, since cells containing pAES7, a high-copy plasmid containing the cgtAV gene and promoter (PCR amplified with primers 5'-GAGGATCCATCATCGTTCGTCAACG and 5'-ATGGATCCGCAAATCACATCGTCT), were trimethoprim sensitive (Tps) (Fig. 2B).
To determine the nature of the trimethoprim resistance, we sequenced cgtAV and the surrounding genomic region. Sequencing was performed at the Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland, or at the University of Michigan Sequencing Core Facility; all oligonucleotide sequences are available upon request. The entire cgtAV coding region was amplified by PCR (polymerase Pfu [Promega] or Red Taq [Sigma]) from BB7 and BB7X (4) colonies or genomic DNA (isolated by using High Pure PCR Template Preparation Kit; Roche) or from pAC1 using primers designed to conserved regions in obg/cgtA genes and by inverse PCR. Our sequence (NCBI AY623050) agrees with the partial 5' sequence (300 bp) of the cgtAV gene (5) that was previously reported. The 5-kb EcoRI fragment of the pAC1 plasmid (5), as well as subclones generated in the present study, was sequenced on both strands by using appropriate oligonucleotide primers. The region upstream of cgtAV was amplified by using a primer complementary to a conserved region of ispB (a gene commonly present upstream of obg/cgtA genes) (5'-AGTTGGGCTTGAATTGTTTCATTCAC) and an internal cgtAV primer (5'-AACTTTTACTACCGCTTCATCTACG). This sequence includes rplU, rpmA (coding for the 50S ribosomal proteins L21 and L27, respectively), and the cgtAV promoter region, and its organization is similar to that found in most bacteria (NCBI DQ180600).
Interestingly, the gene organization downstream of cgtAV (NCBI DQ180601) is quite different from that found in the majority of bacteria, although it is conserved in other Vibrio species. Downstream of cgtAV are two conserved hypothetical genes, yjjP and yjjB, as well as folA (dihydrofolate reductase type 1). Transcribed divergently are apaH (diadenosine tetraphosphatase), apaG, and the C terminus of ksgA (RNA adenine dimethyltransferase) (Fig. 2A). The functions of YjjP and YjjB are unknown, although both are predicted to be transmembrane proteins. In E. coli, the folA, apaH, apaG, and ksgA genes are physically linked but, unlike what we observe in V. harveyi, this gene cluster is not located near cgtAE.
We were struck by the dual observation that the third gene downstream of cgtAV is folA and that V. harveyi FolA is predicted to be 52% identical to E. coli FolA. Although several mechanisms of trimethoprim resistance in E. coli have been described (35), the most common mechanism is an increase in folA expression (3, 11, 28, 36). To determine whether the trimethoprim resistance conferred by pAC1 was caused by expression of V. harveyi folA, we PCR amplified folA from BB7 and BB7X cells by using the primers 5'-CTATCGCCGTGGATCCATAGTTTAA and 5'-TTGCGGTGCTTTCTGCAGTCTATTC and cloned the fragments onto the high-copy plasmids pCR2.1-TOPO and pUC18 and the low-copy plasmid pGD103. DH5 transformants harboring any of these plasmids were Tpr (Fig. 2B), indicating that expression of the V. harveyi folA gene was sufficient to confer trimethoprim resistance to E. coli, even at a relatively low copy number. We observed equivalent Tpr, regardless of whether the source of folA was BB7 or BB7X (Fig. 2B), a finding consistent with our sequence analysis indicating that the folA gene was identical in each strain (data not shown). Thus, we conclude that the Tpr phenotype of E. coli cells harboring pAC1 was due to expression of the V. harveyi folA gene and not due to a transposon insertion in cgtAV, as had previously been reported (5).
The V. harveyi CgtAV protein is associated with the 50S ribosomal particle. In all organisms, a number of GTP-binding proteins, including the Obg/CgtA proteins, are predicted to be involved in some aspect of translation (18). In the case of the bacterial Obg/CgtA proteins, direct association with the large ribosomal subunit has been demonstrated for the E. coli (29, 39), C. crescentus (20), and B. subtilis (31, 41) proteins. Interestingly, the eukaryotic Obg/CgtA proteins are also ribosome associated. In yeast, the mitochondrial GTPase Mtg2p is associated with the large ribosomal subunit (6), the cytosolic GTPases Rbg1p and Rbg2p are associated with translating ribosomes (P. Wout and J. R. Maddock, unpublished), and the nucleolar Nog1p is associated with the pre-60S particle (14, 15). Thus, ribosome association appears to be a conserved feature of the Obg/CgtA family. To determine whether this was also the case for the V. harveyi CgtAV protein, we isolated ribosomes, as previously described (10) from 50 ml of culture of V. harveyi BB7 that were grown at 30°C to an optical density at 600 nm of 0.5. The extract was clarified by centrifugation (10 min, 30,000 x g) and separated on 10 to 30% sucrose gradients (10 ml; 10 mM Tris-HCl [pH 7.5], 30 mM KCl, 5.25 mM magnesium acetate) by centrifugation in a Beckman SW41 Ti rotor at 41,000 rpm for 3 h. Fractions were collected as described previously (20) with the polysome profile monitored by UV absorbance (254 nm). Fractions were precipitated and examined by immunoblotting with purified anti-CgtAC antibodies (27). CgtAV was found in the 50S fractions and at the top of the gradient (Fig. 3). Thus, CgtAV is at least partially associated with the 50S ribosomal particle but not with the 30S particle, the 70S monosome, or with translating ribosomes.
Concluding remarks. In conclusion, we show that in contrast to previous publications (5, 32, 34, 42) cgtAV is an essential gene and that depletion of cgtAV does not lead to a cell elongation phenotype. Moreover, CgtAV is associated with the large ribosomal subunit. It is likely, therefore, that the V. harveyi CgtAV protein plays a cellular role similar to that of other bacterial Obg/CgtA proteins.
ACKNOWLEDGMENTS
We are extremely grateful Susan Sullivan for careful critique of the manuscript and to Patrice Wout for technical assistance. We are also grateful to Grzegorz Wgrzyn for insightful discussions during the course of this study.
This study was supported by a collaborative FIRCA grant (TW006001) from the National Institutes of Health and by NATO funds to J.R.M. and Grzegorz Wgrzyn.
REFERENCES
Arigoni, F., F. Talabot, M. Peitsch, M. D. Edgerton, E. Meldrum, E. Allet, R. Fish, T. Jamotte, M. L. Curchod, and H. Loferer. 1998. A genome-based approach for the identification of essential bacterial genes. Nat. Biotechnol. 16:851-856.
Belas, R., A. Mileham, D. Cohn, M. Hilman, M. Simon, and M. Silverman. 1982. Bacterial bioluminescence: isolation and expression of the luciferase genes from Vibrio harveyi. Science 218:791-793.
Breeze, A. S., P. Sims, and K. A. Tracey. 1975. Trimethoprim-resistant mutants of Escherichia coli K-12: preliminary genetic mapping. Genet. Res. 74:543-556.
Czyz, A., J. Jasiecki, A. Bogdan, H. Szpilewska, and G. Wgrzyn. 2000. Genetically modified Vibrio harveyi strains as potential bioindicators of mutagenic pollution of marine environments. Appl. Environ. Microbiol. 66:599-605.
Czyz, A., R. Zielke, G. Konopa, and G. Wgrzyn. 2001. A Vibrio harveyi insertional mutant in the cgtA (obg, yhbZ) gene, whose homologues are present in diverse organisms ranging from bacteria to humans and are essential genes in many bacterial species. Microbiology 147:183-191.
Datta, K., J. L. Fuentes, and J. R. Maddock. 2005. The yeast GTPase Mtg2p is required for mitochondrial translation and partially suppresses an rRNA methyltransferase mutant, mrm2. Mol. Biol. Cell 16:954-963.
Datta, K., J. M. Skidmore, K. Pu, and J. R. Maddock. 2004. The Caulobacter crescentus GTPase CgtAc is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels. Mol. Microbiol. 54:1379-1392.
Deich, R. A., B. J. Metcalf, C. W. Finn, J. E. Farley, and B. A. Green. 1988. Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus influenzae. J. Bacteriol. 170:489-498.
Dutkiewicz, R., M. Slominska, G. Wgrzyn, and A. Czyz. 2002. Overexpression of the cgtA (yhbZ, obgE) gene, coding for an essential GTP-binding protein, impairs the regulation of chromosomal functions in Escherichia coli. Curr. Microbiol. 45:440-445.
Flardh, K., P. S. Cohen, and S. Kjelleberg. 1992. Ribosomes exist in large excess over the apparent demand for protein synthesis during carbon starvation in marine Vibrio sp. J. Bacteriol. 174:6780-6788.
Flensburg, J., and O. Skold. 1987. Massive overproduction of dihydrofolate reductase in bacteria as a response to the use of trimethoprim. Eur. J. Biochem. 162:473-476.
Foti, J. J., J. Scheienda, V. A. J. Sutera, and S. T. Lovett. 2005. A bacterial G protein-mediated response to replication arrest. Mol. Cell 17:549-560.
Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580.
Jensen, B. C., Q. Wang, C. T. Kifer, and M. Parsons. 2003. The NOG1 GTP-binding protein is required for biogenesis of the 60S ribosomal subunit. J. Biol. Chem. 278:32204-32211.
Kallstrom, G., J. Hedges, and A. Johnson. 2003. The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively. Mol. Cell. Biol. 23:4344-4355.
Kobayashi, G., S. Moriya, and C. Wada. 2001. Deficiency of essential GTP-binding protein ObgE in Escherichia coli inhibits chromosome partition. Mol. Microbiol. 41:1037-1051.
Kok, J., K. A. Trach, and J. A. Hoch. 1994. Effects on Bacillus subtilis of a conditional lethal mutation in the essential GTP-binding protein Obg. J. Bacteriol. 176:7155-7160.
Leipe, D. D., Y. I. Wolf, E. V. Koonin, and L. Aravind. 2002. Classification and evolution of P-loop GTPases and related ATPases. J. Mol. Biol. 317:41-72.
Lin, B., K. L. Covalle, and J. R. Maddock. 1999. The Caulobacter crescentus CgtA protein displays unusual guanine nucleotide binding and exchange properties. J. Bacteriol. 181:5825-5832.
Lin, B., D. A. Thayer, and J. R. Maddock. 2004. The Caulobacter crescentus CgtAC protein cosediments with the free 50S ribosomal subunit. J. Bacteriol. 186:481-489.
MacKenzie, C., M. Chidambaram, E. J. Sodergren, S. Kaplan, and G. M. Weinstock. 1995. DNA repair mutants of Rhodobacter sphaeroides. J. Bacteriol. 177:3027-3035.
Maddock, J., A. Bhatt, M. Koch, and J. Skidmore. 1997. Identification of an essential Caulobacter crescentus gene encoding a member of the Obg family of GTP-binding proteins. J. Bacteriol. 179:6426-6431.
Newman, J. R., and C. Fuqua. 1999. Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene 227:197-203.
Norrander, J., T. Kempe, and J. Messing. 1983. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101-106.
Okamoto, S., M. Itoh, and K. Ochi. 1997. Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation. J. Bacteriol. 179:170-179.
Okamoto, S., and K. Ochi. 1998. An essential GTP-binding protein functions as a regulator of differentiation in Streptomyces coelicolor. Mol. Microbiol. 30:107-119.
Olmsted, J. B. 1981. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J. Biol. Chem. 256:11955-11957.
Rood, J. I., A. J. Laird, and J. W. Williams. 1980. Cloning of the Escherichia coli K-12 dihydrofolate reductase gene following mu-mediated transposition. Gene 8:255-265.
Sato, A., G. Kobayashi, H. Hayashi, H. Yoshida, A. Wada, M. Maeda, S. Hiraga, K. Takeyasu, and C. Wada. 2005. The GTP binding protein Obg homolog ObgE is involved in ribosome maturation. Genes Cells 10:393-408.
Schafer, A., A. Tauch, W. Jager, J. Kalinowski, G. Thierbach, and A. Puchler. 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69-73.
Scott, J. M., J. Ju, T. Mitchell, and W. G. Haldenwang. 2000. The Bacillus subtilis GTP binding protein Obg and regulators of the B stress response transcription factor cofractionate with ribosomes. J. Bacteriol. 182:2771-2777.
Sikora-Borgula, A., M. Slominska, P. Trzonkowski, R. Zielke, A. Mysliwski, G. Wegrzyn, and A. Czyz. 2002. A role for the common GTP-binding protein in coupling of chromosome replication to cell growth and division. Biochem. Biophys. Res. Commun. 292:333-338.
Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Biotechnology 1:784-790.
Slominska, M., G. Konopa, G. Wgrzyn, and A. Czyz. 2002. Impaired chromosome partitioning and synchronization of DNA replication initiation in a Vibrio harveyi insertional mutant in the cgtA gene coding for a common GTP-binding protein. Biochem. J. 362:579-584.
Smith, D. R., and J. M. Calvo. 1982. Nucleotide sequence of dihydrofolate reductase genes from trimethoprim-resistant mutants of Escherichia coli. Evidence that dihydrofolate reductase interacts with another essential gene product. Mol. Gen. Genet. 187:72-78.
Smith, D. R., and J. M. Calvo. 1979. Regulation of dihydrofolate reductase synthesis in Escherichia coli. Mol. Gen. Genet. 175:31-38.
Trach, K., and J. A. Hoch. 1989. the Bacillus subtilis spoOB stage 0 sporulation operon encodes an essential GTP-binding protein. J. Bacteriol. 171:1362-1371.
Vidwans, S. J., K. Ireton, and A. D. Grossman. 1995. Possible role for the essential GTP-binding protein, Obg in regulating the initiation of sporulation in Bacillus subtilis. J. Bacteriol. 177:3308-3311.
Wout, P., K. Pu, S. M. Sullivan, V. Reese, S. Zhou, B. Lin, and J. R. Maddock. 2004. The Escherichia coli GTPase, CgtAE, cofractionates with the 50S ribosomal subunit and interacts with SpoT, a ppGpp synthetase/hydrolase. J. Bacteriol. 186:5249-5257.
Zalacain, M., S. Biswas, K. A. Ingraham, J. Ambrad, A. Bryant., A. F. Chalker, S. Iordanescu, J. Fan, F. Fan, R. D. Lunsford, K. O'Dwyer, L. M. Palmer, C. So, D. Sylvester, C. Volker, P. Warren, D. McDevitt, J. R. Brown, D. J. Holmes, and M. K. Burnham. 2003. A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function. J. Mol. Microbiol. Biotechnol. 6:109-126.
Zhang, S., and W. G. Haldenwang. 2004. Guanine nucleotides stabilize the binding of Bacillus subtilis Obg to ribosomes. Biochem. Biophys. Res. Commun. 322:565-569.
Zielke, R., A. Sikora, R. Dutkiewicz, G. Wgrzyn, and A. Czyz. 2003. Involvement of the cgtA gene function in stimulation of DNA repair in Escherichia coli and Vibrio harveyi. Microbiology 149:1763-1770.(A. E. Sikora, R. Zielke, )
ABSTRACT
It was previously reported that unlike the other obg/cgtA GTPases, the Vibrio harveyi cgtAV is not essential. Here we show that cgtAV was not disrupted in these studies and is, in fact, essential for viability. Depletion of CgtAV did not result in cell elongation. CgtAV is associated with the large ribosomal particle. In light of our results, we predict that the V. harveyi CgtAV protein plays a similar essential role to that seen for Obg/CgtA proteins in other bacteria.
TEXT
All living organisms express one or more Obg/CgtA GTPase. Elucidating the function of these GTPases has been complicated by somewhat contradictory phenotypes associated with specific mutants in different model systems. Some of the confusion stems from the dual function of these proteins (ribosome assembly and stress response) in at least some organisms, as well as from potential species-specific differences that may result in different phenotypes observed for orthologous obg/cgtA mutants. Perhaps the most surprising of these inconsistent reports is that of the nonessential nature of the Vibrio harveyi cgtAV gene (5) and its pleiotropic phenotypes (32, 34, 42), observations at odds with what has been reported for other obg/cgtA mutants. Here we show that, in contrast to these studies, the V. harveyi cgtAV gene is essential. Furthermore, we demonstrate that the trimethoprim resistance associated with a cgtAV clone was conferred by the linked folA gene. We also show that CgtAV is associated with the 50S ribosomal particle. From these studies, we predict that the role of CgtAV is similar to that of other bacterial Obg/CgtA proteins.
Bacterial strains, plasmids, and growth conditions. The strains and plasmids used in the present study are listed in Table 1. Escherichia coli cells were grown at 37°C (unless otherwise indicated) in Luria-Bertani media (LB; 10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl/liter) or LB agar (1.5% agar) containing antibiotics, as required (gentamicin, 30 μg/ml; kanamycin, 30 μg/ml; chloramphenicol, 10 μg/ml; trimethoprim, 100 μg/ml; ampicillin, 100 μg/ml). V. harveyi strains were derived from BB7 (2) and maintained at 30°C on BOSS medium (10 g of Bacto Peptone, 3 g of beef extract, 30 g of NaCl, and 1 ml of 100% glycerol/liter) or BOSS agar (1.5% agar) supplemented with antibiotics (gentamicin, 30 μg/ml; kanamycin, 50 μg/ml).
The V. harveyi cgtAV gene is essential. We were perplexed by the reported viability of the V. harveyi cgtAV transposon insertion mutant BB7X (5) for several reasons. First, in all other organisms examined, obg/cgtA is an essential gene (1, 16, 22, 25, 37, 40). Second, given the sequence similarity among the Obg/CgtA proteins, we would predict that CgtA proteins play similar roles in all bacteria, and yet the viable nature and reported phenotypes of the V. harveyi cgtAV insertional mutant (5, 32, 34, 42) were different from those reported for other obg/cgtA mutant strains (7, 16, 17, 22, 26, 38). Therefore, we reinvestigated the consequences of CgtAV depletion in V. harveyi. Plasmids used in the present study are listed in Table 1 and were introduced into V. harveyi by conjugation with E. coli S17.1. Cloning and plasmid amplification were performed in E. coli DH5.
Initially, we constructed a chromosomal integrant in which expression of cgtAV was controlled by the PBAD promoter. This strain grew in the presence but not the absence of arabinose (data not shown), indicating that either cgtAV or a downstream gene was essential for viability. To rule out possible contributions from downstream genes, we disrupted the chromosomal cgtAV gene by integration of an internal cgtAV fragment and complemented with a plasmid harboring cgtAV (PCR amplified with the primers 5'-TAATCCACGCTAGCAGTAGTCGGAG and 5'-GGCTTAATGACTGCAGTAGCGATTA) controlled by the PBAD promoter (PBAD-cgtAV; BB7AS31) (Table 1). A control strain, BB7AS26, which expressed PBAD-cgtAV in wild-type BB7 cells, was characterized in parallel. BB7AS31 cells grew in both liquid culture (Fig. 1A) and on plates (Fig. 1C) supplemented with 0.01% arabinose. Under these conditions, the cellular level of CgtAV in BB7AS31 and BB7AS26 was similar and was two- to fivefold more than that of wild-type V. harveyi (Fig. 1B). In the absence of arabinose, however, BB7AS31 cells showed a reduction in cell growth in liquid medium (Fig. 1A) and a reduction in cell viability (Fig. 1C). Furthermore, in the absence of CgtAV expression, no colonies formed on plates (Fig. 1C). We conclude that, in contrast to prior reports (5, 32, 34, 42), cgtAV is an essential gene.
In E. coli, overexpression of CgtAE results in elongated cells with aberrant chromosomal segregation (9, 16). Furthermore, overexpression of Obg in S. griseus impairs differentiation (25). To determine whether overexpression of CgtAV resulted in a growth phenotype, we induced expression of the episomal copy of cgtAV in wild-type cells (strain BB7AS26) with various amounts of arabinose. In the absence of arabinose, growth of BB7AS26 cells was identical to that of the wild-type controls. Up to 10-fold induction of CgtAV protein (0.1% arabinose) had no significant effect on cell growth in liquid medium or on plates (data not shown). Thus, a modest overexpression of CgtAV is not deleterious to V. harveyi. In contrast, in the absence of arabinose the levels of CgtAV in BB7AS31 were significantly reduced after 1.5 h and CgtAV was not detectable after 4.5 h (Fig. 1B). In addition, we note a reduction in the level of CgtAV in the control strain, BB7AS26, as cells entered stationary phase (Fig. 1B). A reduction in the levels of CgtA protein upon entry into stationary phase has been previously been reported for Streptomyces coelicolor (26) and E. coli (16).
The Obg/CgtA proteins have been implicated in cell cycle and/or DNA replication control in a number of bacteria. In E. coli, cgtAE temperature-sensitive mutant cells grown to stationary phase and shifted to the nonpermissive temperature become filamentous and have defects in chromosome partitioning (16). A transposon fusion mutant of CgtAE displays defects in the coordinate timing of DNA replication initiation (12). In the developmental bacterium Caulobacter crescentus, cgtAC(ts) mutants arrest prior to the onset to DNA replication (7). In V. harveyi, however, we did not observe a change in cell morphology, as judged by light microscopy, or DNA partitioning, as judged by DAPI (4',6'-diamidino-2-phenylindole) staining, upon depletion of CgtAV (data not shown). Thus, depletion of CgtAV does not result in either a cell elongation or DNA partitioning phenotype. Therefore, it is likely that a role in cell division and/or DNA replication is not a core function for all Obg/CgtA proteins. One possible explanation is that cell division and DNA replication defects are species-specific downstream consequences of obg/cgtA depletion. In addition, discrepancies between the phenotypes of different cgtA mutants may be due to the nature of the lesion (temperature sensitive, depletion or protein fusion) and/or the status of the cells during analysis (stationary versus exponentially growing cells).
The previously reported cgtA::Tn5TpMCS mutant, BB7X, encodes a wild-type cgtAV gene. There has been a series of studies describing the phenotype of BB7X (5, 32, 34, 42), a strain reported to harbor a cgtA::Tn5TprMCS allele. The plasmid pAC1, encoding at least part of the cgtAV gene, was obtained by digestion of BB7X DNA with EcoRI, ligation to pUC19, and selection for both trimethoprim and ampicillin resistance (5). The expectation was that trimethoprim-resistant transformants would harbor both the Tn5 transposon (encoding dhfrII, the dihydrofolate reductase gene [21]) and flanking chromosomal DNA. Using a primer complementary to the IS50 element of Tn5TprMCS (Tn, 5'-TTCAGGACGCTACTTGTGTA-3'), the sequence of the N-terminal 99 amino acids of cgtAV was obtained, and it was concluded that BB7X contained a cgtAV::Tn5TprMCS insertion mutation (5).
In contrast to this conclusion, we demonstrate through several lines of evidence that BB7X encodes a wild-type cgtAV gene. First, immunoblot analysis of cell extracts from BB7 and BB7X with anti-CgtA antibodies resulted in the detection of a protein band that migrates at ca. 48 kDa, 5 kDa larger than the expected size of CgtAV. A slightly slower migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been previously noted for the C. crescentus (19) and E. coli (39) CgtA proteins. This immunoreactive band migrates at the same position as purified CgtAV protein (data not shown). Second, we designed a primer to a conserved region of rpmA (5'-GAGGATCCATCATCGTTCGTCAACG), the gene predicted to be upstream of cgtAV based on conservation of gene organization in most bacteria, including Vibrio species. PCR amplification of BB7 and BB7X chromosomal DNA with this primer and a primer downstream of cgtAV (5'-ATGGATCCGCAAATCACATCGTCT) produced a 1.6-kb PCR product regardless of the source of chromosomal DNA. Sequence analysis of the PCR products shows that the DNA is identical and encodes a full-length cgtAV gene (data not shown). Third, we sequenced the entire 5-kb EcoRI DNA fragment of pAC1 on both strands. This DNA insert did not contain the Tn5TprMCS transposon (Fig. 2A). Finally, examination of the cgtAV sequence reveals that nucleotides 315 to 326 are 83% complementary to the last 12 bases of the Tn primer used to sequence the N-terminal region of pAC1 (5). We propose that the complementarity between the Tn primer and this region of cgtAV was sufficient to generate the DNA sequence reported previously (5). We conclude, therefore, that BB7X encodes a wild-type cgtAV gene and that publications referring to the characterization of the V. harveyi BB7X mutant (5, 32, 34, 42) do not describe a bona fide cgtAV mutant. The true nature of the Tn5TprMCS insertion in BB7X was not explored further.
Expression of the V. harveyi folA gene in E. coli results in trimethoprim resistance. Because pAC1 did not contain a Tn5TpMCS transposon, we investigated the nature of the trimethoprim resistance conferred by the pAC1 plasmid. Introduction of pAC1, but not the high-copy vector pUC18 or the low-copy plasmid pGD103, conferred trimethoprim resistance to E. coli DH5 cells (Fig. 2B). Trimethoprim resistance was not due to expression of the V. harveyi cgtAV gene, since cells containing pAES7, a high-copy plasmid containing the cgtAV gene and promoter (PCR amplified with primers 5'-GAGGATCCATCATCGTTCGTCAACG and 5'-ATGGATCCGCAAATCACATCGTCT), were trimethoprim sensitive (Tps) (Fig. 2B).
To determine the nature of the trimethoprim resistance, we sequenced cgtAV and the surrounding genomic region. Sequencing was performed at the Institute of Biochemistry and Biophysics, Polish Academy of Science, Warsaw, Poland, or at the University of Michigan Sequencing Core Facility; all oligonucleotide sequences are available upon request. The entire cgtAV coding region was amplified by PCR (polymerase Pfu [Promega] or Red Taq [Sigma]) from BB7 and BB7X (4) colonies or genomic DNA (isolated by using High Pure PCR Template Preparation Kit; Roche) or from pAC1 using primers designed to conserved regions in obg/cgtA genes and by inverse PCR. Our sequence (NCBI AY623050) agrees with the partial 5' sequence (300 bp) of the cgtAV gene (5) that was previously reported. The 5-kb EcoRI fragment of the pAC1 plasmid (5), as well as subclones generated in the present study, was sequenced on both strands by using appropriate oligonucleotide primers. The region upstream of cgtAV was amplified by using a primer complementary to a conserved region of ispB (a gene commonly present upstream of obg/cgtA genes) (5'-AGTTGGGCTTGAATTGTTTCATTCAC) and an internal cgtAV primer (5'-AACTTTTACTACCGCTTCATCTACG). This sequence includes rplU, rpmA (coding for the 50S ribosomal proteins L21 and L27, respectively), and the cgtAV promoter region, and its organization is similar to that found in most bacteria (NCBI DQ180600).
Interestingly, the gene organization downstream of cgtAV (NCBI DQ180601) is quite different from that found in the majority of bacteria, although it is conserved in other Vibrio species. Downstream of cgtAV are two conserved hypothetical genes, yjjP and yjjB, as well as folA (dihydrofolate reductase type 1). Transcribed divergently are apaH (diadenosine tetraphosphatase), apaG, and the C terminus of ksgA (RNA adenine dimethyltransferase) (Fig. 2A). The functions of YjjP and YjjB are unknown, although both are predicted to be transmembrane proteins. In E. coli, the folA, apaH, apaG, and ksgA genes are physically linked but, unlike what we observe in V. harveyi, this gene cluster is not located near cgtAE.
We were struck by the dual observation that the third gene downstream of cgtAV is folA and that V. harveyi FolA is predicted to be 52% identical to E. coli FolA. Although several mechanisms of trimethoprim resistance in E. coli have been described (35), the most common mechanism is an increase in folA expression (3, 11, 28, 36). To determine whether the trimethoprim resistance conferred by pAC1 was caused by expression of V. harveyi folA, we PCR amplified folA from BB7 and BB7X cells by using the primers 5'-CTATCGCCGTGGATCCATAGTTTAA and 5'-TTGCGGTGCTTTCTGCAGTCTATTC and cloned the fragments onto the high-copy plasmids pCR2.1-TOPO and pUC18 and the low-copy plasmid pGD103. DH5 transformants harboring any of these plasmids were Tpr (Fig. 2B), indicating that expression of the V. harveyi folA gene was sufficient to confer trimethoprim resistance to E. coli, even at a relatively low copy number. We observed equivalent Tpr, regardless of whether the source of folA was BB7 or BB7X (Fig. 2B), a finding consistent with our sequence analysis indicating that the folA gene was identical in each strain (data not shown). Thus, we conclude that the Tpr phenotype of E. coli cells harboring pAC1 was due to expression of the V. harveyi folA gene and not due to a transposon insertion in cgtAV, as had previously been reported (5).
The V. harveyi CgtAV protein is associated with the 50S ribosomal particle. In all organisms, a number of GTP-binding proteins, including the Obg/CgtA proteins, are predicted to be involved in some aspect of translation (18). In the case of the bacterial Obg/CgtA proteins, direct association with the large ribosomal subunit has been demonstrated for the E. coli (29, 39), C. crescentus (20), and B. subtilis (31, 41) proteins. Interestingly, the eukaryotic Obg/CgtA proteins are also ribosome associated. In yeast, the mitochondrial GTPase Mtg2p is associated with the large ribosomal subunit (6), the cytosolic GTPases Rbg1p and Rbg2p are associated with translating ribosomes (P. Wout and J. R. Maddock, unpublished), and the nucleolar Nog1p is associated with the pre-60S particle (14, 15). Thus, ribosome association appears to be a conserved feature of the Obg/CgtA family. To determine whether this was also the case for the V. harveyi CgtAV protein, we isolated ribosomes, as previously described (10) from 50 ml of culture of V. harveyi BB7 that were grown at 30°C to an optical density at 600 nm of 0.5. The extract was clarified by centrifugation (10 min, 30,000 x g) and separated on 10 to 30% sucrose gradients (10 ml; 10 mM Tris-HCl [pH 7.5], 30 mM KCl, 5.25 mM magnesium acetate) by centrifugation in a Beckman SW41 Ti rotor at 41,000 rpm for 3 h. Fractions were collected as described previously (20) with the polysome profile monitored by UV absorbance (254 nm). Fractions were precipitated and examined by immunoblotting with purified anti-CgtAC antibodies (27). CgtAV was found in the 50S fractions and at the top of the gradient (Fig. 3). Thus, CgtAV is at least partially associated with the 50S ribosomal particle but not with the 30S particle, the 70S monosome, or with translating ribosomes.
Concluding remarks. In conclusion, we show that in contrast to previous publications (5, 32, 34, 42) cgtAV is an essential gene and that depletion of cgtAV does not lead to a cell elongation phenotype. Moreover, CgtAV is associated with the large ribosomal subunit. It is likely, therefore, that the V. harveyi CgtAV protein plays a cellular role similar to that of other bacterial Obg/CgtA proteins.
ACKNOWLEDGMENTS
We are extremely grateful Susan Sullivan for careful critique of the manuscript and to Patrice Wout for technical assistance. We are also grateful to Grzegorz Wgrzyn for insightful discussions during the course of this study.
This study was supported by a collaborative FIRCA grant (TW006001) from the National Institutes of Health and by NATO funds to J.R.M. and Grzegorz Wgrzyn.
REFERENCES
Arigoni, F., F. Talabot, M. Peitsch, M. D. Edgerton, E. Meldrum, E. Allet, R. Fish, T. Jamotte, M. L. Curchod, and H. Loferer. 1998. A genome-based approach for the identification of essential bacterial genes. Nat. Biotechnol. 16:851-856.
Belas, R., A. Mileham, D. Cohn, M. Hilman, M. Simon, and M. Silverman. 1982. Bacterial bioluminescence: isolation and expression of the luciferase genes from Vibrio harveyi. Science 218:791-793.
Breeze, A. S., P. Sims, and K. A. Tracey. 1975. Trimethoprim-resistant mutants of Escherichia coli K-12: preliminary genetic mapping. Genet. Res. 74:543-556.
Czyz, A., J. Jasiecki, A. Bogdan, H. Szpilewska, and G. Wgrzyn. 2000. Genetically modified Vibrio harveyi strains as potential bioindicators of mutagenic pollution of marine environments. Appl. Environ. Microbiol. 66:599-605.
Czyz, A., R. Zielke, G. Konopa, and G. Wgrzyn. 2001. A Vibrio harveyi insertional mutant in the cgtA (obg, yhbZ) gene, whose homologues are present in diverse organisms ranging from bacteria to humans and are essential genes in many bacterial species. Microbiology 147:183-191.
Datta, K., J. L. Fuentes, and J. R. Maddock. 2005. The yeast GTPase Mtg2p is required for mitochondrial translation and partially suppresses an rRNA methyltransferase mutant, mrm2. Mol. Biol. Cell 16:954-963.
Datta, K., J. M. Skidmore, K. Pu, and J. R. Maddock. 2004. The Caulobacter crescentus GTPase CgtAc is required for progression through the cell cycle and for maintaining 50S ribosomal subunit levels. Mol. Microbiol. 54:1379-1392.
Deich, R. A., B. J. Metcalf, C. W. Finn, J. E. Farley, and B. A. Green. 1988. Cloning of genes encoding a 15,000-dalton peptidoglycan-associated outer membrane lipoprotein and an antigenically related 15,000-dalton protein from Haemophilus influenzae. J. Bacteriol. 170:489-498.
Dutkiewicz, R., M. Slominska, G. Wgrzyn, and A. Czyz. 2002. Overexpression of the cgtA (yhbZ, obgE) gene, coding for an essential GTP-binding protein, impairs the regulation of chromosomal functions in Escherichia coli. Curr. Microbiol. 45:440-445.
Flardh, K., P. S. Cohen, and S. Kjelleberg. 1992. Ribosomes exist in large excess over the apparent demand for protein synthesis during carbon starvation in marine Vibrio sp. J. Bacteriol. 174:6780-6788.
Flensburg, J., and O. Skold. 1987. Massive overproduction of dihydrofolate reductase in bacteria as a response to the use of trimethoprim. Eur. J. Biochem. 162:473-476.
Foti, J. J., J. Scheienda, V. A. J. Sutera, and S. T. Lovett. 2005. A bacterial G protein-mediated response to replication arrest. Mol. Cell 17:549-560.
Hanahan, D. 1983. Studies on transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557-580.
Jensen, B. C., Q. Wang, C. T. Kifer, and M. Parsons. 2003. The NOG1 GTP-binding protein is required for biogenesis of the 60S ribosomal subunit. J. Biol. Chem. 278:32204-32211.
Kallstrom, G., J. Hedges, and A. Johnson. 2003. The putative GTPases Nog1p and Lsg1p are required for 60S ribosomal subunit biogenesis and are localized to the nucleus and cytoplasm, respectively. Mol. Cell. Biol. 23:4344-4355.
Kobayashi, G., S. Moriya, and C. Wada. 2001. Deficiency of essential GTP-binding protein ObgE in Escherichia coli inhibits chromosome partition. Mol. Microbiol. 41:1037-1051.
Kok, J., K. A. Trach, and J. A. Hoch. 1994. Effects on Bacillus subtilis of a conditional lethal mutation in the essential GTP-binding protein Obg. J. Bacteriol. 176:7155-7160.
Leipe, D. D., Y. I. Wolf, E. V. Koonin, and L. Aravind. 2002. Classification and evolution of P-loop GTPases and related ATPases. J. Mol. Biol. 317:41-72.
Lin, B., K. L. Covalle, and J. R. Maddock. 1999. The Caulobacter crescentus CgtA protein displays unusual guanine nucleotide binding and exchange properties. J. Bacteriol. 181:5825-5832.
Lin, B., D. A. Thayer, and J. R. Maddock. 2004. The Caulobacter crescentus CgtAC protein cosediments with the free 50S ribosomal subunit. J. Bacteriol. 186:481-489.
MacKenzie, C., M. Chidambaram, E. J. Sodergren, S. Kaplan, and G. M. Weinstock. 1995. DNA repair mutants of Rhodobacter sphaeroides. J. Bacteriol. 177:3027-3035.
Maddock, J., A. Bhatt, M. Koch, and J. Skidmore. 1997. Identification of an essential Caulobacter crescentus gene encoding a member of the Obg family of GTP-binding proteins. J. Bacteriol. 179:6426-6431.
Newman, J. R., and C. Fuqua. 1999. Broad-host-range expression vectors that carry the L-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene 227:197-203.
Norrander, J., T. Kempe, and J. Messing. 1983. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26:101-106.
Okamoto, S., M. Itoh, and K. Ochi. 1997. Molecular cloning and characterization of the obg gene of Streptomyces griseus in relation to the onset of morphological differentiation. J. Bacteriol. 179:170-179.
Okamoto, S., and K. Ochi. 1998. An essential GTP-binding protein functions as a regulator of differentiation in Streptomyces coelicolor. Mol. Microbiol. 30:107-119.
Olmsted, J. B. 1981. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J. Biol. Chem. 256:11955-11957.
Rood, J. I., A. J. Laird, and J. W. Williams. 1980. Cloning of the Escherichia coli K-12 dihydrofolate reductase gene following mu-mediated transposition. Gene 8:255-265.
Sato, A., G. Kobayashi, H. Hayashi, H. Yoshida, A. Wada, M. Maeda, S. Hiraga, K. Takeyasu, and C. Wada. 2005. The GTP binding protein Obg homolog ObgE is involved in ribosome maturation. Genes Cells 10:393-408.
Schafer, A., A. Tauch, W. Jager, J. Kalinowski, G. Thierbach, and A. Puchler. 1994. Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69-73.
Scott, J. M., J. Ju, T. Mitchell, and W. G. Haldenwang. 2000. The Bacillus subtilis GTP binding protein Obg and regulators of the B stress response transcription factor cofractionate with ribosomes. J. Bacteriol. 182:2771-2777.
Sikora-Borgula, A., M. Slominska, P. Trzonkowski, R. Zielke, A. Mysliwski, G. Wegrzyn, and A. Czyz. 2002. A role for the common GTP-binding protein in coupling of chromosome replication to cell growth and division. Biochem. Biophys. Res. Commun. 292:333-338.
Simon, R., U. Priefer, and A. Puhler. 1983. A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria. Biotechnology 1:784-790.
Slominska, M., G. Konopa, G. Wgrzyn, and A. Czyz. 2002. Impaired chromosome partitioning and synchronization of DNA replication initiation in a Vibrio harveyi insertional mutant in the cgtA gene coding for a common GTP-binding protein. Biochem. J. 362:579-584.
Smith, D. R., and J. M. Calvo. 1982. Nucleotide sequence of dihydrofolate reductase genes from trimethoprim-resistant mutants of Escherichia coli. Evidence that dihydrofolate reductase interacts with another essential gene product. Mol. Gen. Genet. 187:72-78.
Smith, D. R., and J. M. Calvo. 1979. Regulation of dihydrofolate reductase synthesis in Escherichia coli. Mol. Gen. Genet. 175:31-38.
Trach, K., and J. A. Hoch. 1989. the Bacillus subtilis spoOB stage 0 sporulation operon encodes an essential GTP-binding protein. J. Bacteriol. 171:1362-1371.
Vidwans, S. J., K. Ireton, and A. D. Grossman. 1995. Possible role for the essential GTP-binding protein, Obg in regulating the initiation of sporulation in Bacillus subtilis. J. Bacteriol. 177:3308-3311.
Wout, P., K. Pu, S. M. Sullivan, V. Reese, S. Zhou, B. Lin, and J. R. Maddock. 2004. The Escherichia coli GTPase, CgtAE, cofractionates with the 50S ribosomal subunit and interacts with SpoT, a ppGpp synthetase/hydrolase. J. Bacteriol. 186:5249-5257.
Zalacain, M., S. Biswas, K. A. Ingraham, J. Ambrad, A. Bryant., A. F. Chalker, S. Iordanescu, J. Fan, F. Fan, R. D. Lunsford, K. O'Dwyer, L. M. Palmer, C. So, D. Sylvester, C. Volker, P. Warren, D. McDevitt, J. R. Brown, D. J. Holmes, and M. K. Burnham. 2003. A global approach to identify novel broad-spectrum antibacterial targets among proteins of unknown function. J. Mol. Microbiol. Biotechnol. 6:109-126.
Zhang, S., and W. G. Haldenwang. 2004. Guanine nucleotides stabilize the binding of Bacillus subtilis Obg to ribosomes. Biochem. Biophys. Res. Commun. 322:565-569.
Zielke, R., A. Sikora, R. Dutkiewicz, G. Wgrzyn, and A. Czyz. 2003. Involvement of the cgtA gene function in stimulation of DNA repair in Escherichia coli and Vibrio harveyi. Microbiology 149:1763-1770.(A. E. Sikora, R. Zielke, )