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Xanthomonas campestris pv. campestris katG encodes a catalase-peroxidase that has a role in protecting the bacterium against micromolar concentrations of H2O2. A knockout mutation in katG that causes loss of catalase-peroxidase activity correlates with increased susceptibility to H2O2 and a superoxide generator and is avirulent in a plant model system. katG expression is induced by oxidants in an OxyR-dependent manner.
Catalase-peroxidases (KatGs) are heme-containing enzymes that possess catalatic and substantial peroxidatic activities. KatGs belong to class I of the superfamily of bacterial, fungal, and plant peroxidases, and they are present in a variety of prokaryotes and some ascomycete fungi (21). These enzymes function as H2O2 scavengers, protecting cells from H2O2 toxicity. In addition, in Mycobacterium tuberculosis, KatG is also involved in the activation of the frontline antituberculosis drug isoniazid (32). A number of isoniazid-resistant strains are the consequence of mutations within katG (32). katG also has roles in bacterial pathogenicity (31).
In several enteric and soil bacteria, katG is a member of the OxyR regulon, and its expression is inducible by peroxides (13, 15). Reduced and oxidized OxyR regulons bind differently to the target promoters, and both repress or induce transcription of the target genes (28). We have been investigating the OxyR-mediated response to peroxide stress in Xanthomonas campestris (2, 5, 14, 17, 18). The expression of ahpCF and katA, which encode an alkyl hydroperoxide reductase and a monofunctional catalase, respectively, are governed by OxyR. H2O2, organic hydroperoxides, and superoxide generators are potent inducers for genes in the OxyR regulon (17). Analysis of the recently published genome sequence of X. campestris pv. campestris (7) revealed an additional gene annotated as Xcc1205, encoding a bifunctional catalase-peroxidase, KatG, in addition to katA and katE, encoding atypical and stationary-phase-dependent monofunctional catalases, respectively. In this study, the physiological roles of X. campestris pv. campestris katG in bacterial stress protection and its gene regulation were characterized. Furthermore, the role of katG in bacterial virulence was investigated.
To evaluate the role of the putative katG gene annotated as Xcc1205 in the X. campestris pv. campestris genome (7), a katG knockout mutant was constructed by insertional inactivation using pKNOCK (1). The 300-bp EcoRI fragment of katG was cloned into pKNOCK-Km digested with EcoRI. The recombinant plasmid was subsequently transferred into X. campestris pv. campestris by electroporation, and recipient cells were selected by kanamycin resistance. The X. campestris pv. campestris katG mutant was verified by PCR and Southern blot analysis (data not shown). Next, the physiological role of the gene in oxidative stress protection was evaluated. The extent of resistance to H2O2 in the katG mutant and the X. campestris pv. campestris wild type was validated primarily using the plate sensitivity assay (23), in which bacterial cultures were serially diluted and plated on Silva Buddenhagen (SB) medium (17) containing 200 μM H2O2. Bacterial colonies capable of growing on H2O2-containing medium were counted after a 48-h incubation. The katG mutant was 104-fold less resistant to H2O2 than the isogenic wild-type strain (Fig. (Fig.1A1A).
We have previously observed that an X. campestris pv. phaseoli katA mutant is also less resistant to H2O2 (5). Nonetheless, the roles of each of these two catalase genes in protecting the bacteria from H2O2 have not been evaluated and compared. Therefore, an X. campestris pv. campestris katA knockout mutant was constructed using the pKNOCK system. A 200-bp katA (Xcc3949) DNA fragment was PCR amplified from X. campestris pv. campestris genomic DNA with BT2237 and BT2238 primers (Table (Table1).1). The resulting PCR products were cloned into pGEM-T Easy (Promega) before an EcoRI fragment was subcloned into pKNOCK-Gm. The resultant recombinant plasmid (pKNOCK-katA) was electroporated into X. campestris pv. campestris. The gentamicin-resistant transformants were screened for katA inactivation. The katA mutant was verified using both PCR and Southern analysis. A katA katG double mutant was also constructed by electroporating pKNOCK-katA into a katG mutant. Next, the levels of resistance to a low dose (200 μM) of H2O2 in katG and katA mutants and the parental strain were determined by the plate sensitivity assay. The results show that the katG mutant was roughly 500-fold less resistant to 200 μM of H2O2 than the katA mutant. The katA mutant was 50-fold less resistant to H2O2 than a wild-type strain (Fig. (Fig.1A).1A). This suggests that katG has a principal role in protecting the X. campestris pv. campestris cells from a low dose of H2O2.
Next, we determined the levels of catalase in various kat mutants. The katG mutant had reduced total catalase activity (4.7 ± 0.5 U mg−1 protein) compared to that in an isogenic wild-type strain (6.0 ± 0.4 U mg−1 protein). The katA mutant had a drastically lower catalase activity (1.2 ± 0.3 U mg−1 protein). There are no direct correlations between resistance to 200 μM of H2O2 and total catalase activities in various strains; KatA is a major source of catalase activity in exponentially growing X. campestris pv. campestris cells. The katG mutant has 4-fold-higher levels of catalase and yet 500-fold less resistance to 200 μM H2O2 than the katA mutant, supporting the idea that KatG catalase-peroxidase has a primary role for bacterial survival under micromolar concentrations of H2O2. Generally, degradations of H2O2 by KatG catalase-peroxidase and KatA monofunctional catalase are two-step reactions. The first step involves heme oxidation by H2O2 to form an oxyferryl species, compound I. The second reaction is a reduction of compound I by H2O2 to regenerate the resting state enzyme, water, and oxygen. While the apparent Km values for H2O2 in the heme oxidation of both KatG catalase-peroxidases and KatA monofunctional catalases are somewhat similar (~ 300 μM), the apparent Km values of KatGs for the reduction of compound I (2.4 to 4.5 mM) are much lower than those of KatA (38 to 200 mM) (26, 27). This reflects a higher affinity of KatG for H2O2 degradation.
Additionally, the katA katG double mutant was hypersensitive to H2O2. This mutant was unable to grow on medium containing 200 μM H2O2 (Fig. (Fig.1A).1A). Nevertheless, the katA katG double mutant had no aerobic growth defects when grown in either liquid or solid enriched medium (SB). This is unlike a Bradyrhizobium japonicum katG mutant that has defects in aerobic growth, suggesting that the gene has a primary role in the detoxification of H2O2 generated from aerobic life (22). We have shown in X. campestris pv. phaseoli that an ahpC (alkyl hydroperoxide reductase) mutant has reduced ability to form colonies on an agar plate, but this ability could be rescued by the addition of a peroxide scavenger, 0.1% pyruvate, to the medium (19). In Xanthomonas, AhpC has a primary role in scavenging physiologically generated H2O2 analogous to that of Escherichia coli AhpC (25).
If catalatic activity is crucial for detoxification of high concentrations of H2O2, one would anticipate that a katA mutant is less resistant to treatment with a high concentration of H2O2 than a katG mutant. We performed additional experiments using the H2O2 killing assay (5). In this procedure, bacterial cultures are subjected to lethal doses of H2O2 (5, 10, and 20 mM) for 30 min. Cells surviving the treatments were determined by viable cell count. As illustrated in Fig. Fig.1B,1B, compared to the X. campestris pv. campestris wild type, the katA mutant was 103-fold less resistant to 20 mM H2O2 killing. The katG mutant was 500-fold more resistant to the same concentration of H2O2 than the katA mutant and only 5-fold less resistant than a wild-type strain. This supports the role of KatA as a major catalase responsible for protecting X. campestris pv. campestris against high levels of H2O2, whereas katG has only a minor role in the process (Fig. (Fig.1B).1B). Additionally, the levels of resistance to high concentrations of H2O2 show a correlation with total catalase activities of the cells. As expected, the double mutant, which produced no detectable level of catalase activity, was the most sensitive to killing by a high concentration of H2O2 (Fig. (Fig.1B).1B). These data support the hypothesis that the catalatic activity of KatA as well as that of KatG is responsible for the protection of Xanthomonas from killing by millimolar concentrations of H2O2.
We extended the investigation by assessing the ability of high levels of expression of either katA or katG from an expression vector to complement the H2O2-hypersensitive phenotype of the katA katG double mutant. The double mutant harboring pKatA or pKatG (pBBR1MCS4 expression vector  containing X. campestris pv. campestris katA or katG [full-length]) produced catalase activities of 195 ± 23 and 73 ± 8 U mg−1 protein, respectively. Although the katA katG double mutant harboring pKatA had more than twofold-higher total catalase activity, it failed to fully complement the H2O2-hypersensitive phenotype, as determined by the plate sensitivity assay using 200 μM H2O2 (Fig. (Fig.1A),1A), but the strain was highly resistant to the high-concentration H2O2 killing treatment (Fig. (Fig.1B).1B). On the other hand, a high-level expression of katG fully restored the H2O2-hypersensitive phenotype of the katA katG double mutant determined using both the 200 μM H2O2 plate sensitivity assay (Fig. (Fig.1A)1A) and millimolar H2O2 killing treatments (Fig. (Fig.1B).1B). The results further support the role of KatG in protection against micromolar concentrations of H2O2. In addition, the resistance to millimolar concentrations of H2O2 shows correlation with total catalase activity.
Taken together, the results of H2O2 resistance tests and the catalase levels indicate a departure from a current paradigm for the roles of different catalases. In X. campestris, both katG and katA have primary roles in protecting the bacteria against different levels of H2O2. The basis of the mechanism is the enzymes’ different biochemical properties (bifunctional peroxidase/catalase versus monofunctional catalase) and not the activities of each enzyme.
The katG mutant showed a 10-fold increase in resistance to tert-butyl hydroperoxide compared with the wild-type strain, and the phenotype could be complemented by pKatG (Fig. (Fig.1A).1A). Inactivation of X. campestris pv. phaseoli katA has been shown to produce a compensatory increase in ahpC expression along with enhanced bacterial tolerance to organic hydroperoxides (4). To test whether the level of ahpC expression is altered, an end-point reverse transcription (RT)-PCR was performed with RNA prepared from the X. campestris pv. campestris wild type, the katG mutant, and a katG-complemented strain by using ahpC-specific primers (BT2684 and BT2685 [Table [Table1]).1]). The level of ahpC transcripts in the katG mutant was higher than the level in either the X. campestris pv. campestris wild type or the katG-complemented strain (Fig. (Fig.2A).2A). Thus, this compensatory increase in ahpC expression accounted for, at least in part, the enhanced resistance to organic hydroperoxide observed in the katG mutant. However, the precise mechanism responsible for this resistance is not yet clear. Likewise, inactivation of ahpC leads to compensatory upregulation of katG in X. campestris pv. phaseoli (19).
We also determined the resistance levels of a superoxide generator, menadione, in the X. campestris pv. campestris wild type and the katG mutant by using the plate sensitivity assay with 200 μM menadione. The results in Fig. Fig.1A1A illustrate that the katG mutant was 100-fold less resistant to menadione than the wild-type strain. Furthermore, the phenotype could be complemented by pKatG. The superoxide anions formed are dismutated to yield H2O2 and other reactive oxygen species. Efficient degradation of H2O2 in part contributes to protection against menadione toxicity. Thus, impaired H2O2 detoxification in the katG mutant is likely responsible for its reduced menadione resistance phenotype.
X. campestris pv. campestris is a causative agent of black rot disease in cruciferous crops. Oxidative stress is an important component of the plant defense response against microbes (12). Hence, we determined the effects of katG inactivation on the virulence of the bacteria on a host plant. The X. campestris pv. campestris wild type, the katG mutant, and the katG-complemented strain (katG/pKatG) were inoculated into Chinese radish (Raphanus sativus) leaves by using the leaf-clipping method (9). The results in Fig. Fig.1C1C reveal that the katG mutant is avirulent on the radish, as shown by the lack of detectable lesions in all tests, while the X. campestris pv. campestris wild type and the katG-complemented strain (katG/pKatG) produced similarly sized lesions (10.7 ± 2.7 mm for X. campestris pv. campestris and 9.8 ± 2.4 mm for the katG/pKatG strain), and no significant differences in the length of lesions (P > 0.05 by paired t test) could be detected. The results suggest that katG is required for full virulence of X. campestris pv. campestris on the Chinese radish host plant. A study of the sugar beet plant defense response reveals that the levels of H2O2 in the oxidative burst of the phase I response peaked at 2 mM in elicited plant leaves (3). The phase II burst occurs at approximately 2 h postinoculation and peaks at 4 mM H2O2. These levels of H2O2 would have lethal effects on bacterial pathogens, including Xanthomonas. We speculate that the avirulent phenotype of the katG mutant is due in part to a reduction in the ability of the bacteria to cope with plant-generated H2O2. Our speculation is supported by the in vitro experiments that show hypersensitivity of the katG mutant to H2O2 (Fig. 1A and B). In addition, we found that the X. campestris pv. campestris katA mutant showed an avirulent phenotype on the Chinese radish host, indicating that monofunctional catalase, which contributes to the protection of millimolar levels of H2O2, also plays a crucial role in Xanthomonas pathogenicity (S. Mongkolsuk, S. Buranajitpakorn, and P. Vattanaviboon, unpublished data).
The expression profile of katG in X. campestris pv. campestris was investigated using end-point RT-PCR. The experiments were performed using total RNAs extracted from untreated X. campestris pv. campestris cultures and cultures treated with various oxidants and katG-specific primers (BT2239 and BT2240 [Table [Table1]).1]). Representative results demonstrate that the levels of katG transcripts were markedly increased in cells treated with a superoxide generator, menadione (100 μM), and to a lesser extent in those treated with organic hydroperoxides (100 μM tert-butyl or cumene hydroperoxides) or H2O2 (100 μM) (Fig. (Fig.2B).2B). The katG expression profiles in response to oxidants resemble typical OxyR-dependent gene expression in X. campestris (5, 14, 17). The RT-PCR experiments were repeated in an X. campestris pv. campestris oxyR knockout mutant that was constructed using pKNOCK-Gm containing a 210-bp oxyR (Xcc0832) fragment amplified with BT1413 and BT1414 primers (Table (Table1).1). The oxidant's induction of katG expression was abolished in the oxyR mutant (Fig. (Fig.2B).2B). This indicates that OxyR regulates the peroxide-inducible expression of katG and that the gene is a member of the OxyR regulon, as in several bacteria (6, 16, 20).
OxyR commonly regulates transcription of a target gene through binding to specific motif sequences located in close proximity to the −35 promoter region. Primer extension was performed to map the 5′ end of katG mRNA and to localize the promoter by using a labeled BT2652 primer (Table (Table1)1) and total RNA extracted from uninduced and menadione-induced X. campestris pv. campestris cultures. The transcription start site (+1) based on the primer extension product was mapped to the A located 21 nucleotides upstream of the ATG codon (Fig. (Fig.3A).3A). The putative −35 and −10 elements were identified as TTCCAC and GCAGCT, respectively, and were separated by a suboptimal distance of 20 nucleotides. Both motifs share low sequence identity to the proposed consensus σ70 binding sequences for X. campestris promoters that consist of the −35 element, TTGTNN, and the −10 element, T/AATNAA/T (10). The poor promoter sequence and structure of katG are a likely reason for a relatively low expression level of the gene. A putative OxyR binding site could be identified immediately upstream of the −35 element (Fig. (Fig.3A).3A). This proposed binding site poorly matches (6 of 16) the consensus sequence of the E. coli OxyR binding site (ATAGN7CTATN7ATAGN7CTAT) (28). This is quite surprising because we have shown that the putative oxyR binding sites in the katA and ahpC promoters in X. campestris pv. phaseoli highly match (10 of 16) the E. coli consensus sequence (5, 14). Nonetheless, in several microorganisms, the OxyR binding sequences of the target promoters are diverse and differ from the consensus sequence (20, 30).
We next tested the binding of X. campestris OxyR to the katG promoter fragment by using a gel mobility shift assay. Purified X. campestris pv. phaseoli OxyR (14), which shares 99% homology with X. campestris pv. campestris OxyR, was mixed with radioactively labeled katG promoter DNA fragments in the binding buffer. The results clearly illustrate that purified OxyR bound to the katG promoter fragment (Fig. (Fig.3B).3B). The binding showed high specificity, as shown by competition experiments; only cold katG promoter DNA fragments could compete with the labeled probe in the binding reaction. The results support a notion that OxyR binds in the vicinity of the katG promoter and regulates its expression.
X. campestris pv. campestris has katA, katG, katE, and ahpC genes that are involved in the degradation of H2O2. A major puzzle in the overall bacterial peroxide stress response is whether these peroxide-scavenging genes are redundant or whether they serve disparate roles in the process. katE has no protective roles during the exponential phase of growth but is involved in protecting the bacteria from H2O2 in the stationary phase (29). The remaining three genes are regulated by OxyR and are presumably activated by similar concentrations of H2O2. Hence, the physiological roles of these genes could not be distinguished at the transcription activation level. Induction of three distinct types of peroxide-degrading enzymes enables the bacteria to detoxify H2O2 at a wide range of concentrations as well as other organic peroxides. AhpC is associated with the detoxification of low-concentration H2O2 generated from normal aerobic metabolism and organic hydroperoxides (25). At intermediate levels, KatG is better suited for detoxification of H2O2 at high micromolar levels. KatA, being the most abundant σ70 and also having a high apparent Km, is responsible for protection against millimolar concentrations of H2O2.
We thank Weerachai Tanboon, Supa Utamapongchai, and Aekkapol Mahavihakanont for technical assistance.
The research was supported by grants from the National Center for Genetic Engineering and Biotechnology (BIOTEC) and from Mahidol University. T.J. was supported by a Royal Golden Jubilee Scholarship, PHD/0222/2547, from the Thailand Research Fund.
Published ahead of print on 25 September 2009.