Laboratory of Molecular Biology (affiliated with the University of Gdask), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, K
adki 24, 80-822 Gda
sk, Poland1
Department of Molecular Biology, University of Gdask, Kladki 24, 80-822 Gda
sk, Poland2
Marine Biology Center, Polish Academy of Sciences, w. Wojciecha 5, 81-347 Gdynia, Poland3
Author for correspondence: Grzegorz Wgrzyn. Tel: +48 58 346 3014. Fax: +48 58 301 0072. e-mail: wegrzyn{at}biotech.univ.gda.pl
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ABSTRACT |
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Keywords: cgtA gene, GTP-binding protein, bioluminescence, signal transduction
Abbreviations: DAPI, 4',6-diamidino-2-phenylindole
The GenBank accession number for the sequence reported in this paper is AF247677.
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INTRODUCTION |
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The best-studied prokaryotic GTP-binding protein is Era (for Escherichia coli Ras-like protein). This protein is essential for bacterial growth, and mutants in the era gene have pleiotropic phenotypes, including alterations in the regulation of carbon metabolism, the stringent response, and cell division (Lerner & Inouye, 1991 ; Britton et al., 1997
, 1998
). Recent studies have demonstrated that Era bears an RNA-binding motif (Chen et al., 1999
) and binds to the 30S ribosomal subunit (Sayed et al., 1999
).
Apart from Era, a subfamily of small GTP-binding proteins was discovered recently in bacteria. Interestingly, members of this subfamily have homologues in diverse organisms ranging from bacteria to humans (Okamoto & Ochi, 1998 ). This subfamily is Obg/Gtp1, and examples of bacterial members of it are Obg from Bacillus subtilis, Streptomyces griseus and S. coelicolor, CgtA from Caulobacter crescentus, and YhbZ from Escherichia coli and Haemophilus influenzae. Among this group, the B. subtilis obg gene product is the best-studied protein. Genetic studies led to proposals that Obg may regulate initiation of sporulation (Trach & Hoch, 1989
; Vidwans et al., 1995
), may be involved in the control of DNA replication (Kok et al., 1994
), and is necessary for stress-dependent activation of transcription factor
B (Scott & Haldenwang, 1999
). It was proposed that Obg can function by sensing the intracellular GTP level (Kok et al., 1994
) and may be required to stimulate the activity of the phosphorelay system (Vidwans et al., 1995
). Therefore, it is likely that Obg may be involved in signal transduction.
Functions of obg and cgtA genes have been investigated to date in bacteria that display a development programme, i.e. in B. subtilis, S. griseus, S. coelicolor and C. crescentus. However, members of the Obg/Gtp1 subfamily, with a high homology to Obg and CgtA, were found to be essential also in bacteria that do not sporulate and do not differentiate. For example, the yhbZ gene of E. coli was demonstrated to be essential (Arigoni et al., 1998 ), whereas its role in this bacterium is unknown. Investigation of the role of an essential gene is complicated by the fact that it is not possible to obtain viable null mutants. In the case of B. subtilis, apart from a temperature-sensitive obg mutant (Kok et al., 1994
), a strain in which the only functional obg gene copy is under control of an IPTG-inducible promoter has been constructed (Vidwans et al., 1995
). This strain is viable only in the presence of the inducer in the medium. Without IPTG-induced transcription of obg, the levels of the Obg protein were depleted during the cell growth; however, cells appeared to grow normally for several generations before there was any significant change relative to the same strain grown in the presence of IPTG (Vidwans et al., 1995
). This allowed investigation of the influence of obg gene function on sporulation, but such a phenotype would cause difficulties in studies on many other processes potentially affected by this gene function. Moreover, using an IPTG-inducible transcription of a given gene, it is difficult to obtain a level of the gene product comparable to that found in the wild-type strain.
Vibrio harveyi is a Gram-negative, free-living, luminescent marine bacterium. Here we describe a V. harveyi mutant which bears a transposon insertion in the cgtA (obg, yhbZ) gene. To our knowledge, this is the first reported viable bacterial mutant in which a gene belonging to the Obg/Gtp1 subfamily has been disrupted by insertion.
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METHODS |
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Culture media.
LuriaBertani (LB) medium (used for E. coli cultivation) and BOSS medium (a rich medium used for V. harveyi cultivation) have already been described by Sambrook et al. (1989) and Klein et al. (1998)
, respectively. Minimal medium 3 (W
grzyn & Taylor, 1992
) was used, but the concentration of NaCl was 3%, and the following carbon sources were employed: 1% glucose plus 1% Casamino acids; 1% glucose; 1% (v/v) glycerol; 1% sodium succinate; or 1% sodium acetate. If not indicated, V. harveyi strains were cultivated at 30 °C.
DNA sequencing.
A fragment of plasmid pAC1 (bearing the V. harveyi BB7X DNA region flanking the site of transposon insertion) was sequenced automatically using a Perkin-Elmer ABI 310 sequencer. The ABI Prism BigDye Terminator Cycle Sequencing Reaction Kit with Ampli Taq DNA Polymerase FS was employed. The primers used for sequencing reactions were as follows: primer TN, 5'-TTC AGG ACG CTA CTT GTG TA-3'; and primer pUCR, 5'-AGC GAA TAA CAA TTT CAC ACA GG-3'.
Electron microscopy.
V. harveyi cells were examined by electron microscopy using two techniques: negative staining with phosphotungstic acid (PTA), and ultrathin sectioning. For the first technique, cells were prepared by negative staining with 0·21% PTA (neutralized with KOH to pH 7·5) on carbon-coated copper grids according to Quintarelli et al. (1971) . Briefly, the grid was placed on the top of a drop of bacterial culture and left for 30 s, and then transferred to the top of a drop of the PTA solution for 30 s. Excess stain was removed with filter paper and the grid was dried at room temperature. For the second technique, the basic methods used to fix and embed cells for thin sectioning were according to Spurr (1969)
. Sections were stained with saturated uranyl acetate dissolved in 50% ethanol, and then with lead citrate for 2 min at room temperature. The grid was dried at room temperature. In both techniques, the grids were examined and photographed using a Philips CM 100 electron microscope operating at 60 kV.
Light microscopy.
V. harveyi cells were examined using differential interference contrast under a Nikon Eclipse E800 microscope. Staining with 4',6-diamidino-2-phenylindole (DAPI) was carried out essentially as described previously by Hause et al. (1993) . Briefly, culture samples were mixed with DAPI solution (25 µg ml-1) and left in the dark for 20 min. For staining with ethidium bromide, culture samples were mixed with the dye solution (100 µg ml-1) and left in the dark for 20 min. In both staining procedures, before examination under a microscope, the stained cells were placed on a 0·5% agar layer on a microscope slide and covered with a coverslip.
Southern blotting.
Southern-blotting analysis was performed according to Sambrook et al. (1989) using the Random Primer Fluorescein Labelling Kit with Antifluorescein-AP (NEN Life Science Products) for probe labelling and detection. The templates for preparation of probes were the EcoRIPvuII fragment of plasmid pSupTn5pMCS (encompassing a trimethoprim-resistance gene from the transposon) and the EcoRVBglII fragment of plasmid pAC1 (encompassing a part of the V. harveyi cgtA gene).
Measurement of bioluminescence.
Bacteria were grown to high cell density in BOSS medium. Then the cultures were diluted 10000-fold in the fresh medium and cultivation was continued. Samples were withdrawn at intervals; each sample was centrifuged (2000 g, 5 min) and the pellet was resuspended in an equal volume of 3% NaCl. The number of bacteria was determined by plating and luminescence was monitored in a scintillation counter using chemiluminescence mode as described previously (Bassler et al., 1994 ). The relative light units were calculated as counts min-1 ml-1 per cell.
Survival of cells under starvation conditions.
Bacterial cultures growing in BOSS medium were centrifuged; pellets were washed with 3% NaCl and resuspended in an equal volume of this solution. The suspensions were incubated at 30 °C; samples were withdrawn at indicated times and the number of living cells was determined by plating on BOSS plates.
UV sensitivity assay.
Bacteria were cultivated in BOSS medium, centrifuged and resuspended in 3% NaCl. Then 1x108 cells were irradiated with different UV doses followed by incubation in BOSS medium in the dark for 2 h and titration on BOSS plates (the plates were incubated overnight in the dark).
Measurement of RNA synthesis.
The experiments were performed as described previously by Wgrzyn et al. (1991)
. Briefly, bacteria were grown in BOSS medium to mid-exponential phase; [3H]uridine was added to 2 µCi ml-1 (74 kBq ml-1) and incubation was continued. At indicated times samples (50 µl each) were withdrawn to paper filters and transferred immediately to ice-cold 10% trichloroacetic acid (TCA). After 5 min incubation in an ice bath, the filters were transferred to ice-cold 5% TCA for 5 min, and then washed twice in 96% ethanol. After drying at room temperature, the radioactivity of the samples was measured in a scintillation counter. To induce the stringent response, serine hydroxamate was added to the culture up to 1 mg ml-1.
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RESULTS |
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Analysis of the putative amino acid sequence of the N-terminal 99 amino acids of the investigated gene product indicated a high percentage of identity/similarity to small GTP-binding proteins from diverse organisms ranging from bacteria to humans (Fig. 1). This analysis indicated that the gene product belongs to the Obg/Gtp1 subfamily. The best-investigated bacterial members of this subfamily of proteins are Obg (for Spo0B-associated GTP-binding protein) from B. subtilis, S. griseus and S. coelicolor and CgtA (for Caulobacter GTP-binding protein) from C. crescentus, although our knowledge of the roles of these proteins and the corresponding genes is very limited. To avoid further confusion in the nomenclature of homologous proteins, we propose to keep the name CgtA for the V. harveyi member of the Obg/Gtp1 subfamily (however, the name CgtA would be for common GTP-binding protein rather than for Caulobacter GTP-binding protein).
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One could argue that V. harveyi might contain a homologue of the cgtA gene whose expression could partially suppress effects of cgtA dysfunction, making the insertional mutant described in this report viable, in contrast to other bacteria devoid of such a homologue. To test such a possibility, we performed Southern-blotting analysis using EcoRI-digested chromosomal DNA of the wild-type V. harveyi (strain BB7). Following agarose gel electrophoresis, transfer of DNA to nylon membrane, and hybridization with a fluorescein-labelled probe prepared using the evolutionarily conserved 5' region of the cgtA gene of V. harveyi as a template (the EcoRVBglII fragment of plasmid pAC1), we observed a single band (data not shown). This makes the hypothesis of the presence of a homologue of the cgtA gene in V. harveyi chromosome less likely.
Morphology of colonies and cells of the cgtA mutant
The cgtA::Tn5TpMCS mutant formed considerably smaller colonies on plates with nutrient agar medium relative to the otherwise isogenic wild-type strain (data not shown). Moreover, the colonies of the mutant were less luminescent than those of the wild-type V. harveyi (data not shown).
The morphology of mutant cells also differed from that of the wild-type bacteria. This was revealed using electron microscopy and light microscopy with interference contrast and after staining with ethidium bromide and DAPI (Fig. 2). Generally, the cgtA mutant cells were significantly longer than normal V. harveyi cells, i.e. they had a tendency to form filaments. Such a morphology of cells may suggest problems with cell division and/or regulation of DNA replication. The long V. harveyi cgtA::Tn5TpMCS cells stained quite uniformly with the DNA-binding dyes, suggesting that there are problems with regulation of DNA replication rather than with segregation of genomes to daughter cells. Frequent formation of chains of cells (Fig. 2j
) supports the suggestion of problems with cell division in the mutant.
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Wild-type V. harveyi and the cgtA mutant were grown to high cell density (to allow efficient luminescence), and then the cultures were diluted 10000-fold in fresh medium. Cultivation was continued, and luminescence and cell density were measured at intervals. In the culture of the wild-type cells, the luminescence decreased after dilution and then became more effective due to the increase in the cell density of the culture (Fig. 3). The same phenomenon was observed in the culture of the cgtA mutant; however a significantly higher cell density had to be achieved to stimulate luminescence (Fig. 3
). These results indicate that the quorum-sensing regulation may be less efficient in the cgtA mutant in comparison to the wild-type strain.
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Survival of the cgtA mutant during starvation
Because of the inhibition of growth of the cgtA mutant in minimal media, we measured its viability under starvation conditions. Bacteria were incubated in 3% NaCl (this salt concentration is optimal for V. harveyi) for several days and the number of survivors was measured by plating of the cell suspensions. Viability of the cgtA mutant cells was dramatically decreased under these conditions relative to the wild-type (Fig. 5).
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We also investigated the response of the cgtA mutant to another stress agent, increase in temperature. It was demonstrated previously that V. harveyi shows a typical heat-shock response, although at temperatures somewhat lower than E. coli (Klein et al., 1995 ). However, we failed to detect any difference in the heat-shock response between the cgtA mutant and the wild-type strain (data not shown).
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DISCUSSION |
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The V. harveyi cgtA mutant revealed multiple defects in several cellular processes. Formation of cell filaments (Fig. 2) may suggest problems with regulation of cell division and/or DNA replication at least under certain conditions. In fact, it was proposed previously that the Obg protein of B. subtilis may be involved in the regulation of DNA replication (Kok et al., 1994
). The same B. subtilis protein was recently demonstrated to be involved in activation of the
B factor (Scott & Haldenwang, 1999
). This factor is the general stress-response
subunit of RNA polymerase that is activated when intracellular ATP levels fall or the bacterium experiences environmental stress. Activity of
B is regulated by a set of kinases and phosphatases, called the Rsb proteins, which catalyse the release of
B from an anti-
factor. It was suggested that Obg may interact with one or more
B regulators (Scott & Haldenwang, 1999
). The cgtA mutant of V. harveyi is not able to grow in minimal media and its viability in 3% NaCl is dramatically decreased (Fig. 5
), suggesting that induction of expression of certain genes that are normally active under conditions of starvation and energy deprivation may be impaired. Therefore, one may speculate that activation of an appropriate
factor (e.g. a homologue of E. coli
S) could be dependent on the cgtA gene function, similarly to the obg-dependent activation of B. subtilis
B. However, it is worth noting that in the case of the B. subtilis obg gene dysfunction the
B factor was no longer activated in response to environmental stress, but it retained the ability to be activated by the ATP-responsive pathway (Scott & Haldenwang, 1999
). Thus, we should also consider that the mechanism of cgtA-dependent V. harveyi growth in minimal media and survival under starvation conditions might be significantly different from that of B. subtilis.
Colonies of the cgtA mutant of V. harveyi were less luminescent than those of the wild-type. However, calculation of the relative luminescence per single bacterium in a liquid culture of high cell density revealed significantly smaller differences (Fig. 3). This could suggest defects in the quorum-sensing regulation rather than in the luminescence reaction itself. Indeed, the cgtA mutant required significantly higher cell density for efficient quorum-sensing-dependent induction of luminescence relative to the wild-type strain (Fig. 3
). The quorum-sensing regulation in V. harveyi requires activation of kinases and phosphatases (Freeman & Bassler, 1999a
, b
; Freeman et al., 2000
). Therefore, it seems likely that the cgtA gene product may be involved in the process of signal transduction during the quorum-sensing regulation.
Currently it is difficult to explain the mechanism of decreased efficiency of DNA repair in the cgtA mutant (Fig. 4, and Czy
et al., 2000a
). One possibility is that decreased efficiency of light emission in this mutant results in less efficient photoreactivation, since it was proposed recently that bioluminescence may stimulate photolyase activity (Czy
et al., 2000b
). On the other hand, CgtA might be involved in the regulation of expression of genes encoding DNA repair proteins.
In conclusion, the cgtA::Tn5TpMCS mutant of V. harveyi reveals multiple defects in important cellular processes, although it is still viable in rich media, in contrast to null mutants in the homologous genes of other bacteria investigated to date, which are not viable. Most, if not all, of the processes affected by the cgtA dysfunction in V. harveyi and reported in this article involve regulation of gene expression in response to various environmental signals. The V. harveyi cgtA gene product is a potential GTP-binding protein as it has a high homology to such proteins identified in many other organisms (Fig. 1). Therefore, it seems likely that V. harveyi CgtA protein is involved in signal-transduction processes. According to this speculation, bacterial responses to environmental stresses that apparently do not require protein phosphorylation/dephosphorylation-mediated signal transduction, e.g. the stringent response and the heat-shock response, appear to be normal in the cgtA mutant of V. harveyi.
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ACKNOWLEDGEMENTS |
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Received 15 May 2000;
revised 11 September 2000;
accepted 19 September 2000.