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Involvement of the cgtA gene function in stimulation of DNA repair in Escherichia coli and Vibrio harveyi

Rafa Dutkiewicz²

Agata Czy⁴

¹ Department of Molecular Biology, University of Gdask, Kadki 24, 80-822 Gdask, Poland
² Department of Molecular and Cellular Biology, Institute of Biotechnology, Intercollegiate Faculty of Biotechnology of the University of Gdask and Medical University of Gdask, Kadki 24, 80-822 Gdask, Poland
³ Institute of Oceanology, Polish Academy of Sciences, w. Wojciecha 5, 81-347 Gdynia, Poland
⁴ Laboratory of Molecular Biology (affiliated with the University of Gdask), Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Kadki 24, 80-822 Gdask, Poland

Correspondence
Agata Czy
czyz{at}biotech.univ.gda.pl

	ABSTRACT

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CgtA is a member of the Obg/Gtp1 subfamily of small GTP-binding proteins. CgtA homologues have been found in various prokaryotic and eukaryotic organisms, ranging from bacteria to humans. Nevertheless, despite the fact that cgtA is an essential gene in most bacterial species, its function in the regulation of cellular processes is largely unknown. Here it has been demonstrated that in two bacterial species, Escherichia coli and Vibrio harveyi, the cgtA gene product enhances survival of cells after UV irradiation. Expression of the cgtA gene was found to be enhanced after UV irradiation of both E. coli and V. harveyi. Moderate overexpression of cgtA resulted in higher UV resistance of E. coli wild-type and dnaQ strains, but not in uvrA, uvrB, umuC and recA mutant hosts. Overexpression of the E. coli recA gene in the V. harveyi cgtA mutant, which is very sensitive to UV light, restored the level of survival of UV-irradiated cells to the levels observed for wild-type bacteria. Moreover, the basal level of the RecA protein was lower in a temperature-sensitive cgtA mutant of E. coli than in the cgtA⁺ strain, and contrary to wild-type bacteria, no significant increase in recA gene expression was observed after UV irradiation of this cgtA mutant. Finally, stimulation of uvrB gene transcription under these conditions was impaired in the V. harveyi cgtA mutant. All these results strongly suggest that the cgtA gene product is involved in DNA repair processes, most probably by stimulation of recA gene expression and resultant activation of RecA-dependent DNA repair pathways.

	INTRODUCTION

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The Obg/Gtp1 subfamily of small GTP-binding proteins consists of proteins that occur in diverse organisms ranging from bacteria to humans (Czy et al., 2001 and references therein). The most intensively studied protein from this subfamily is the obg gene product of Bacillus subtilis. This protein is involved in the regulation of sporulation initiation (Vidwans et al., 1995), possibly controls DNA replication (Kok et al., 1994), is required for stress-dependent activation of transcription factor ^B (Scott & Haldenwang, 1999) and may interact with ribosomes (Scott et al., 2000). However, even in the case of the Obg protein, our knowledge about the function of this GTPase is highly incomplete.

Biochemical analysis of the Caulobacter crescentus Obg homologue, the CgtA protein, revealed its unusual nature of GTP binding and exchange parameters. Namely, it binds guanine nucleotides with moderate affinity and has rapid GDP and GTP exchange rate constants (Maddock et al., 1997; Lin et al., 1999, 2001; Lin & Maddock, 2001).

Genetic analysis of cgtA (obg) function is complicated by the fact that this gene is essential for most bacterial species, including the best investigated model bacterium, Escherichia coli (Arigoni et al., 1998). Since in most cases cgtA-null mutants are not viable, our knowledge about cgtA function is very limited. Nevertheless, a viable insertional cgtA mutant of the marine bacterium Vibrio harveyi has been isolated (Czy et al., 2001). Since an analogous E. coli mutant could not be constructed, it seems that special features of this marine bacterium, rather than putative residual activity of CgtA in the V. harveyi mutant, are responsible for its viability in the absence of functional cgtA (Dutkiewicz et al., 2002).

Using the insertional cgtA mutant of V. harveyi, the function of this gene has been studied (Czy et al., 2000a, 2001; Dutkiewicz et al., 2002; Sikora-Borgula et al., 2002; Somiska et al., 2002). These studies indicated that this mutant reveals multiple phenotypes, including slower growth in a rich medium, completely inhibited growth in minimal medium, dramatically reduced survival in a physiological saline and impairment in some chromosome functions (chromosome partitioning, synchronization of DNA replication initiation and coupling of chromosome replication to cell growth and cell division). Interestingly, an increased sensitivity of the cgtA mutant to UV irradiation and its enhanced mutagenicity upon treatment with different mutagens were observed (Czy et al., 2001). Although the mechanism of such sensitivity is completely unknown, this mutant has been successfully used in the development of a new assay for detection of mutagenic compounds (Czy et al., 2000b, 2002).

An E. coli strain with a deleted chromosomal cgtA gene can grow only in the presence of a copy of the wild-type allele on a plasmid. Such a system has been used to construct a temperature-sensitive cgtA mutant (Kobayashi et al., 2001). However, the mutation may be ‘leaky’ and it is difficult to obtain comparable intracellular levels of wild-type CgtA and CgtA(ts) proteins in bacteria with cgtA-containing plasmids (our unpublished data). Thus, most information about E. coli cgtA gene function comes from experiments in which wild-type CgtA protein has been overproduced in cells. These studies suggested that the cgtA gene product plays a role in synchronization of DNA replication initiation and partitioning of daughter chromosomes after a replication round (Dutkiewicz et al., 2002). Moreover, overexpression of the cgtA gene could suppress defects in the rrmJ (ftsJ) gene encoding an rRNA methyltransferase (Tan et al., 2002).

Recent DNA microarray analysis has revealed that transcription of the cgtA gene (described previously as yhbZ) in E. coli increases upon UV irradiation (Courcelle et al., 2001). This information, together with increased UV sensitivity of the V. harveyi cgtA insertional mutant (Czy et al., 2001), suggested that the CgtA protein may be involved in the regulation of DNA repair in both these bacterial species. Therefore, we aimed to investigate the nature of such regulation. We have used the cgtA mutant of V. harveyi to investigate the effects of dysfunction of this gene, whereas the effects of cgtA overexpression were studied in E. coli, though a temperature-sensitive cgtA mutant of E. coli was also employed. Since both cgtA genes are highly homologous (Czy et al., 2001; Sikora-Borgula et al., 2002), we assumed that this experimental strategy might give us data which could be helpful in understanding the function of small GTP-binding proteins from the Obg/Gtp1 subfamily.

	METHODS

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Bacterial strains and plasmids.
E. coli and V. harveyi strains are presented in Table 1. Plasmids are listed in Table 2. Plasmid pSSTuvrAcm was constructed by insertion of the cat-containing BsaAI–BsaAI fragment of pACYC184 into the SmaI site of pSST. For construction of plasmid pTZ18uvrBcm, the same fragment of pACYC184 was inserted into the ScaI site (located in the bla gene) of pTZ18uvrB. Plasmid pMMM1 was constructed by introduction of the SalI–PstI fragment of pFF1, encompassing oriT and cat, into corresponding sites of pUC19. For construction of pOLA1 and pOLA2 plasmids, an XbaI–HindIII fragment of plasmid pAIR79, encompassing the recA gene, was treated with the Klenow fragment of DNA polymerase I (Fermentas) and inserted into the SmaI site of plasmid pMMM1. The resultant plasmids, pOLA1 and pOLA2, bear the same DNA fragment harbouring the recA gene; however, orientations of this gene are different, such that effective transcription of recA is possible from pOLA1 but not from pOLA2. All plasmids were introduced into E. coli cells by transformation and into V. harveyi strains by conjugation using E. coli S17.1 strain as a donor.

Western blotting.
Western-blotting experiments were performed generally according to a procedure described previously (Wgrzyn et al., 1995a). Rabbit anti-CgtA antibodies were kindly provided by J. Tan and J. C. Bardwell (University of Michigan, Ann Arbor, USA). Mouse anti-RecA mAbs (IgG₁, clone ARM191) and goat anti-mouse IgG-HRP (F_ab-specific) horseradish peroxidase conjugate were purchased from StressGen Biotechnologies. The SuperSignal West Pico kit (Perbio) was used for signal detection.

UV sensitivity assay.
Bacteria were cultured in either LB medium (E. coli) or BOSS medium (V. harveyi) to mid-exponential phase, centrifuged and resuspended in 0·9 % NaCl (E. coli) or 3 % NaCl (V. harveyi). Following UV irradiation of 1x10⁸ cells, bacteria were incubated in LB medium (E. coli) or BOSS medium (V. harveyi) in the dark for 2 h and titrated on analogous plates.

Determination of mRNA levels in V. harveyi cells.
Samples (1x10⁹ cells) of exponentially growing bacterial cultures were withdrawn, cells were harvested by centrifugation, washed with 3 % NaCl and resuspended in MM3 salts (Wgrzyn & Taylor, 1992) containing 3 % NaCl. Cell suspensions were transferred to Petri dishes and irradiated with UV at the indicated doses. Following the addition of BOSS medium and cultivation for 15 min at 30 °C, total RNA was isolated from bacterial cells using the Total RNA Prep Plus kit (A&A Biotechnology). Dot-blot hybridization was performed according to Sambrook et al. (1989), using a fluorescein-labelled oligonucleotide probe (5'-GGCAGAAGACTTAACCGAATACCTGCACGAGCACG-3') and CDP-Star chemiluminescence reagent. Intensity of dots was quantified by densitometry using the Quantity One system (Bio-Rad).

	RESULTS

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Increased levels of the CgtA protein in UV-irradiated E. coli and V. harveyi
Using DNA microarray technology, Courcelle et al. (2001) demonstrated that in UV-irradiated E. coli cells, transcription of the cgtA gene (called in that article yhbZ, according to previous nomenclature) is increased. We measured levels of CgtA in UV-irradiated E. coli and V. harveyi wild-type strains, at different times after irradiation with doses inducing the SOS response. Different UV doses were used for these two bacterial species as a significantly higher UV sensitivity was reported previously for V. harveyi relative to E. coli (Czy et al., 2000a).

We found that the level of the CgtA protein increased significantly upon UV irradiation of both E. coli and V. harveyi (Fig. 1). Moreover, in both cases, a dose-response correlation was clear, i.e. higher UV doses led to higher levels of the CgtA protein (Fig. 2). These results are compatible with the finding of increased levels of cgtA mRNA in UV-irradiated E. coli cells (Courcelle et al., 2001) and might suggest that CgtA can be involved in some processes coupled to bacterial response to DNA damage. Such a suggestion was supported by the fact that the V. harveyi cgtA : : Tn5TpMCS mutant is more sensitive to UV irradiation than a wild-type strain of this bacterium (Czy et al., 2000a).

View larger version (31K): [in this window] [in a new window]   Fig. 1. Relative level of the CgtA protein in E. coli (circles) and V. harveyi (squares) wild-type cells after UV irradiation (10 J m-2). Bacteria were irradiated at time 0, samples of cultures were withdrawn at indicated times and the amounts of CgtA protein were estimated by Western blotting (see lower part of figure for examples) and densitometry. Mean results from three measurements, with error bars, are shown.

View larger version (35K): [in this window] [in a new window]   Fig. 2. Dose-response curves of relative CgtA protein levels in E. coli (circles) and V. harveyi (squares) wild-type cells after UV irradiation at various doses. CgtA protein levels were estimated by Western blotting (see lower part of figure for examples) and densitometry in samples of cultures withdrawn 60 min after irradiation. Mean results from three measurements, with error bars, are shown.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Sensitivity of (a) E. coli bearing either a control plasmid pRD4B (circles) or cgtA-overexpressing plasmid pRD4A (triangles), and of (b) V. harveyi wild-type (squares) and the cgtA mutant (inverted triangles), to UV irradiation. Mean results from three measurements (performed as described in Methods) are shown. In each case SD was below 15 %.

The dnaQ mutant reveals a high frequency of mutations due to impaired correction of mis-incorporated nucleotides during DNA replication (Linn, 1996). We observed an increased resistance of this mutant to UV irradiation upon moderate overexpression of cgtA, similar to the ‘wild-type’ (control) strain (Table 3). However, analogous cgtA overexpression in uvrA and uvrB mutants, encoding components of the excision repair system, as well as in the umuC mutant, defective in the error-prone DNA repair system, had no significant effect on survival of UV-irradiated cells (Table 3). Similar results were obtained with the recA gene mutant, whose function is required for expression of both DNA repair systems mentioned above. These results suggest that recA-dependent DNA repair pathways may be stimulated by the cgtA gene product.

Overexpression of the E. coli recA gene in wild-type V. harveyi cells had no significant effect on their survival after UV irradiation. Some moderate differences were observed only at the highest UV doses used, when survival of recA-overexpressing bacteria was up to twofold higher than that of the control strain (data not shown). However, recA-overexpressing cgtA mutant cells were considerably more resistant to UV irradiation than bacteria used in control experiments, in which a plasmid analogous to that used for recA overexpression but bearing the recA gene cloned in an orientation precluding its transcription was used (Fig. 4).

View larger version (15K): [in this window] [in a new window]   Fig. 4. UV sensitivity of V. harveyi cgtA mutant bearing either a control plasmid pOLA2 (inverted triangles) or recA-overexpressing plasmid pOLA1 (squares). Mean results from three measurements (performed as described in Methods) are shown. In each case SD was below 15 %.

To test whether expression of the recA gene depends on cgtA gene function, we used a temperature-sensitive cgtA mutant of E. coli, described previously by Kobayashi et al. (2001). Although this mutant is not easy to investigate (see Introduction), we chose such an experimental system because (i) it is the only E. coli cgtA mutant available, (ii) it was not possible to detect a V. harveyi RecA protein using mAbs recognizing E. coli RecA (data not shown) and (iii) the recA gene of V. harveyi has not been sequenced.

We found that the basal level of the RecA protein was lower in the cgtA(ts) mutant growing at 43 °C than in an otherwise isogenic cgtA⁺ strain (Fig. 5). Even more significant differences between these strains in the level of RecA were observed after UV irradiation. While relatively low doses of UV caused an increase in the amount of RecA shortly after irradiation of cgtA⁺ bacteria, no stimulation of RecA protein synthesis was observed under these conditions in cgtA(ts) cells (Fig. 5). These results indicate that expression of the recA gene is impaired in cells defective in CgtA function.

View larger version (16K): [in this window] [in a new window]   Fig. 5. Levels of RecA protein in E. coli cgtA+ (GN5002) and cgtA(ts) (GN5003) strains growing at 43 °C before and after UV irradiation. UV doses (in J m-2) are shown above each lane. Bacteria were cultivated for 10 min after UV irradiation and the amounts of RecA were estimated by Western blotting.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Levels of uvrB mRNA in V. harveyi wild-type (squares) and the cgtA mutant (inverted triangles) after UV irradiation. Samples of cultures for RNA isolation and dot-blotting were withdrawn 15 min after irradiation. Results were quantified by densitometry. Mean results from three measurements, with error bars, are shown.

	DISCUSSION

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There are many phenotypes associated with either dysfunction or gain of function of Obg-like proteins (see Introduction). One of them, described previously in an insertional cgtA mutant of V. harveyi (the only known viable chromosomal null mutant in a gene encoding a member of bacterial proteins from the Obg/Gtp1 subfamily), is increased sensitivity to UV irradiation (Czy et al., 2000a, b, 2002). Since the mechanism of the effect of cgtA dysfunction on UV sensitivity is completely unknown, we aimed to answer the question what is the role of CgtA in the protection of cells against UV irradiation? The use of both E. coli and V. harveyi strains allowed us to employ various experimental systems (i.e. loss of function and gain of function). Due to the high similarity of CgtA proteins from E. coli and V. harveyi, results obtained in experiments with each of these bacterial species could be generalized.

The results of our experiments indicated that CgtA stimulates some, but not all, DNA repair processes. DNA repair pathways involving products of the uvrA, uvrB and umuC genes are affected by CgtA function. These pathways, as part of the SOS response, depend on activity of the RecA protein (Walker, 1996). Increased sensitivity of the V. harveyi cgtA mutant to UV irradiation may be caused by deficiency of RecA as overexpression of the E. coli recA gene restores UV resistance in this mutant to the wild-type level. Even more striking was the discovery that an increase in the RecA protein level, normally observed after UV irradiation of wild-type bacteria, does not occur in cells defective in cgtA gene function. In this light, a lack of induction of transcription of the uvrB gene in the UV-irradiated V. harveyi cgtA mutant may suggest either a direct effect of CgtA on uvrB transcription or an indirect effect, via impairment in recA gene expression.

Impaired expression of recA and uvrB genes in cgtA mutants indicates that Obg-like proteins may be specific regulators of gene expression. It remains to be elucidated whether CgtA influences transcription of recA and uvrB or modulates translation of recA and thus indirectly influences uvrB transcription. In fact, it has been reported that the Obg protein of B. subtilis is required for stress-dependent activation of transcription factor ^B (Scott & Haldenwang, 1999) and may interact with ribosomes (Scott et al., 2000). Moreover, DNA-binding properties have been found in the E. coli CgtA protein (Kobayashi et al., 2001).

Apart from being a regulator of expression of at least some SOS genes (e.g. recA and uvrB), CgtA is synthesized more efficiently upon UV irradiation. Interestingly, increased transcription of the cgtA gene in UV-irradiated E. coli cells was found to be independent of lexA gene function (Courcelle et al., 2001). Therefore, it is unlikely that the cgtA gene is a member of the SOS regulon.

	ACKNOWLEDGEMENTS

 
We thank Professor C. Wada and Dr G. Kobayashi for providing E. coli strains GN5002 and GN5003. We are very grateful to Dr Marian Sktas (Department of Microbiology, University of Gdask) and Dr Hanna Szpilewska (Institute of Oceanology, Polish Academy of Sciences) for providing plasmids, and to Dr Roel M. Schaaper (National Institute of Environmental Health Sciences, NC) and Dr Iwona Fijakowska (Institute of Biochemistry and Biophysics, Polish Academy of Sciences) for providing bacterial strains. We thank Dr Janine Maddock for fruitful discussions. This work was supported by the Polish State Committee for Scientific Research (project grant 6 P04B 022 20 to A. C.), NATO Science Programme (grant LST.CLG.978855) and NIH (grant no. TW6001). G. W. and A. C. also acknowledge financial support from the Foundation for Polish Science (subsidy 14/2000 and a stipend, respectively).

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Received 10 February 2003; revised 3 April 2003; accepted 7 April 2003.

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