Genetic and physiological analysis of the major OxyR-regulated katA from Xanthomonas campestris pv. phaseoli

Nopmanee Chauvatcharin1,2,{dagger}, Sopapan Atichartpongkul1, Supa Utamapongchai1, Wirongrong Whangsuk1, Paiboon Vattanaviboon1 and Skorn Mongkolsuk1,2

1 Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok 10210, Thailand
2 Department of Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand

Correspondence
Skorn Mongkolsuk
skorn{at}tubtim.cri.or.th


   ABSTRACT
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katA encodes the major catalase that accounts for 90 % of the total catalase activity present in Xanthomonas campestris pv. phaseoli. katA is located upstream of an ORF designated ankA encoding a cytoplasmic membrane protein homologous to eukaryotic ankyrin. Transcriptional analysis of katA and ankA identified two katA transcripts: a major monocistronic katA transcript and a minor bicistronic katA–ankA transcript. KatA expression was induced in the presence of various oxidants including H2O2, organic hydroperoxides and the superoxide-generating agent menadione, in an OxyR-dependent manner. Analysis of the katA promoter region showed a putative OxyR binding site located upstream of an Escherichia coli-like {sigma}70 –35 region that is likely to be responsible for transcription activation in response to oxidant treatment. Gel mobility shift experiments confirmed that purified OxyR specifically binds to the katA promoter. A katA mutant was highly sensitive to H2O2 during both the exponential and stationary phases of growth. This phenotype could be complemented by functional katA, confirming the essential role of the gene in protecting X. campestris from H2O2 toxicity. Unexpectedly, inactivation of ankA also significantly reduced resistance to H2O2 and the phenotype could be complemented by plasmid-borne expression of ankA. Physiological analyses showed that katA plays an important role in, but is not solely responsible for, both the adaptive and menadione-induced cross-protective responses to H2O2 killing in X. campestris.


Abbreviations: tBOOH, tert-butyl hydroperoxide; NEM, N-ethylmaleimide

{dagger}Present address: Center for Vectors and Vector-Borne Diseases, Mahidol University, Bangkok 10400, Thailand.


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Catalase is recognized as a principal enzyme in the protection of organisms from H2O2 toxicity through its ability to catalyse the conversion of H2O2 to oxygen and water. Elimination of H2O2 thereby reduces the potential for transition-metal-mediated hydroxyl radical formation via the Fenton reaction (Farr & Kogoma, 1991). Bacteria have several unrelated forms of catalase (Klotz et al., 1997). Inactivation of the genes encoding catalases leads in most cases to increased susceptibility to H2O2, and in some cases to a defect in the ability to colonize a host (Visick & Ruby, 1998; Xu et al., 2001). A novel role for catalase in protection against electrophile toxicity has also been reported (Vattanaviboon et al., 2001).

The identity of key regulators mediating the expression of bacterial catalases can be inferred from observations in Escherichia coli and Salmonella typhimurium showing that the synthesis of catalase-peroxidase (hydroperoxidase I encoded by katG) is regulated by OxyR whereas expression of the monofunctional catalase (hydroperoxidase II encoded by katE) is under the regulation of a stationary-phase-specific {sigma}S (Storz & Altuvia, 1994). OxyR is a peroxide sensor and global transcriptional regulator of the peroxide stress response (Toledano et al., 1994; Zheng et al., 1998). The precise role of OxyR in the regulation of catalases seems to vary in different bacteria, particularly in non-enteric species. The expression of monofunctional catalase, encoded by katB, in Pseudomonas aeruginosa is activated by OxyR (Ochsner et al., 2000), whereas expression of the Neisseria gonorrhoeae catalase, encoded by kat, is presumably repressed by OxyR (Tseng et al., 2003). Streptomyces coelicolor produces multiple catalase isozymes, none of which is regulated by OxyR (Hahn et al., 2002).

Xanthomonas campestris is an important bacterial phytopathogen. Treatment of X. campestris pv. phaseoli with sublethal concentrations of H2O2 or a superoxide-generating agent (menadione) induced elevated levels of total catalase activity during both exponential and stationary phase (Vattanaviboon & Mongkolsuk, 2000). The level of total catalase activity correlates with the ability to resist H2O2 toxicity (Fuangthong & Mongkolsuk, 1997; Mongkolsuk et al., 1997a). X. campestris pv. phaseoli produces two detectable isozymes of monofunctional catalase, denoted KatA and KatE, that are encoded by katA and katE, respectively. KatA is the major catalase produced during all phases of growth, while KatE is detected only as cells enter the stationary phase or under nutrient-starved conditions (Vattanaviboon & Mongkolsuk, 2000). More recently, the X. campestris pv. phaseoli katA gene, encoding the major catalase, was cloned and characterized (Chauvatcharin et al., 2003). Its putative amino acid sequence is highly homologous (87 % identity) to the clade I catalase from Pseudomonas syringae, CatF, whose crystal structure has been solved (Carpena et al., 2003). In this paper, we report expression analysis and demonstrate the physiological importance of katA in protection against H2O2 toxicity. The involvement of ankA, encoding an ankyrin homologue, in the H2O2 resistance of X. campestris pv. phaseoli is also demonstrated.


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Growth conditions and media.
All Xanthomonas strains were grown aerobically at 28 °C in Silva–Buddenhagen (SB) medium containing the appropriate antibiotics (Mongkolsuk et al., 1997b). All E. coli strains were grown aerobically at 37 °C in Luria–Bertani (LB) medium. Induction experiments were performed with exponential-phase X. campestris pv. phaseoli cultures (OD600 0·6) treated with 100 µM H2O2, menadione, tert-butyl hydroperoxide (tBOOH) or N-ethylmaleimide (NEM) (Vattanaviboon et al., 2001) for 15 min for Northern analysis and 30 min for enzyme assays (Mongkolsuk et al., 1997b).

Nucleic acid manipulations.
All nucleic acid manipulations were performed using standard molecular biology techniques (Sambrook et al., 1989) or according to the manufacturers' recommendations. The labelling of DNA probes with [{alpha}-32P]dCTP was performed using a DNA labelling bead (Amersham Pharmacia Biotech). Southern and Northern blot analyses were performed as previously described (Mongkolsuk et al., 1997b).

Catalase activity gels and assays.
Cell lysate preparation and catalase activity gel staining were performed as previously described (Vattanaviboon & Mongkolsuk, 2000). Bacterial cells were lysed in 50 mM sodium phosphate buffer, pH 7·0, containing 1 mM PMSF by brief sonication followed by centrifugation at 10 000 g for 10 min. Supernatants were used for catalase activity gels and catalase isozymes were visualized on native PAGE gels as previously described (Vattanaviboon & Mongkolsuk, 2000). Catalase activity appeared as colourless bands against a dark brown background. The catalase assay was carried out spectrophotometrically, according to the method of Beers & Sizer (1952). One unit of catalase was defined as the amount of enzyme required to decompose 1·0 µmol H2O2 at 25 °C at pH 7·0.

Cloning of full-length ankA.
The full-length ankA gene in pKat29 (Chauvatcharin et al., 2003) was amplified using the oligonucleotide primers BT176 and BT177 (see Table 1). The 620 bp PCR product was cloned into pGemT-easy and subsequently subcloned into the broad-host-range plasmid pBBR1MCS-5 (Kovach et al., 1995) to generate the ankA overexpression plasmid, pAnkA (see Table 1).


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Table 1. Bacterial strains, plasmids and primers

 
Construction of katA (Xp20) and ankA (Xp21) insertion mutants.
The X. campestris katA mutant, Xp20, was constructed by insertional mutagenesis using pKat111, consisting of the non-replicative plasmid pGemT containing a 344 bp katA internal DNA fragment (Chauvatcharin et al., 2003; see Table 1). pKat111 was transferred into X. campestris pv. phaseoli wild-type strain (Xp) by electroporation. Homologous recombination between the katA fragment on pKat111 and its counterpart on the chromosome resulted in the Ampr strain Xp20 containing an insertionally inactivated katA. Inactivation of katA was confirmed by catalase activity gel staining and Southern blots of Xp20 chromosomal DNA digested with EcoRI or SalI and hybridized to a katA-specific probe (data not shown). The ankA mutant Xp21 was constructed by insertional inactivation using the pKNOCK system (Alexeyev, 1999). A blunt-ended 160 bp BssHII fragment from pAnkA was ligated to SmaI-digested pKNOCK-Km to form pKNOCKankA. pKNOCKankA was introduced into Xp by conjugation, and the Kanr ankA mutant, Xp21, was selected and confirmed by Southern blot hybridization. The katA ankA double mutant, Xp31, was constructed by transferring Xp20 genomic DNA into Xp21 by electroporation and selecting for Kanr Ampr exconjugants.

Gel mobility shift assay.
32P-labelled DNA fragments were prepared by PCR using the oligonucleotide primers BT151 and BT150 (see Table 1) and pKat29 (Chauvatcharin et al., 2003) as the template to generate a 254 bp fragment spanning the katA promoter region. Gel mobility shift reactions were performed by adding 3 fmol labelled probe in 25 µl reaction buffer [20 mM Tris pH 7·0, 50 mM KCl, 1 mM EDTA, 5 %, v/v, glycerol, 50 µg BSA ml–1, 5 µg calf thymus DNA ml–1, 0·5 mg poly(dI/dC) ml–1]; 400 ng purified OxyR (Loprasert et al., 2000) was added and the reaction was incubated at 25 °C for 15 min. Protein–DNA complexes were separated by electrophoresis on 6 % non-denaturing polyacrylamide gel in 0·5x Tris/borate/EDTA buffer (TBE) at 4 °C.

RT-PCR of katA–ankA mRNA.
Reverse transcription (RT) of katA–ankA mRNA was performed to confirm the bicistronic transcriptional organization of these genes. Total RNA was isolated from X. campestris pv. phaseoli cultures using the hot acid/phenol method (Mongkolsuk et al., 2002). Purified RNA was treated with 10 U RNase-free DNase I for 30 min to remove contaminating DNA. Primer BT149 (located within ankA; see Table 1) was mixed with 10 µg RNA, and 200 U cloned Moloney murine leukaemia virus (MMLV) reverse transcriptase (Promega) was added. The mixture was incubated at 42 °C for 60 min. Five microlitres of the mixture was added to a PCR reaction containing primers BT149 and BT148 (located in katA; see Table 1). PCR was performed for 35 cycles under the following conditions: denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 30 s. The PCR products were analysed by agarose gel electrophoresis.

Primer extension.
Total RNA was isolated from uninduced and menadione-induced X. campestris pv. phaseoli cultures. Primer extension experiments were performed using 32P-labelled oligonucleotide primer BT150 (see Table 1), 5 µg total RNA and 200 U superscript II MMLV reverse transcriptase (Promega). Extension products were sized on sequencing gels next to dideoxy sequencing ladders generated using a PCR sequencing kit with labelled BT150 primer and pKat29 plasmid as the template (Chauvatcharin et al., 2003).

Determination of oxidant resistance.
Analysis of the killing effects of various reagents on X. campestris pv. campestris strains was performed using inhibition zone assays as described by Mongkolsuk et al. (1998a). Briefly, overnight cultures were subcultured as 5 % inocula into fresh SB broth and incubated at 28 °C with shaking. One millilitre of exponential-phase cells (4 h, culture OD600 ~0·5) was mixed with 10 ml molten top agar (SB containing 0·7 % agar) held at 50 °C, and overlaid onto SB plates (14 cm diameter Petri dishes containing 40 ml SB agar). The plates were left at room temperature for 15 min to let the top agar solidify. Sterile 6 mm diameter paper discs soaked with 5 µl H2O2 (0·5 M), tBOOH (0·5 M) or menadione (1·0 M) were placed on top of the cell lawn and the diameters of the inhibition zones were measured after 24 h incubation at 28 °C.

Determination of adaptive and cross-protective resistance to H2O2.
The induced adaptive or cross-protective resistance to H2O2 killing was measured by adding H2O2 or menadione (100 µM), respectively, to exponential-phase cultures of X. campestris pv. campestris strains prior to treatment with lethal concentrations of H2O2 (10, 20, 30 mM) for 30 min. After treatment, cells were removed and washed once with fresh SB medium and cell survival was determined by plating appropriate dilutions on SB agar plates. Colonies were counted after 48 h incubation at 28 °C. The surviving fraction was defined as the number of c.f.u. recovered after treatment divided by the number of c.f.u. prior to treatment. Three independent experiments were performed in each case and representative data are shown.


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Gene and transcription organization of katA–ankA
The cloning and sequencing of katA encoding the clade I monofunctional catalase from X. campestris pv. phaseoli (Xp) has been reported previously (Chauvatcharin et al., 2003). Additional sequencing of DNA surrounding katA (GenBank accession no. AF461425) revealed a 600 bp ORF capable of encoding a 199 amino acid protein with a calculated molecular mass of 20·5 kDa. The deduced amino acid sequence was searched against the GenBank database using the BLAST program (Altschul et al., 1997), revealing that the putative protein has a high degree of sequence identity (60 %) with an ankylin-like protein encoded by ankB from P. aeruginosa (Howell et al., 2000). This ORF was subsequently designated ankA. katA and ankA are located in the same orientation and separated by 59 bp. Analyses of the gene organization of the kat loci in various micro-organisms reveal that the kat–ank gene organization is found not only in Pseudomonas and Xanthomonas, but also in several other bacteria such as Vibrio cholerae (AE004235), Campylobacter jejuni (AL139078) and Streptomyces coelicolor (AL939105). The conservation of kat–ank organization suggests that these genes could have related functions that are of physiological significance.

The transcriptional organization of katA and ankA was determined by Northern blots. The results showed that a katA probe hybridized to two mRNA bands at 1·6 kb and 2·2 kb that corresponded to the predicted sizes of monocistronic katA and bicistronic katA–ankA mRNAs respectively (Fig. 1a). Northern blot analysis using an ankA-specific probe detected a single positively hybridizing band at 2·2 kb (Fig. 1a), suggesting that ankA is co-transcribed with katA. This was confirmed by RT-PCR experiments that were performed using two specific primers, one located near the 3' end of the katA coding region and another located near the 5' end of ankA. RT-PCR using an RNA sample prepared from an uninduced Xp culture gave rise to a 550 bp product that corresponded to the expected size of a product derived from a katA–ankA mRNA template (Fig. 1b). This 550 bp band was absent when the RNA sample was first treated with RNaseA, indicating that the product was not the result of priming to contaminating DNA (Fig. 1b). Thus, katA and ankA are transcribed as a bicistronic mRNA of 2·2 kb. Densitometer analyses of katA Northern blots indicated that the monocistronic katA mRNA made up 85 % of the katA-encoding mRNA while 15 % consisted of the katA–ankA bicistronic message (Fig. 1b). Examination of the DNA sequence in the katA–ankA intergenic region shows the presence of an inverted repeat sequence spanning 32 bp followed by a run of three T residues (Fig. 1c). The stem–loop structure resembles a typical rho-independent transcription terminator, suggesting that the major 1·5 kb katA monocistronic transcripts are the result of rho-independent transcription termination at this site. Moreover, the minor 2·2 kb katA–ankA bicistronic mRNA is likely to result from transcriptional readthrough at this site. The mechanism responsible for the antitermination at the katA terminator is not known. It could be mediated by a regulator, as with AmiR in the amidase operon (Wilson et al., 1996). Alternatively, readthrough may be the result of the intrinsic efficiency of the terminator itself (Weisberg & Gottesman, 1999), such that it permits 15 % readthrough transcription into ankA.



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Fig. 1. Expression of katA–ankA. (a) Northern blot analysis of total RNA prepared from exponential-phase cultures of X. campestris pv. phaseoli under uninduced (UN), tBOOH-induced (tBOOH), menadione-induced (MD) and H2O2-induced (H2O2) conditions hybridized with 32P-labelled katA or ankA DNA probes. (b) RT-PCR analysis of a total RNA sample (S) extracted from a X. campestris pv. phaseoli culture as described in Methods. C1, positive control generated using genomic DNA as the template; C2, negative control in which RNase was added prior to the PCR reaction; M, DNA size marker. (c) Nucleotide sequence of the katA–ankA intergenic region. The stop codon of katA and the start codon of ankA are indicated in bold capitals. The inverted repeats of a putative rho-independent transcription terminator sequence are in bold lower case and indicated by arrows.

 
Oxidant induction of KatA requires a functional OxyR
It has been previously shown that X. campestris pv. phaseoli produces two monofunctional catalase isozymes, namely KatA and KatE (formerly Kat2) (Chauvatcharin et al., 2003; Vattanaviboon & Mongkolsuk, 2000). The KatA level was found to be high during the exponential phase of growth and declined slightly when cells entered stationary phase. By contrast, KatE levels increased as cells entered stationary phase. In many micro-organisms, catalase activity is induced by exposure to low concentrations of H2O2. In order to determine if this was true in X. campestris pv. phaseoli, total catalase activity and the levels of KatA were determined after exponential-phase cultures were treated with various oxidants. Total catalase activity showed that menadione was the most potent inducer and stimulated an increase in total catalase activity of 9·5-fold. H2O2, tBOOH and NEM also induced catalase activity, but to a lesser degree: twofold, fourfold and fourfold, respectively (Fig. 2a). The effect of oxidants on the expression of the two catalases was also assessed using catalase activity-stained gels (Fig. 2a). While oxidant treatments did not significantly change the levels of KatE (data not shown), KatA levels increased in response to the oxidant treatments (Fig. 2a). The magnitude of induction as judged by densitometer analyses of the KatA-specific activity-stained bands showed good correlation with the degree of induction observed using total catalase activity measurements: i.e. menadione was the most potent inducer of KatA, while H2O2, tBOOH and NEM stimulated lower levels of induction (Fig. 2a). The results supported the idea that an increase in KatA activity was responsible for the observed increase in total catalase levels following oxidant treatments. Furthermore, the results of Northern blot analyses showing oxidant-induced synthesis of katA mRNA suggested that the increased KatA activity was due to increased transcript levels of katA (Fig. 1a). Similar oxidant-induced expression of monofunctional catalase has also been observed in Xanthomomas oryzae pv. oryzae, where menadione was found to be the most potent inducer, followed by H2O2 and other oxidants (Mongkolsuk et al., 1996).



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Fig. 2. Effect of oxidant treatment on the expression of katA. X. campestris pv. phaseoli (Xp, a) and an oxyR mutant (Xp01, b) were grown to exponential phase and the cells were then induced with H2O2, menadione (MD), tBOOH and NEM, and extracts were prepared as descibed in Methods. Crude protein (50 µg for Xp and 100 µg for Xp01) was separated using native PAGE and subjected to catalase activity gel staining (upper panels). The total catalase activity was also determined (lower panels). UN, uninduced control.

 
Since menadione, H2O2, tBOOH, and NEM are known to be potent inducers of other genes in the OxyR regulon in X. campestris pv. phaseoli (Loprasert et al., 2000; Mongkolsuk et al., 1997b), it was hypothesized that OxyR may also regulate the oxidant-induced expression of katA. In order to test this hypothesis, the total catalase activity was measured in the oxyR mutant, Xp01, grown in the presence and absence of oxidants. Strain Xp01 showed no oxidant-dependent increases in either total catalase activity or KatA synthesis as determined using activity-stained gels (Fig. 2b). The results indicated that oxidant-dependent induction of KatA synthesis during exponential phase requires a functional oxyR.

Analysis of OxyR regulation of the katA promoter
A more detailed characterization of OxyR-regulated expression of katA was performed. First, the transcriptional start site of the katA–ankA operon was mapped. The results of primer extension analyses using total RNA samples prepared from uninduced and menadione-induced cultures showed a single extension product of 78 bases, indicating that katA transcription is initiated at the C residue located 21 nucleotides upstream of the katA translational start codon (Fig. 3). The proposed {sigma}70-type RNA polymerase consensus binding sequence for a X. campestris promoter consists of the –35 element, TTGTNN, separated by 16 to 24 nucleotides from the –10 element, T/AATNAA/T (Katzen et al., 1996). Examination of the sequence upstream of the transcriptional start site revealed the presence of two sequence motifs, TTCTCA (–34 to –29) and GATGAT (–11 to –6), that are separated by 17 bp, and that closely matched the –35 and –10 consensus promoter sequences, respectively (Fig. 3). A significant amount of katA primer extension products was detected in the absence of inducer (Fig. 3, uninduced sample) indicating that the gene is constitutively transcribed. This is consistent with other observations (Fig. 2) indicating that even in the absence of oxidant inducers, katA is highly expressed during exponential-phase growth. The amount of primer extension products in the menadione-treated sample was fivefold higher than that in the uninduced sample (Fig. 3). This reinforced the Northern blot hybridization results and confirmed that menadione induction of KatA activity is the result of increased katA transcript levels.



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Fig. 3. Localization of the katA promoter. Results of primer extention mapping of the transcription start site of the X. campestris pv. phaseoli katA–ankA operon using total RNA prepared from uninduced (UN) and menadione-induced (MD) X. campestris pv. phaseoli cultures, sized against a dideoxy sequencing ladder generated using the same primer (upper panel). The X. campestris pv. phaseoli katA promoter sequence (lower panel) showing the mapped transcription start site (+1) as well as E. coli-like {sigma}70 –10 and –35 promoter elements (large bold type). A putative OxyR binding site is shown in bold lower-case type and is compared to the E. coli consensus OxyR binding sequence.

 
The results obtained using the oxyR mutant, Xp01, indicated that a functional oxyR was required to mediate oxidant-dependent induction of katA expression. In E. coli, OxyR-mediated activation of gene expression requires the binding of oxidized OxyR to an extended DNA recognition sequence (ATAGntnnnanCTATnnnnnnnATAGntnnnanCTAT; Toledano et al., 1994) located immediately upstream of the –35 region of the promoter. When bound to this site, OxyR facilitates the binding of RNA polymerase to the promoter, resulting in increased transcription of the gene (Toledano et al., 1994). A DNA sequence that is highly similar (62 % identity) to the E. coli consensus OxyR DNA-binding motif, located immediately upstream of the –35 region, was identified in the X. campestris pv. phaseoli katA promoter (Fig. 3). This sequence is also similar to a previously characterized OxyR binding site within the X. campestris pv. phaseoli ahpC promoter (Loprasert et al., 2000).

In order to conclusively demonstrate the direct participation of OxyR in the activation of X. campestris pv. phaseoli katA transcription, purified Xp OxyR and a 254 bp DNA fragment spanning the katA promoter, including the putative OxyR binding site, were used in mobility shift assays. The results of these assays demonstrated that OxyR bound to the katA promoter (Fig. 4). OxyR binding was inhibited by the addition of excess unlabelled probe fragment (Fig. 4, UP), but not by the addition of excess nonspecific competitor DNA (pBBR1MCS-5) (Fig. 4, UD), indicating that binding was specific for the katA promoter. Moreover, the unrelated oxidant-sensing transcription repressor, OhrR (Mongkolsuk et al., 2002), did not bind to the katA promoter, indicating that katA expression is not under direct OhrR control (Fig. 4, OhrR). These data, coupled with the in vivo results, support the hypothesis that katA is directly regulated by OxyR.



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Fig. 4. OxyR binding to the katA promoter. A DNA mobility shift assay using a 32P-labelled katA promoter fragment and increasing amounts (0 to 200 ng) of purified OxyR (Loprasert et al., 2000) was performed as described in Methods. UD and UP indicate reactions containing 2 µg unrelated DNA (pBBR1MCS-5 plasmid) and 2 µg unlabelled katA promoter, respectively, in addition to OxyR (200 ng). P represents 32P-labelled katA promoter. OhrR (Mongkolsuk et al., 2002) indicates a reaction containing 2 µg purified Ohr in place of OxyR. F, free probe; B, bound probe.

 
katA and ankA mutants are sensitive to H2O2
In order to understand the physiological roles of KatA and AnkA in the X. campestris pv. phaseoli oxidative stress response, individual katA (Xp20) and ankA (Xp21) mutants were constructed and their resistance levels against various oxidants were evaluated using the growth inhibition zone method. As expected, Xp20 was highly sensitive to H2O2, but not to the superoxide generator menadione (Fig. 5a and data not shown). The increased sensitivity to H2O2 could be complemented by expression of katA from a vector (Fig. 5a). Interestingly, Xp20 was more resistant to tBOOH than the parental strain Xp (data not shown). This probably resulted from a compensatory increase in ahpC expression levels in response to the loss of katA. A similar situation has been previously observed in X. campestris pv. phaseoli, where inactivation of ahpC has been shown to cause an increase in catalase activity (Mongkolsuk et al., 2000). The mechanism of this putative compensatory interaction involving the upregulation of ahpC in response to the inactivation of katA is currently being investigated.



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Fig. 5. Resistance levels to H2O2 and the expression of katA in X. campestris pv. phaseoli mutant strains. (a) Levels of resistance against H2O2 killing in: X. campestris pv. phaseoli (Xp), Xp20 (katA), Xp20 harbouring pKatA (katA/pKatA), Xp21 (ankA), Xp21 harbouring pAnkA (ankA/pAnkA) and Xp31 (katA ankA). Cultures were grown to exponential phase. Values in parentheses indicate the total catalase activity (U mg–1) expressed as the means of triplicate assays. Bars represent the standard deviation. 0, activity not detectable. (b) Catalase activity gel of crude proteins (100 µg) prepared from exponential-phase cultures of Xp and Xp21 (ankA) and separated by native PAGE. (c) Northern blot analysis of total RNA prepared from exponential-phase cultures of X. campestris pv. phaseoli (Xp) and the ankA mutant (Xp21) under uninduced (UN) and menadione-induced (MD) conditions hybridized with 32P-labelled katA DNA probes.

 
Since katA and ankA make up an operon, inactivation of katA is likely to have a polar effect on ankA expression. Although the overexpression of katA in Xp20/pKatA complemented the H2O2 sensitivity of Xp20, the phenotype of Xp20 could have been a result of the loss of both KatA and AnkA. In order to test this idea, a X. campestris pv. phaseoli double katA ankA mutant (Xp31) was constructed and its resistance levels to H2O2 were evaluated. The results showed that Xp31 and Xp20 had identical phenotypes with respect to H2O2 sensitivity and catalase activity (Fig. 5a), suggesting that the observed phenotype of Xp20 was primarily due to the loss of KatA. Unexpectedly, disruption of ankA alone in strain Xp21 caused a small but significant decrease in the total catalase level and rendered the cells more sensitive to H2O2. The decrease in catalase activity and the increased H2O2 sensitivity in Xp21 could be complemented by the constitutive expression of ankA from the plasmid pAnkA (Fig. 5a). The small increase in sensitivity to H2O2 in Xp21 combined with the observation that the katA ankA double mutant, Xp31, did not show increased sensitivity to H2O2 relative to the single katA mutant, Xp20 (Fig. 5a), suggested that the increased H2O2 sensitivity of Xp21 is due to a reduction in the KatA level (Fig. 5b) and not to other independent mechanisms. We also tested whether a mutation in ankA altered the steady-state levels of katA mRNA. Northern experiments performed using total RNA isolated from Xp20 and Xp21 and hybridized to a katA-specific DNA probe indicated that the levels of katA mRNA were similar (Fig. 5c), suggesting that inactivation of ankA affected KatA levels at the post-transcriptional level. It is possible but highly unlikely that AnkA interacts with KatA and affects the Vmax of the enzyme, leading to reduction in catalase activity in the ankA mutant. A more likely interpretation is that AnkA isinvolved in the anchoring or export of KatA. Alignment of X. campestris pv. phaseoli AnkA and P. aeruginosa AnkB revealed that the two proteins shared 60 % amino acid sequence identity (data not shown). Although AnkA from X. campestris pv. phaseoli has an extended hydrophobic N-terminal region containing six repeats of proline-alanine residues, topology prediction of the membrane protein using TopPred (Claros & von Heijne, 1994) (available at http://bioweb.pasteur.fr/seqanal/interfaces/toppred.html) predicted the presence of a cytoplasmic segment at the first 2 amino acids of the N-terminus followed by a single 21 amino acid transmembrane segment. The remaining portion of the protein (168 amino acid residues) was predicted to reside in the periplasm (data not shown). This structural profile agrees with the data reported previously for P. aeruginosa AnkB, suggesting that X. campestris pv. phaseoli AnkA is localized in the same cellular compartment as AnkB (Howell et al., 2000). In P. aeruginosa, AnkB, a membrane-associated protein containing an inner-membrane-spanning motif at the N-terminus, has been proposed to form an antioxidant scaffolding that anchors catalase in the periplasm and provides a protective lattice for effective H2O2 detoxification (Howell et al., 2000). It is not known whether this is true in the case of ankA. In addition, since both P. aeruginosa katB and X. campestris katA contain a putative leader sequences identified by the SignalP program (Bendtsen et al., 2004; available at http://www.cbs.dtu.dk) (data not shown), it is possible that one of the functions of ankyrin is in the export of KatA, and inactivation of ankA would therefore lead to blockage of the process. This could back up KatA in the cytoplasm and interfere with the translation of katA mRNA. The secretion of KatA is being investigated.

katA contributes to H2O2-induced adaptive protection and menadione-induced cross-protection against H2O2 killing treatments
Physiological adaptation to stresses is an important response for bacterial survival under stressful conditions. The process often involves complex alteration in the expression pattern of genes involved in stress protection and repair of stress-induced damage. The oxidative stress-induced physiological adaptation and cross-protection responses are widely distributed in both Gram-negative and Gram-positive bacteria. We have reported the presence of H2O2-induced physiological adaptive and menadione-induced cross-protective responses to lethal concentrations of H2O2 in Xp (Mongkolsuk et al., 1998b). The H2O2-induced adaptive response is completely abolished in an oxyR mutant while the menadione-induced cross-protective response is only partially lost (Mongkolsuk et al., 1998b). The role of OxyR-regulated katA expression in these responses was evaluated. Xp20 and its parental strain Xp were grown to exponential phase before being induced with either H2O2 or menadione (100 µM) for 30 min. The induced cultures were then treated with lethal concentrations of H2O2 for 30 min and the percentage survival relative to an untreated control culture was determined. The results show that Xp20 had significantly impaired H2O2-induced adaptive and menadione-induced cross-protection responses against H2O2 relative to the parental strain (Fig. 6). In the parental strain Xp, pretreatment with H2O2 or menadione induced 100-fold and 1000-fold protection, respectively, against subsequent H2O2 killing treatments (Fig. 6a). In the katA mutant, Xp20, the levels of induced protection against H2O2 killing decreased to 10-fold and 100-fold after induction with H2O2 or menadione, respectively (Fig. 6b). This indicated that katA has a general role in the protection against killing by H2O2 in both uninduced cells and those induced by oxidants. Furthermore, even though uninduced and oxidant-induced Xp20 cells were 100-fold more sensitive to H2O2 killing than the parental strain Xp, strain Xp20 still retained the ability to mount a partial H2O2-induced adaptive response. This observation, combined with the fact that inactivation of oxyR in X. campestris pv. phaseoli has been shown to completely abolish the H2O2-induced adaptive response (Mongkolsuk et al., 1998b), implies that OxyR-regulated genes other than katA must contribute to the H2O2-induced adaptive response in Xp20. In E. coli, the alkylhydroperoxidase AhpC has an essential role in scavenging H2O2 during normal growth (Seaver & Imlay, 2001) and it has been shown that ahpC is highly induced by both H2O2 and menadione (Loprasert et al., 2000). Thus, it is likely that induction of ahpC by oxidants contributed to both the adaptive and menadione-induced cross-protective responses to H2O2 killing in X. campestris pv. phaseoli. The mechanism of menadione-induced cross-protection against H2O2 is more complex. The fact that OxyR was only partially responsible for the process (Mongkolsuk et al., 1998b) suggests that, in addition to katA and ahpC, there must be other genes, independent of OxyR regulation, that are involved. The OxyR-independent menadione induction of these protective systems is currently being investigated.



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Fig. 6. Adaptive and cross-protective responses against H2O2 in Xp20. The survival curves of exponential-phase cultures of Xp (a) and the katA mutant Xp20 (b) pretreated with H2O2 ({triangleup}), or menadione ({circ}) prior to subsequent treatments with lethal concentrations of H2O2 at the indicated concentrations and compared to an uninduced control culture ({bullet}). The values presented are the means and standard deviations of three replicates.

 


   ACKNOWLEDGEMENTS
 
The authors thank Dr J. M. Dubbs for a critical reading of the manuscript and P. Munpiyamit for manuscript preparation. This research was supported by a Research Team Strengthening Grant from the National Center for Genetic Engineering and Biotechnology (BIOTEC), a Senior Research Scholar Grant RTA4580010 from the Thailand Research Fund (TRF) to S. M. and a grant from the ESTEM through the Higher Education Development Project of the Ministry of University Affairs. N. C. was supported by a Royal Golden Jubilee Scholarship (PHD/00194/2541) from the TRF.


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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 1 September 2004; revised 22 October 2004; accepted 5 November 2004.



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