Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand1
Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand2
Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101, USA3
Author for correspondence: Skorn Mongkolsuk. Tel: +66 2 574 0623 ext. 1402. Fax: +66 2 574 2027. e-mail: skorn{at}tubtim.cri.or.th
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ABSTRACT |
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Keywords: Pseudomonas aeruginosa, Deinococcus radiodurans, organic peroxide resistance, osmotic stress
Abbreviations: Ahp, alkyl hydroperoxide reductase; tBOOH, tert-butyl hydroperoxide
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INTRODUCTION |
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Bacteria have evolved complex systems to protect themselves from organic-peroxide toxicity. Alkyl hydroperoxide reductase (Ahp) is the best-characterized bacterial enzyme involved in the metabolism of organic peroxides (Poole, 1996 ; Niimura et al., 1995
). This enzyme consists of two subunits: catalytic subunit C (AhpC) and reductase subunit F (AhpF). AhpC reduces organic peroxides to the corresponding alcohols (Poole & Ellis, 1996
). AhpC belongs to a large family of peroxidases (the AhpC/thiol-specific antioxidant family) found in organisms ranging from bacteria to man (Chae et al., 1994a
). Some organisms express multiple AhpC/thiol-specific antioxidant paralogues, presumably with distinct functions (regulation or cellular localization) (Baillon et al., 1999
; Bsat et al., 1996
; Hillas et al., 2000
).
In the bacterial phytopathogen Xanthomonas campestris pv. phaseoli, the defence against organic-peroxide toxicity is complex (Loprasert et al., 1996 ). In addition to AhpC, there is a recently characterized novel organic hydroperoxide resistance gene, ohr (Loprasert et al., 1997
; Mongkolsuk et al., 1998a
). X. campestris ohr mutants are sensitive to organic peroxides, but not to other oxidants (Mongkolsuk et al., 1998a
). In addition, ohr has a unique pattern of oxidant-induced expression; only organic peroxides induce high levels of expression (Mongkolsuk et al., 1998a
). This unusual pattern of induction distinguishes ohr from other known oxidative stress genes. Analysis of Ohr primary structure shows that it has homology to proteins with unknown functions from both Gram-positive and Gram-negative bacteria, and that it has moderate homology to an osmotically inducible protein (OsmC) from Escherichia coli (Gutierrez & Devedjian, 1991
).
On the basis of sequence analysis of Ohr and OsmC homologues, we propose that these two proteins define two protein subfamilies. In this report, we focus on two organisms with one member of each subfamily: Pseudomonas aeruginosa and Deinococcus radiodurans. Genetic analyses in P. aeruginosa, and expression studies in both organisms, support the hypothesis that these proteins are functionally, as well as structurally, distinct.
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METHODS |
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Quantitative determinations of plating efficiency in the presence of various oxidants of Pseudomonas strains were performed as described previously (Hassett et al., 2000 ; Ochsner et al., 2000
). Essentially, cells from exponential phase cultures were serially diluted and plated on LB agar containing various concentrations of tert-butyl hydroperoxide (tBOOH). The numbers of colonies at the different oxidant concentrations were counted after 24 h incubation at 37 °C. Percentage survival is defined as the percentage ratio between the c.f.u. growing on plates containing tBOOH and those growing on plates without tBOOH.
Alignment and phylogenetic analysis.
Protein sequences related to Ohr and OsmC were retrieved from public sequence databases using the BLAST program (Altschul et al., 1997 ). These amino acid sequences were aligned using the program CLUSTAL W, version 1.7 (Thompson et al., 1994
). A phylogenetic tree was constructed by the neighbour-joining method, using the TREE program from the phylogenetic analysis page of D. L. Robertson, E. Beaudoing & J. M. Claverie (http://igs-server.cnrss-mrs.fr/anrs/phylogenetics). The results were drawn using the program PHYLODENDRON, version 0.8d (D. G. Gilbert, Department of Biology, University of Indiana, USA; http://iubio.bio.indiana.edu).
Stress-induced expression of ohr and osmC.
Exponential phase cultures (OD600=0·4) were divided into flasks and oxidants or other chemicals were added. The following concentrations of chemicals were used: 250 µM H2O2, 200 µM cumene hydroperoxide, 200 µM tBOOH, 100 µM menadione, 2% (w/v) sodium chloride and 4% (v/v) ethanol for P. aeruginosa; 250 µM H2O2, 100 µM tBOOH, 4% (w/v) sodium chloride and 4% (v/v) ethanol for D. radiodurans. Treated and untreated cultures were harvested after 20 min incubation at appropriate temperatures.
Cloning of P. aeruginosa ohr and osmC.
Full-length P. aeruginosa ohr and osmC genes were cloned using PCR. Primers 5'ohrP (5'-TCAGACAGGTGACTCTC-3'), 3'ohrP (5'-AGTCGGAAGCTTCAGAC-3'), 5'osmCP (5'-CGACGCGAGCGGATGTC-3') and 3'osmCP (5'-AGCGTTCCGCTCAGCCG 3') were designed using sequence data obtained from the genome sequence of P. aeruginosa (Stover et al., 2000 ). A primer pair, P. aeruginosa genomic DNA, the PCR reaction mix and 2 U Pfu polymerase were mixed and used to amplify either ohr or osmC genes, under the following conditions: denaturation at 96 °C for 1 min, annealing at 50 °C for 1 min, and extension at 72 °C for 2 min. The 450 bp ohr and 470 bp osmC PCR-generated fragments were cloned into pBBR1MCS-4 and pBBR1MCS-5, respectively (Kovach et al., 1994
), giving two recombinant plasmids, pBBRohrP and pBBRosmCP. The nucleotide sequences of both genes were determined using a BigDye terminator cycle sequencing kit on an automated DNA sequencer (ABI 310).
Construction of ohr and osmC mutants in P. aeruginosa.
Mutants were constructed by insertional inactivation of ohr and osmC genes. Essentially, pBBRohrP was digested with SfiI and SacII. The ends of the 340 bp fragment containing the coding region of ohr were gap-filled by DNA polymerase, and the blunt-ended fragment was cloned into SmaI-digested pKnock-GM (Alexeyev, 1999 ) to give pKnock-ohr. Similarly, pBBRosmCP was digested with SalI and BstEII. The ends of the 240 bp DNA fragment containing part of the osmC coding region were gap-filled by DNA polymerase, and cloned into SmaI-digested pKnock-Ap (Alexeyev, 1999
) to give pKnock-osmCP. The sequences of the cloned DNA in both recombinant plasmids were determined using an automated DNA sequencer (ABI 310). pKnock-ohrP and pKnock-osmCP were conjugated into P. aeruginosa PAOI as described previously (Hassett et al., 2000
). Gentamicin-resistant and carbenicillin-resistant colonies will arise from homologous recombination of the in-coming recombinant plasmid with either ohr or osmC genes on the chromosome, depending on the fragment on the plasmid. Insertion of the plasmid into the chromosome is expected to inactivate the gene. Transconjugants containing pKnock-ohr and pKnock-osmC were selected with gentamicin (15 µg ml-1) and carbenicillin (200 µg ml-1), respectively. Putative mutants were screened by PCR using a universal sequence primer for a site located in pKnock vectors and either the 3'ohr or the 3'osmC primer. The expected insertions resulting in inactivation of ohr and osmC were confirmed by Southern analysis of genomic DNA extracted from the mutants and were probed with gene-specific probes (data not shown).
Northern analysis of ohr and osmC homologues.
Total RNA was extracted from P. aeruginosa and D. radiodurans by using the hot acid phenol method performed as described previously (Mongkolsuk et al., 1997 ). RNA samples were separated by electrophoresis in formaldehyde agarose gels and were then transferred by capillary action to pieces of nylon membrane. Total RNA (10 µg) was loaded into each well. Probes were prepared, and RNA hybridization and membrane washing were performed as described previously (Mongkolsuk et al., 1997
). P. aeruginosa ohr and osmC probes of 300 bp and 375 bp, respectively, were made from MluI-digested pKnock-ohrP and SfiIHindIII-digested pKnock-osmCP. The DNA fragments were separated on an agarose gel, extracted and then purified prior to being radioactively labelled using a random prime DNA-labelling kit. D. radiodurans ohr and osmC probes were made using PCR. Primers corresponding to coding regions of either ohr (5'ohrD, 5'-TGCGGGCGAGGGAATAG-3', and 3'ohrD, 5'-GTGTCTTATTCGCGGAC-3') or osmC (5'osmCD, 5'-CAGCGAGCACACTGGGC-3', and 3'osmCD, 5'-GCTTGAGCGACTCAGCC-3') were designed using the D. radiodurans genome sequence (White et al., 1999
). PCR was performed with D. radiodurans genomic DNA and the gene-specific primers in the PCR reactions noted above, using the following conditions: denaturation at 96 °C for 1 min, annealing at 50 °C for 1 min and extension at 72 °C for 2 min for 35 cycles. The 445 bp ohr and 470 bp osmC PCR-generated fragments were gel-purified, and radioactively labelled probes were made using the random primer DNA-labelling kit.
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RESULTS AND DISCUSSION |
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X. campestris pv. phaseoli Ohr and E. coli OsmC amino acid sequences were used to search the GenBank and bacterial genome databases for related proteins. Homologues of both proteins are widely distributed in both Gram-negative and Gram-positive bacteria, but no homologues were detected in eukaryotes. Amino acid alignments generated using CLUSTAL W (Thompson et al., 1994 ) suggest that the Ohr/OsmC family can be divided into two subfamilies, each being defined by sequence motifs conserved only among Ohr (designated Oh regions) or only among OsmC (designated Os regions) homologues (Fig. 1
). At present, we do not know the biological significance of these different motifs. A notable feature of the primary structure of Ohr and OsmC family members is the two highly conserved cysteine residues. C residues have been shown to be the active site of AhpC, an enzyme that metabolizes organic peroxide (Chae et al., 1994b
). The amino acid sequences around the second C residue are conserved within members of the Ohr and OsmC families but are very diverse between the two families. The conserved amino acid region around C-125 of the Ohr family members contains the sequence motif VCPY (Fig. 1
). This region is not present in members of the OsmC family. The VCPY motif places the cysteine residue in an environment of abnormally strong nucleophilicity that makes it highly susceptible to reactive oxygen species (Lim et al., 1994
). The strongly nucleophilic regions in thiol-specific antioxidant proteins such as AhpC (Chae et al., 1994b) and in the peroxide-scavenging protein ovothiol (Turner et al., 1988
) have been shown to be the catalytic sites for the breakdown of peroxides. This suggests that the C-125 residue in members of the Ohr family could participate in peroxide reduction. This idea is being investigated. The amino acid sequences were used to construct a phylogenetic tree (Fig. 2
); it clearly shows that there are two separate groups of proteins, defined here as the Ohr and OsmC subfamilies.
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Unexpectedly, several bacteria have homologues from both subfamilies. Several Gram-negative bacteria (P. aeruginosa, Pseudomonas putida) and a Gram-positive bacterium (D. radiodurans) have one member each from the OsmC and Ohr subfamilies. Other Gram-positive bacteria, such as B. subtilis and Streptomyces coelicolor, have one member of the OsmC family and two or more members of the Ohr family. Multiple Ohr homologues have not been identified in genomes from Gram-negative bacteria. At present, the functions of the multiple Ohr homologues are unknown but are the subject of further investigation.
Ohr and OsmC homologues have different physiological roles
The separation of Ohr and OsmC homologues into two subfamilies raises an important question: do these two subfamilies have distinct or overlapping functions? Bacteria such as P. aeruginosa, having one member each from the ohr and osmC subfamilies, offer an attractive model system for investigating this question. Using insertional inactivation, we generated mutants of the P. aeruginosa ohr and osmC genes. The P. aeruginosa ohr mutant, but not the osmC mutant, has a much reduced (more than 100 times lower) plating efficiency on agar containing 500 µM tBOOH when compared with the parent strain (Fig. 3). No changes in the plating efficiency in the presence of H2O2 or menadione for either mutant were observed (data not shown). Mutations in oxidative stress genes can lead to decreased aerobic growth rate and plating efficiency (Mongkolsuk et al., 1998b
; Hassett et al., 2000
). However, both mutants had the same growth rate as the parent strain in rich medium, and no deficiency in aerobic plating was detected (data not shown).
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ohr and osmC homologues have different expression patterns
X. campestris pv. phaseoli ohr has a unique expression pattern in that its expression is induced only by organic peroxide, and not by menadione or H2O2 (Mongkolsuk et al., 1998a ). In contrast, E. coli osmC is under both growth-phase (RpoS) and osmotic-stress regulation (Bouvier et al., 1998
; Gordia & Gutierrez, 1996
). This suggests that members of the ohr and osmC subfamilies may have different patterns of stress-inducible expression.
We used Northern blotting experiments to determine the expression patterns of ohr and osmC homologues in response to osmotic and oxidative stresses in P. aeruginosa and D. radiodurans bacteria, each of which has one gene from each subfamily. In both organisms, ohr was strongly induced by low concentrations of organic peroxides (cumene hydroperoxide and tBOOH) (Fig. 4) but not by other oxidants such as menadione (not shown for D. radiodurans) or H2O2. Neither osmotic stress (a high salt concentration) nor ethanol induced expression of the ohr homologues. In contrast, expression of osmC homologues in both bacteria was induced by ethanol, while salt stress induced osmC expression only in P. aeruginosa; none of the oxidants tested induced the gene expression (Fig. 4
). Thus, the patterns of ohr and osmC expression in P. aeruginosa and D. radiodurans are consistent with the known regulation of X. campestris pv. phaseoli ohr (Mongkolsuk et al., 1998a
) and E. coli osmC (Gutierrez & Devedjian, 1991
). The ohr and osmC mRNAs in both bacterial species were each approximately 0·7 kb in length, indicating that these genes are transcribed as monocistronic mRNAs. Expression of both genes is different: ohr and osmC are induced by organic peroxide and osmotic stress, respectively. At present, well-characterized regulators of stress-induced gene expression such as OxyR, SoxRS and RpoS cannot account for the ohr and osmC patterns of expression, implying that these genes are regulated by novel regulators.
It was noticeable that basal levels of ohr and osmC from P. aeruginosa and D. radiodurans varied greatly, ranging from barely detectable amounts in the former to moderately high levels in the latter. In addition, the degree of induction varied significantly between these bacteria: D. radiodurans showed a lower magnitude of induction than P. aeruginosa. It remains to be seen if these differences in basal level expression and degree of induction are related to the ability of each bacterium to cope with organic peroxide stress or are simply indicative of the differences between Gram-negative and Gram-positive bacteria. It is remarkable that the patterns of stress-induced expression of ohr and osmC homologues are highly conserved in a diverse range of bacteria. This suggests that both genes might have important functions.
Concluding remarks
Members of the Ohr family are widely distributed in both Gram-negative and Gram-positive bacteria. Analysis of primary structure, the physiological characterization of mutants and expression patterns show that Ohr and OsmC proteins belong to different, but related, subfamilies. We have shown, in P. aeruginosa and X. campestris pv. phaseoli (Mongkolsuk et al., 1998a ), that mutations in ohr increase susceptibility to organic peroxides. This phenotype, coupled with the specific induction of ohr by organic peroxides, suggests that ohr represents a novel organic peroxide protection system. Recent results from Ochsner et al. (2001)
confirm our finding that mutation in P. aeruginosa ohr results in increased organic-peroxide sensitivity. More studies are needed to discover the physiological function of OsmC. The osmotically inducible expression of the gene suggests that it could have some kind of role in the bacterial osmotic-stress response. Recently, Conter et al. (2001)
reported contradictory results that E. coli osmC mutants showed increase sensitivity to tBOOH but not to cumene hydroperoxide.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389-3402.
Baillon, M. L., van Vliet, A. H., Ketley, J. M., Constantinidou, C. & Penn, C. W. (1999). An iron-regulated alkyl hydroperoxide reductase (AhpC) confers aerotolerance and oxidative stress resistance to the microaerophilic pathogen Campylobacter jejuni. J Bacteriol 181, 4798-4804.
Baker, C. J. & Orlandi, E. W. (1995). Active oxygen in plant pathogenesis. Annu Rev Phytopathol 33, 299-321.
Bouvier, J., Gordia, S., Kampmann, G., Lange, R., Hengge-Aronis, R. & Gutierrez, C. (1998). Interplay between global regulators of Escherichia coli: effect of RpoS, Lrp and H-NS on transcription of the gene osmC. Mol Microbiol 28, 971-980.[Medline]
Bsat, N., Chen, L. & Helmann, J. D. (1996). Mutation of the Bacillus subtilis alkyl hydroperoxide reductase (ahpCF) operon reveals compensatory interactions among hydrogen peroxide stress genes. J Bacteriol 178, 6579-6586.[Abstract]
Chae, H. Z., Robison, K., Poole, L. B., Church, G., Storz, G. & Rhee, S. G. (1994a). Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes. Proc Natl Acad Sci USA 91, 7017-7021.[Abstract]
Chae, H. Z., Uhm, T. B. & Rhee, S. G. (1994b). Dimerization of thiol-specific antioxidant and the essential role of cysteine 47. Proc Natl Acad Sci USA 91, 7022-7026.[Abstract]
Conter, A., Gangneux, C., Suzanne, M. & Gutierrez, C. (2001). Survival of Escherichia coli during long-term starvation: effects of aeration, NaCl, and the rpoS and osmC gene products. Res Microbiol 152, 17-26.[Medline]
Fraser, C. M., Gocayne, J. D., White, O. & 25 other authors (1995). The minimal gene complement of Mycoplasma genitalium. Science 270, 397403.
Gonzalez-Flecha, B. & Demple, B. (1997). Homeostatic regulation of intracellular hydrogen peroxide concentration in aerobically growing Escherichia coli. J Bacteriol 179, 382-388.[Abstract]
Gordia, S. & Gutierrez, C. (1996). Growth-phase-dependent expression of the osmotically inducible gene osmC of Escherichia coli K-12. Mol Microbiol 19, 729-736.[Medline]
Gutierrez, C. & Devedjian, J. C. (1991). Osmotic induction of gene osmC expression in Escherichia coli K12. J Mol Biol 220, 959-973.[Medline]
Halliwell, B. & Gutteridge, J. M. (1984). Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 219, 1-14.[Medline]
Hassett, D. J., Alsabbagh, E., Parvatiyar, K., Howell, M. L., Wilmott, R. W. & Ochsner, U. A. (2000). A protease-resistant catalase, KatA, released upon cell lysis during stationary phase is essential for aerobic survival of a Pseudomonas aeruginosa oxyR mutant at low cell densities. J Bacteriol 182, 4557-4563.
Hillas, P. J., del Alba, F. S., Oyarzabal, J., Wilks, A. & Ortiz De Montellano, P. R. (2000). The AhpC and AhpD antioxidant defense system of Mycobacterium tuberculosis. J Biol Chem 275, 18801-18809.
Koonin, E. V., Arvind, L. & Galperin, M. Y. (2000). A comparative-genomic view of the microbial stress response. In Bacterial Stress Response , pp. 417-444. Edited by G. Storz & R. Hengge-Aronis. Washington, DC:American Society for Microbiology.
Kovach, M. E., Phillips, R. W., Elzer, P. H., Roop, R. M.II & Peterson, K. M. (1994). pBBR1MCS: a broad-host-range cloning vector. Biotechniques 16, 800-802.[Medline]
Levine, A., Tenhaken, R., Dixon, R. & Lamb, C. (1994). H2O2 from the oxidative burst orchestrates the plant hypersensitive disease resistance response. Cell 79, 583-593.[Medline]
Lim, Y. S., Cha, M. K., Kim, H. K. & Kim, I. H. (1994). The thiol-specific antioxidant protein from human brain: gene cloning and analysis of conserved cysteine regions. Gene 140, 279-284.[Medline]
Loprasert, S., Vattanaviboon, P., Praituan, W., Chamnongpol, S. & Mongkolsuk, S. (1996). Regulation of the oxidative stress protective enzymes, catalase and superoxide dismutase, in Xanthomonas a review. Gene 179, 33-37.[Medline]
Loprasert, S., Atichartpongkun, S., Whangsuk, W. & Mongkolsuk, S. (1997). Isolation and analysis of the Xanthomonas alkyl hydroperoxide reductase gene and the peroxide sensor regulator genes ahpC and ahpF-oxyR-orfX. J Bacteriol 179, 3944-3949.[Abstract]
Mongkolsuk, S., Loprasert, S., Whangsuk, W., Fuangthong, M. & Atichartpongkun, S. (1997). Characterization of transcription organization and analysis of unique expression patterns of an alkyl hydroperoxide reductase C gene (ahpC) and the peroxide regulator operon ahpF-oxyR-orfX from Xanthomonas campestris pv. phaseoli. J Bacteriol 179, 3950-3955.[Abstract]
Mongkolsuk, S., Praituan, W., Loprasert, S., Fuangthong, M. & Chamnongpol, S. (1998a). Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with a novel pattern of oxidative stress regulation from Xanthomonas campestris pv. phaseoli. J Bacteriol 180, 2636-2643.
Mongkolsuk, S., Sukchawalit, R., Loprasert, S., Praituan, W. & Upaichit, A. (1998b). Construction and physiological analysis of a Xanthomonas mutant to examine the role of the oxyR gene in oxidant-induced protection against peroxide killing. J Bacteriol 180, 3988-3991.
Niimura, Y., Poole, L. B. & Massey, V. (1995). Amphibacillus xylanus NADH oxidase and Salmonella typhimurium alkyl-hydroperoxide reductase flavoprotein components show extremely high scavenging activity for both alkyl hydroperoxide and hydrogen peroxide in the presence of S. typhimurium alkyl-hydroperoxide reductase 22-kDa protein component. J Biol Chem 270, 25645-25650.
Ochsner, U. A., Vasil, M. L., Alsabbagh, E., Parvatiyar, K. & Hassett, D. J. (2000). Role of the Pseudomonas aeruginosa oxyR-recG operon in oxidative stress defense and DNA repair: OxyR-dependent regulation of katB-ankB, ahpB, and ahpC-ahpF. J Bacteriol 182, 4533-4544.
Ochsner, U. A., Hassett, D. J. & Vasil, M. L. (2001). Genetic and physiological characterization of ohr, encoding a protein involved in organic hydroperoxide resistance in Pseudomonas aeruginosa. J Bacteriol 183, 773-778.
Poole, L. B. (1996). Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium. 2. Cystine disulfides involved in catalysis of peroxide reduction. Biochemistry 35, 65-75.[Medline]
Poole, L. B. & Ellis, H. R. (1996). Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium. 1. Purification and enzymatic activities of overexpressed AhpF and AhpC proteins. Biochemistry 35, 56-64.[Medline]
Stover, C. K., Pham, X. Q., Erwin, A. L. & 23 other authors (2000). Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406, 959964.[Medline]
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.[Abstract]
Turner, E., Hager, L. J. & Shapiro, B. M. (1988). Ovothiol replaces glutathione peroxidase as a hydrogen peroxide scavenger in sea urchin eggs. Science 242, 939-941.[Medline]
Volker, U., Andersen, K. K., Antelmann, H., Devine, K. M. & Hecker, M. (1998). One of two osmC homologs in Bacillus subtilis is part of the sigma B-dependent general stress regulon. J Bacteriol 180, 4212-4218.
White, O., Eisen, J. A., Heidelberg, J. F. & 29 other authors (1999). Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1. Science 286, 15711577.
Received 29 December 2000;
revised 16 March 2001;
accepted 2 April 2001.