Only one catalase, katG, is detectable in Rhizobium etli, and is encoded along with the regulator OxyR on a plasmid replicon

María del Carmen Vargas1, Sergio Encarnación1, Araceli Dávalos1, Agustín Reyes-Pérez1, Yolanda Mora1, Alejandro García-de los Santos2, Susana Brom2 and Jaime Mora1

1 Programa de Ingeniería Metabólica, Centro de Investigación sobre Fijación de Nitrógeno, Universidad Nacional Autónoma de México, Apdo. Postal 565-A, Cuernavaca, Morelos, CP62210, Mexico
2 Programa de Genética Molecular de Plásmidos Bacterianos, Centro de Investigación sobre Fijación de Nitrógeno, Universidad Nacional Autónoma de México, Apdo. Postal 565-A, Cuernavaca, Morelos, CP62210, Mexico

Correspondence
Sergio Encarnación
encarnac{at}cifn.unam.mx


   ABSTRACT
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The plasmid-borne Rhizobium etli katG gene encodes a dual-function catalase-peroxidase (KatG) (EC 1.11.1.7) that is inducible and heat-labile. In contrast to other rhizobia, katG was shown to be solely responsible for catalase and peroxidase activity in R. etli. An R. etli mutant that did not express catalase activity exhibited increased sensitivity to hydrogen peroxide (H2O2). Pre-exposure to a sublethal concentration of H2O2 allowed R. etli to adapt and survive subsequent exposure to higher concentrations of H2O2. Based on a multiple sequence alignment with other catalase-peroxidases, it was found that the catalytic domains of the R. etli KatG protein had three large insertions, two of which were typical of KatG proteins. Like the katG gene of Escherichia coli, the R. etli katG gene was induced by H2O2 and was important in sustaining the exponential growth rate. In R. etli, KatG catalase-peroxidase activity is induced eightfold in minimal medium during stationary phase. It was shown that KatG catalase-peroxidase is not essential for nodulation and nitrogen fixation in symbiosis with Phaseolus vulgaris, although bacteroid proteome analysis indicated an alternative compensatory mechanism for the oxidative protection of R. etli in symbiosis. Next to, and divergently transcribed from the catalase promoter, an ORF encoding the regulator OxyR was found; this is the first plasmid-encoded oxyR gene described so far. Additionally, the katG promoter region contained sequence motifs characteristic of OxyR binding sites, suggesting a possible regulatory mechanism for katG expression.


Abbreviations: SOD, superoxide dismutase

The katG and oxyR sequences discussed in this study have been deposited in GenBank under accession number AF486647.


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Cellular metabolism of molecular oxygen produces reactive and potentially toxic oxygen species such as superoxide, hydroxyl radicals and hydrogen peroxide (H2O2) (Halliwell & Gutteridge, 1986). For defence against reactive oxygen species, organisms contain antioxidants and enzymes that prevent or repair oxidative damage. Catalases are haem-containing enzymes which break down H2O2 to O2 and H2O. These enzymes play an important role in reducing the formation of the highly reactive hydroxyl radical which arises from the degradation of H2O2 via the Fenton reaction (Halliwell & Gutteridge, 1986). The response of bacteria to oxidative stress has been most extensively studied in Escherichia coli (Farr & Kogoma, 1991), which synthesizes two types of catalase: a bifunctional catalase-peroxidase (HPI) encoded by katG (Triggs-Raine et al., 1988) and a monofunctional catalase (HPII) encoded by katE (von Ossowski et al., 1991). These two kat genes are regulated differently in terms of growth phase and in response to oxidative stress (Loewen & Hengge-Aronis, 1994).

Catalase activity is often described as essential for aerobic life, but only recently has it been realized that there are at least three unrelated sequence families that code for enzymes with catalase activity. These are ‘true catalase’, ‘catalase-peroxidase’ and ‘Mn-catalase’. All three of these sequence families are found in the prokaryotic world. Catalase-peroxidases are a distinct subclass of the haem peroxidase superfamily of enzymes (Welinder, 1991), and share no significant sequence homology with proteins of the ubiquitous ‘true catalase’ family found in bacteria, eukarya and archaea. Catalase-peroxidases have been implicated as virulence factors in mycobacteria (Zhang et al., 1992) and Agrobacterium tumefaciens (Xu & Pan, 2000).

During symbiosis and free life, the cellular metabolism of molecular oxygen by Rhizobium spp. produces reactive and potentially toxic oxygen species (Halliwell & Gutteridge, 1986). In Sinorhizobium meliloti, the katA gene encodes an H2O2-inducible catalase (KatA) (Herouart et al., 1996). Two additional catalases, a monofunctional catalase (KatC) and a bifunctional catalase-peroxidase (KatB), are produced in S. meliloti in stationary phase on rich medium. A katA : : Tn5 mutant showed a dramatic sensitivity to H2O2, and KatA appeared to be the major component of the H2O2 adaptative response. Neither nodulating capacity nor nitrogen fixing activity was impaired in the katA mutant, suggesting that KatA is not essential for nodulation and nitrogen fixation. In contrast, a dramatic decrease in nitrogen fixation capacity was observed in a katA katC double mutant (Herouart et al., 1996). Three distinct catalase activities have also been detected in free-living Rhizobium leguminosarum bv. phaseoli (Crockford et al., 1995).

In prokaryotes, catalases are usually chromosomally encoded. Only two plasmid-encoded catalases have been described, a catalase-peroxidase from E. coli O157 : H7 (Brunder et al., 1996) and KatA from Pseudomonas fluorescens (Peters et al., 2001). Both of these bacteria additionally contain one or more chromosomally encoded catalases.

A key regulator of the adaptive responses to oxidative stress is the OxyR transcription factor, which induces the expression of antioxidant activities in response to H2O2 stress (Bauer et al., 1999; Storz & Imlay, 1999). A variety of biochemical assays, the characterization of strains carrying mutations and computational approaches to identify regulator binding sites, together with transcriptional profiling, have shown that in some ways the oxidative stress responses and resistance to HOCl, organic solvents and reactive nitrogen species (Storz & Imlay, 1999) are intimately coupled to OxyR. The expression of many of the H2O2-inducible activities is regulated by the OxyR transcription factor. These include hydroperoxidase I (catalase, katG), two subunits of an alkyl hydroperoxidase reductase (ahpCF), glutaredoxin 1 (grxA), glutathione reductase (gorA) and the Fur repressor (fur), and new OxyR-activated genes, including hemH, the six-gene suf operon and four genes of unknown function, and several genes, including uxuA, encoding mannonate hydrolase, whose expression might be repressed by OxyR (Calcutt et al., 1998; Mongkolsuk et al., 1998; Sherman et al., 1995). These regulatory capacities indicate an important role of OxyR as a global regulator in the adaptation to H2O2 and to different kinds of stresses.

Here, we report the cloning of a gene encoding an inducible catalase-peroxidase, KatG, from Rhizobium etli CE3 and present a characterization of its role in oxygen stress resistance. Adjacent to katG, we encountered a gene encoding the global regulator OxyR, and this is the first example of oxyR being cloned from a member of the Rhizobiaceae. Remarkably, both katG and oxyR are encoded on plasmid f of R. etli CE3, which carries several fix genes involved in nitrogen fixation (Girard et al., 2000).


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Bacterial strains, plasmids and growth conditions.
The bacterial strains and plasmids used in this study are listed in Table 1. Culture media and growth conditions for Rhizobium etli and Escherichia coli were as reported previously (Encarnación et al., 1995).


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Table 1. Bacterial strains and plasmids used in this study

 
DNA manipulations and sequence analysis.
Cloning, restriction mapping, transformation, plasmid isolation, random priming, Southern blotting and hybridization were performed according to standard protocols (Sambrook et al., 1989). DNA fragments to be sequenced were subcloned into pBluescript and sequenced using a model 373A automated DNA sequencer and a dye terminator cycle-sequencing FS Ready-Reaction kit (Applied Biosystems). Nucleotide sequences were assembled with the GENEWORKS package (release 2.3.1; IntelliGenetics). Nucleotide and protein sequence homology searches were made using the BLAST program (Altschul et al., 1997) via the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov/). The sequences of katG and oxyR have been deposited in the GenBank database under accession number AF486647.

Transposon mutagenesis.
R. etli CE3 was mutagenized with transposon Tn5 as described previously (Dunn et al., 1996). Transconjugants were tested for their inability to produce gas (O2) upon addition of H2O2. One clone containing the Tn5 insertion was selected and was designated VEM1673.

Cloning, construction and complementation of a katG mutant.
Two different complementations were done. In the first complementation, we isolated recombinant plasmids that complemented the R. etli catalase-negative mutant VEM1673, using an R. etli CFN42 pLAFR1 genomic library of 1200 recombinant clones generated in E. coli HB101, ensuring 99·9 % probability of complete representation of the R. etli genome (Huerta-Zepeda et al., 1997). The library cosmids were transconjugated into strain VEM1673 by triparental crosses using pRK2013 as a helper and selecting for nalidixic acid- and tetracycline-resistant colonies. In the second complementation, we cloned the full-length katG gene and its promoter region. We designed primers (upKat, 5'-AGAATTCGCCGAGCTTATCGCAGCA -3'; lowKat, 5'-TGAATTCGGCAAAATTGGCGGAACC-3') to anneal to the up- and downstream sequences, respectively, of the katG gene. Both primers introduced EcoRI sites that flanked katG to facilitate subsequent cloning. Total DNA from R. etli CE3 was used as a PCR template, resulting in the amplification of a 2346 bp fragment. The PCR product was digested with EcoRI and ligated into the promoterless vector pTR101, to create pTCV5K. Plasmid pTCV5K was introduced by triparental mating into VEM1673 to create strain VEM1673-5K (Table 1). Complementing clones were identified using the bubble assay, in which H2O2 was poured onto one replica set of colonies, to detect catalase production mediated through breakdown of H2O2 to H2O and O2.

Analysis of transposon insertion.
This was performed using Inverse PCR methodology, as described previously (Beard et al., 1997), with the following modifications. Genomic DNA from strain VEM1673 was digested with XhoI overnight at 37 °C, then the enzyme was inactivated by incubation at 65 °C for 15 min. The ligation reaction was prepared in a total volume of 0·03 ml and incubated at 12 °C for 48 h. The ligated DNA was used directly as a template for PCR amplification using the oligonucleotide primers XhoI-3 (5'-CCGCAATCCCAACGTTTCTGCCGAGGCG-3') and XhoI-5 (5'-CGCCTTCTTGCATGGCTTAACTACCCTCTG-3'). Amplification was carried out in a thermocycler using the following program: one cycle of 5 min at 95 °C, followed by 30 cycles of 1 min at 94 °C, 1 min at 60 °C and 5 min at 72 °C, with a final cycle of 10 min at 72 °C. The PCR products were purified from a low-melting-point-agarose gel and used directly as a template for automated sequencing.

Catalase assay.
Catalase activity was observed via degradation of H2O2 as determined by a decrease in UV light (240 nm) absorbance over time, as reported previously (Herouart et al., 1996). Measurements of absorbance were taken at 20, 40, 60 and 80 s after addition of the lysate to the H2O2 buffer, and specific activity was expressed as nmol min-1 (mg protein)-1. Cell lysate samples were assayed in triplicate.

Detection of catalase activity in gels.
Bacteria were grown for 6 and 24 h on peptone/yeast extract (PY) broth or on minimal medium (MM) with succinate and NH4Cl as carbon and nitrogen source, respectively. Cell-free supernatants were prepared as described previously (Herouart et al., 1996), and 100 µg of total protein were electrophoresed on 7·5 % (w/v) native polyacrylamide gels. Catalase and peroxidase activities were visualized using the activity staining procedure described by Gregory & Fridovich (1974).

H2O2 sensitivity assays
Liquid culture challenge.
To determine the survival rates of R. etli in the presence of H2O2, overnight cultures (14–16 h) were diluted in MM to a final cell density (OD540) of 0·2 and tested as described by Xu & Pan (2000). After treatment, samples were diluted into PY broth containing 1 mg bovine catalase ml-1, and different dilutions were plated onto PY medium in duplicate. Colony-forming units (c.f.u.) were determined after 3 days incubation at 30 °C. Survival rates were expressed as the percentage of c.f.u. recovered from H2O2-containing media compared with untreated control samples.

Halo assay.
Bacteria were grown overnight (14–16 h) at 30 °C in PY broth and tested as described by Xu & Pan (2000). Each experiment was done in triplicate and repeated at least three times.

Growth rates.
R. etli CE3 and mutant strain VEM1673 were grown in PY or MM broth as described previously (Encarnación et al., 1995). Aliquots of the cultures were removed at 6, 12, 24, 48 and 96 h post-inoculation, and growth was monitored spectrophotometrically at OD540, and by determining the protein concentration by the method of Lowry et al. (1951). Viable counts were determined at each time point by plating aliquots of the cultures onto PY medium.

Filter blot hybridization and plasmid profiles.
Genomic DNA was isolated, digested with EcoRI, electrophoresed in 1 % (w/v) agarose gels, blotted onto nitrocellulose membranes and hybridized under stringent conditions as described by Flores et al. (1987). Plasmid patterns were visualized by the Eckhardt technique (Eckhardt, 1978), blotted onto nylon membranes and hybridized similarly (Flores et al., 1987). Plasmid pAGS3, containing an internal fragment of the R. etli katG gene, was purified by the alkaline lysis method (Sambrook et al., 1989) and labelled with [{alpha}-32P]dCTP using the random primer labelling system (Amersham Pharmacia Biotech). The 5' region of the oxyR gene was isolated by melting the DNA band from an ethidium-bromide-stained, low-melting-point-agarose gel, and labelled as described above.

Two-dimensional (2D)-PAGE.
Freshly harvested bean nodules (1·5 g fresh weight) were crushed in a mortar at 4 °C in 3 ml of homogenization medium containing 50 mM potassium phosphate buffer (pH 7·4) and insoluble polyvinylpyrrolidone. The homogenate was filtered through a 200 µm nylon mesh. A first centrifugation (1500 g, 5 min, 4 °C) eliminated cell debris and polyvinylpyrrolidone residues. Bacteroids were pelleted during a second centrifugation (8000 g, 8 min, 4 °C). The pellet was washed twice in 50 mM phosphate buffer (pH 7·4) containing 2 mM magnesium sulfate and 0·3 M sucrose and then resuspended in 1 ml of extraction buffer containing 50 mM phosphate buffer (pH 7) and 1 mM EDTA. Methods used for sample preparation, 2D-PAGE and N-terminal sequencing have been described previously (Encarnación et al., 2002). The gel image was analysed using a Pdi image analysis system and PDQUEST software (Pdi, Huntington Station, NY, USA).

Nodulation and nitrogen fixation assays.
The symbiotic phenotypes of the R. etli strains in combination with Phaseolus vulgaris L. cv. Negro Jamapa were analysed as described previously (Cevallos et al., 1996). Nodule occupancy was determined by plating nodule homogenates onto PY medium with or without kanamycin to differentiate the strains. The values obtained were analysed for significance with the Student's t-test. These experiments were performed six times.


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Analysis of R. etli, S. meliloti and Rhizobium tropici extracts in catalase activity gels
To investigate the number of catalase activities in representative strains of the Rhizobiaceae family, cell extracts of R. etli CE3, S. meliloti 1021, R. tropici subgroup A strain CFN 299 and R. tropici subgroup B strain CIAT 899 grown on PY medium were electrophoresed through a native polyacrylamide gel and stained for catalase activity (data not shown) as described in Methods. The S. meliloti and R. tropici subgroup A and B extracts contained two catalase activities, while extracts from R. etli contained only one catalase activity. In S. meliloti, it is known that these two catalase activities are the products of separate genes (Herouart et al., 1996; Sigaud et al., 1999). Therefore, a more detailed analysis was performed in R. etli CE3, to find out whether only one catalase was expressed in different conditions. The results showed that only one catalase was present in the following conditions tested: during growth on PY medium (Fig. 1) or on MM throughout the exponential, early- and late-stationary growth phases, and also after induction with H2O2 (data not shown). These studies indicated that there was an important difference between R. etli and the other organisms studied so far (including some from the Rhizobiaceae) showing that while R. etli seems to have only one catalase activity, S. meliloti (Herouart et al., 1996), R. leguminosarum bv. phaseoli (Crockford et al., 1995), R. tropici, A. tumefaciens (Xu & Pan, 2000), E. coli (Brunder et al., 1996), Pseudomonas syringae (Klotz & Hutcheson, 1992) and Bacillus subtilis (Loewen & Switala, 1987) are known to have more than one.



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Fig. 1. Detection of catalase and peroxidase activities in native gels of wild-type R. etli CE3 and the katG mutant (VEM1673) after 10 h growth on PY medium. Catalase (lanes 1 and 2) and peroxidase (lanes 3 and 4) containing extracts of wild-type strain CE3 (lanes 1 and 3) and mutant strain VEM1673 (lanes 2 and 4). Catalase activity appeared as an achromatic band against a dark background. All samples contained 0·1 mg protein.

 
Generation and characterization of an isogenic katG mutant strain
In R. etli CE3, screening of approximately 7000 transconjugants resulted in the identification of one catalase mutant, which was designated VEM1673. The inability of strain VEM1673 to produce a functional catalase was immediately obvious, since the colonies did not produce gas (O2) upon the addition of H2O2. This result was further confirmed using the spectrophotometric analysis described in Methods. Lysates prepared from cells of wild-type (KatG+) strain CE3 grown on MM had a catalase activity of 18·7 nmol min-1 (mg protein)-1, while extracts prepared from VEM1673 contained no detectable catalase activity. R. etli CE3 and strain VEM1673 grown on PY (rich medium) for 10 h were analysed for catalase and peroxidase activities in native polyacrylamide gels (Gregory & Fridovich, 1974). These enzyme assays revealed that R. etli CE3 produced a bifunctional catalase-peroxidase which was absent in the mutant strain (Fig. 1).

In order to map the insertion site of the Tn5 transposon, the nucleotide sequences flanking the 3' and 5' ends of this element were determined. The entire sequences (105 and 103 bp for 3' and 5' sequences, respectively) were identical to segments of the R. etli katG gene (GenBank accession no. AF486647). The Tn5 insertion in strain VEM1673 was found to be localized following nucleotide 2118 of a 2181 bp katG ORF. These data confirmed the insertion of Tn5 in the coding region of the catalase-peroxidase gene (Fig. 2). The resulting katG mutant showed an increased sensitivity to H2O2 and a loss of peroxidase and catalase activities (Figs 1 and 4b).



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Fig. 2. Genetic map orienting katG relative to oxyR in R. etli CE3. The locations and relative orientations of the ORFs are shown above the map. The position of the Tn5 transposon insertion is indicated on the map by the ‘lollipop’ symbol. The vertical lines indicate the restriction sites in the fragment. B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; P, PstI; S, SalI; X, XhoI.

 


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Fig. 4. Effect of a katG mutation on total catalase activity during growth on MM (circles) and PY medium (squares) of the R. etli wild-type strain CE3 (closed symbols) and the katG mutant strain VEM1673 (open symbols). Samples were taken from cultures at the indicated times. Bacterial growth (expressed in terms of protein content) was monitored (a), and total catalase activity was measured (b). Values represent the means of at least three experiments; error bars show standard deviations.

 
Sequence analysis of the R. etli katG and oxyR genes
To isolate recombinant plasmids that complemented the catalase activity in VEM1673 (katG mutant), cosmids from the R. etli gene library were conjugated into VEM1673. Transconjugants were tested for catalase activity as described in Methods. Six catalase-positive clones were identified. Cosmids isolated from these clones shared a 4·2 kb band, which was subsequently subcloned and sequenced. Nucleotide sequence analysis of the insert in pRCV59, which restored catalase activity to the catalase mutant VEM1673 (VEM1673-59), revealed at least two potential ORFs. One ORF encodes a gene product of 727 aa with a calculated molecular mass of 79 967 Da and a theoretical pI value of 6·45. The translation start codon is preceded by a potential ribosome-binding site sequence (GAAGGAG) located six nucleotides upstream of the coding region, and also by putative -10 and -35 promoter sequences of TAAAAT and TTATCG, respectively. Comparison of known OxyR binding sites with the sequence in the R. etli katG promoter region showed that this region also contains a four-part sequence motif characteristic of OxyR binding sites reported by Kim & Mayfield (2000) (data not shown).

The deduced amino acid sequence of this ORF had high identity with bifunctional catalase-peroxidases (KatG). The multiple amino acid sequence alignment using the BLAST algorithm (Altschul et al., 1997) revealed a strong relationship between the R. etli KatG protein and the group of catalase-peroxidases produced by many prokaryotes. These included two members of the Rhizobiaceae; a katG-like gene occurs in the Mesorhizobium loti chromosome and in S. meliloti plasmid A (psymA) whose product has an identity to the KatG proteins of 75 and 65 %, respectively, suggesting that katG is conserved in this group of bacteria. In contrast, the R. etli KatG protein does not exhibit significant amino acid identity (<10 %) with monofunctional Rhizobiaceae catalases.

Upstream of the catalase promoter we found another ORF transcribed divergently from katG and putatively encoding the regulatory protein OxyR (Fig. 2). The deduced amino acid sequence of this ORF had 39 and 36 % identity with the OxyR proteins of Brucella abortus and E. coli, respectively. The R. etli genes are oriented such that the promoters of the catalase and oxyR genes most likely overlap. The predicted start codons are separated by 146 nt, and potential promoter sequences for both genes are present. The ORF predicts a 305 aa polypeptide with a molecular mass of 33 934 Da and a pI value of 11·36.

Prediction of the secondary structures of the R. etli OxyR protein and homologous sequences in S. meliloti and M. loti at the BMERC server (http://bmerc-www.bu.edu) showed the presence of potential helix–turn–helix DNA-binding motifs in the N-terminal domain of these OxyR proteins (data not shown). OxyR is activated through oxidation by H2O2 and then induces the transcription of genes necessary for the bacterial defence against oxidative stress (Zheng et al., 1998). The activation of OxyR by H2O2 occurs by the formation of an intramolecular disulfide bond between Cys-199 and Cys-208, most likely via the oxidation of Cys-199 to a sulfenic acid intermediate (Zheng et al., 1998). Sequence alignment of OxyR-like proteins (data not shown) revealed that the R. etli OxyR sequence contains the conserved cysteine residues corresponding to positions 199 and 208 of the E. coli OxyR sequence.

The oxyR gene family is extensive among prokaryotes (Bauer et al., 1999; Brunder et al., 1996; Geissdorfer et al., 1999; Mongkolsuk et al., 1998; Sherman et al., 1995; Zheng et al., 1998), where it plays an important role in adaptation to H2O2. Its potential regulatory capacity was shown in E. coli, where a computational approach identified OxyR binding sites and suggested the possibility that OxyR binds to more than 100 sites in the genome (Zheng et al., 2001). The presence of a four-part sequence motif in the katG promoter region of R. etli, characteristic of OxyR binding sites (Kim & Mayfield, 2000), and preliminary results obtained by this group, suggested OxyR regulation over the katG gene.

Studies in progress will define the role of OxyR in free-living R. etli and in the plant–R. etli symbiotic system.

Cloning and complementation analysis of the catalase gene from R. etli
To demonstrate clearly that katG, and not oxyR, was able to complement strain VEM1673, a plasmid harbouring only the katG gene expressed from its own promoter was transformed into VEM1673. Plasmid pTCV5K, which contained a functional R. etli catalase-peroxidase (katG) structural gene and its own promoter region, was constructed (see Methods). The plasmid did indeed complement VEM1673 (VEM 1673-5K) to a bubble-positive phenotype (which was tested in native gels), confirming that it encoded a catalase structural gene from R. etli (data not shown).

Catalase encoded by katG is essential for R. etli H2O2 resistance in vitro
We determined the in vitro H2O2 sensitivity of the wild-type strain (CE3) and the katG mutant strain (VEM1673). Survival curves were determined for both strains exposed to H2O2 in PY broth, as described in Methods. As shown in Fig. 3, 0·50 mM H2O2 was sufficient to kill 100 % of cells from mutant strain VEM1673, whereas 2·0 mM H2O2 was required to kill 100 % of the population of wild-type cells. The LD50 of strain VEM1673 was 0·15 mM H2O2, while that of the wild-type strain was 0·8 mM H2O2. These results were consistent with inhibition zone tests on plates (data not shown).



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Fig. 3. Sensitivity of R. etli CE3 ({blacksquare}), katG mutant strain VEM1673 ({bullet}), complemented strain VEM1673-5K ({blacktriangleup}) and R. etli CE3 after 30 min exposure to 200 µM H2O2 previous to challenge ({blacktriangledown}) with H2O2. Cells were challenged with various concentrations of H2O2 as described in Methods. The cultures were then diluted and plated onto PY medium to determine cell viability. Data points are the means of duplicates from a representative experiment.

 
To verify that the effects associated with the katG insertion were due specifically to changes in katG, strain VEM1673-5K was exposed to H2O2, like the other strains. This strain was more resistant to H2O2 than wild-type strain CE3 (Fig. 3). This may be the result of high KatG levels as a consequence of the high copy number of the plasmid harbouring the katG gene. The present studies with the R. etli katG mutant are consistent with findings in other organisms, in that the disruption of catalase production generates bacteria that are hypersensitive to H2O2.

Importance of katG in stationary-phase survival
Decreases in stationary-phase plating efficiency of several orders of magnitude have been reported for catalase-peroxidase null mutants of Rhodobacter capsulatus (Hochman et al., 1992) and Caulobacter crescentus (Steinman et al., 1997). A similar decrease in stationary-phase survival was found in the Rhizobium etli katG mutant, where 4 days incubation on PY decreased survival 100-fold; after 5 days incubation, no c.f.u. were detected, compared to a titre of >105 cells ml-1 found in the wild-type strain under the same conditions. The survival deficit was completely reversed in strain VEM1673-5K, indicating that this phenotype was attributable to katG (data not shown).

Regulation of catalase-peroxidase
Expression of katG [specific activity, nmol min-1 (mg of protein)-1] in the wild-type strain (CE3) increased from exponential to early- and late-stationary phase in MM cultures (Fig. 4a, b). The zymograms were consistent with the activity determinations in demonstrating an increase in KatG activity during early- and late-stationary phase on MM (data not shown). During growth on PY medium, catalase-specific activity was approximately the same at all time points tested, even in the exponential phase (Fig. 4a, b). The induction on MM suggested positive regulation in stationary phase. We observed that when R. etli CE3 was cultured on MM, an overexpression of superoxide dismutase (SOD) proteins was apparent. This metabolic modification was not observed when R. etli was cultured on PY medium, a condition correlated with greater sensitivity to oxidative stress (S. Encarnación & J. Mora, unpublished data). In this context, it is significant that during stationary phase on MM, R. etli is responding to oxidative stress by induction of the katG gene. In addition, catalase enzyme activity increased approximately fourfold [6 to 25·2 nmol min-1 (mg of protein)-1] after 30 min exposure to 200 µM H2O2 in PY broth. This resulted in a greater resistance to H2O2, allowing the bacteria to withstand normally lethal levels of oxidative stress (Fig. 3).

As katG was required by R. etli to detoxify H2O2, we tested whether the katG mutation affected bacterial viability. Therefore, growth of the wild-type (CE3) and katG mutant strains was monitored in PY and MM broth, where decreases in growth rate (Fig. 4a) and viable cell counts (data not shown) were found in the katG mutant compared with the wild-type strain. This suggested that the mutation in katG affected the ability of the bacteria to grow even in the absence of exogenous H2O2.

In some cases, catalase genes exhibit sequence homology but they are regulated differently (Schellhorn & Hassan, 1988). Low sequence similarity was observed between the R. etli KatG and E. coli HPII proteins, yet both catalases are upregulated on MM during stationary phase. However, only the R. etli KatG protein is induced by exogenous H2O2 (Loewen et al., 1985). In S. meliloti, KatA is induced by H2O2 but is not upregulated in stationary phase, in contrast to KatC catalase, which is upregulated in stationary phase but not induced by H2O2. Alternatively, S. meliloti KatB, which has high homology to R. etli KatG, considerably reduces the impact of KatC or KatA induction on total catalase activity during early and stationary phase (Sigaud et al., 1999). The R. etli KatG protein was induced by H2O2, similar to S. meliloti KatA and E. coli HPI, and was upregulated during stationary phase, like S. meliloti KatC.

In E. coli, HPI activity increased during exponential growth, correlating with an enhancement in H2O2 production (González-Flecha & Demple, 1997). In R. etli, the increase in KatG activity during stationary phase on MM could be a consequence of the high concentration of reductive power observed under these growth conditions (Encarnación et al., 1995). This may provoke the formation of a highly reactive hydroxyl radical, which arises from H2O2 degradation via the Fenton reaction (Halliwell & Gutteridge, 1986). For this reason, an increase in catalase activity would be important in promoting a higher increase in resistance to exogenous H2O2 under these growth conditions.

The difference in catalase regulation between the R. etli KatG protein and the regulation of catalases of other bacteria could be explained by the existence of only one catalase in R. etli, in contrast to the multiple catalase activities present in other bacteria. This unique catalase must maintain the H2O2 concentration at low levels throughout the growth phase.

The katG and oxyR genes are located on R. etli plasmid pCFN42f
In addition to the chromosome, R. etli CE3 contains six plasmids (pCFN42a to f) ranging in size from 150 to 630 kb. An 840 bp PstI–BamHI internal fragment of the katG gene and a 333 bp HindIII–EcoRI fragment from the 5' region of the oxyR gene were used as radiolabelled probes in hybridizations against plasmid blots from R. etli CE3. A strong hybridization signal was observed on the largest plasmid, pCFN42f (Fig. 5). To further support these results, similar hybridization experiments were carried out using strain CFNX186, which is cured of plasmid pCFN42f (García-de los Santos et al., 1996) and lacks catalase activity. Fig. 5 shows that no hybridizing bands were observed in either the plasmid or the genomic DNA blots from strain CE3 cured of plasmid pCFN42f. These data clearly demonstrate that katG and oxyR are located on pCFN42f. Furthermore, Southern analysis also showed that there were no additional copies of these genes in other replicons.



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Fig. 5. Plasmid location of the katG and oxyR genes as determined by hybridization analysis. (a, b) Plasmid profiles of R. etli CE3 (lane 1 in a and b) and its derivative CFNX186 cured of plasmid pCFN42f (lane 3 in a and b) stained with ethidium bromide. Southern blots of each plasmid profile probed with an 840 bp PstI–BamHI internal fragment from the katG gene (lanes 2 and 4 in a) or with a 333 bp HindIII–EcoRI fragment from the 5' region of the oxyR gene (lanes 2 and 4 in b). The letters to the left of the plasmid profiles in (a) indicate the positions of the plasmids pCFN42a (150 kb), pCFN42b (170 kb), pCFN42c (270 kb), pCFN42d (390 kb), pCFN42e (510 kb) and pCFN42f (630 kb). (c) Southern blots of EcoRI-digested genomic DNA from R. etli CE3 (lanes 1 and 3) and its derivative CFNX186 cured of plasmid pCFN42f (lanes 2 and 4), hybridized with the katG probe (lanes 1 and 2) or with the oxyR probe (lanes 3 and 4). The size of the hybridizing band was estimated using {lambda}DNA digested with EcoRI and HindIII.

 
In spite of the fact that katG and oxyR may be an essential part of the oxidative stress response, they are plasmid-encoded, similar to E. coli O157 : H7 (Brunder et al., 1996), Pseudomonas fluorescens (Peters et al., 2001), M. loti (Kaneko et al., 2000) and S. meliloti (Sigaud et al., 1999). Evidence for another antioxidant activity (SOD) located on a plasmid (pRtrW8-7e) has been described in R. leguminosarum bv. trifolii W8-7 (Baldani et al., 1992).

In addition, the oxyR gene has always been reported to be present on the chromosome; analysis of the M. loti database (http://www.kazusa.or.jp/rhizobase) and the S. meliloti database (http://sequence.toulouse.inra.fr/meliloti.html) showed that oxyR homologues are present on the chromosome of these two species. Therefore, to the best of our knowledge, the R. etli oxyR gene is the first plasmid-encoded oxyR to be described.

Symbiotic phenotype of the katG mutant in bean plants, and proteome analysis of the katG mutant in symbiosis
To investigate the role of catalase-peroxidase in the development of functional nodules and nitrogen fixation, Phaseolus vulgaris L. bv. Negro Jamapa host plants were inoculated with strain VEM1673 (KatG mutant) or wild-type strains. Nitrogen fixation activity was determined at different times by C2H2 reduction, as described previously (Cevallos et al., 1996).

We observed that there were no significant differences between the strains in relation to nodulation efficiency and nitrogen fixation, apparently indicating that KatG has a minor protective role in the nitrogen fixation process. These experiments suggested that in R. etli CE3, KatG has a central role in protecting the bacterium from endogenous H2O2 generated during aerobic respiration in the free-living state, as well as in protecting it against exogenous H2O2. It is possible that in symbiosis other defences exist which are induced in the katG null mutant.

With the aim of finding alternative mechanisms for the oxidative protection of R. etli in symbiosis, a proteome analysis from strains CE3 and VEM1673 was developed. Extracts of proteins were prepared from bacteroids obtained from nodules at the point of maximum nitrogen fixation (18 days after inoculation). Up to 600 proteins were resolved from the wild-type strain, and up to 651 were resolved from the katG mutant strain, by 2D-PAGE. Of these 651 proteins, 594 were also present in strain CE3. One of the most striking differences revealed in the protein patterns of the two strains was that 57 proteins present in VEM1673 were absent (Fig. 6), or overexpressed, with respect to the wild-type strain, CE3. During the routine N-terminal sequencing of the R. etli CE3 proteins isolated from the 2D-PAGE gels, we identified four proteins with high identity to SOD (spots 6 and 7, Fig. 6), DnaK (spot 28, Fig. 6) and a putative member of the peroxiredoxin 2 family (spot 30). These proteins were present in the proteomes of the wild-type and mutant strains. The molecular masses of the proteins determined from the gels were similar to the molecular masses deduced from their nucleotide sequences (Table 2). These proteins have been described as belonging to the oxidative stress response regulon in micro-organisms (Barr & Gedamu, 2001; Echave et al., 2002; Storz & Imlay, 1999).



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Fig. 6. Proteome map of bacteroids (18 days) from R. etli CE3 (wild-type) and strain VEM1673 (mutant) displayed by using PDQUEST software. The colour coding of the protein spots is as follows. Blue, R. etli CE3 proteins; red, VEM1673 proteins; green, proteins present in both of the strains being compared.

 

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Table 2. N-terminal sequences of differentially displayed katG mutant bacteroid proteins and FASTA matches to a non-redundant database

 
Oxygen protection might be essential for efficient infection if rhizobia, like many pathogens, induce an oxidative burst on plant-cell infection, but little is known about the response of symbiotic soil bacteria to environmental stresses. The results of the R. etli bacteroid proteome analysis suggest that in the katG mutant the presence of SODs, DnaK and peroxiredoxin, in addition to new and overexpressed proteins, may be directly involved in protection from oxidative stress in symbiosis. Most bacteria contain SODs (EC 1.15.1.1) which detoxify radicals and are normally produced in biological systems. Recent results suggest that, in addition to the known role of protecting cells against heat stress, DnaK also protects numerous kinds of proteins from oxidative damage (Echave et al., 2002). Peroxiredoxins, also known as thiol-specific antioxidants, compose a family of antioxidants that have recently been discovered in numerous prokaryotes and eukaryotes. The substrates of peroxiredoxins include H2O2, alkyl hydroperoxides (e.g. cumene and t-butyl hydroperoxides) and peroxynitrite (ONOO-) (Barr & Gedamu, 2001). Furthermore, the results of the bacteroid proteome analysis suggest that the peroxiredoxin protein which is encoded by a gene belonging to a symbiotic plasmid in R. etli is expressed only in symbiotic conditions (S. Encarnación & J. Mora, unpublished data). The newly identified overexpressed proteins may also be directly engaged in the compensation mechanism of protection from oxidative stress in symbiosis. As published previously, a number of ORFs in the S. meliloti genome (Galibert et al., 2001) may be involved in oxidative stress responses, and these include one hydroperoxidase, two haloperoxidases, two SODs, 17 glutathione S-transferases (GSTs) (the same number have been described in the sequence of M. loti) (Kaneko et al., 2000) and eight rpoE, all of which might be included in this new or overexpressed pattern of proteins identified on the proteome which contribute to protection against oxygen or other reactive molecular species in symbiosis.

However, many experiments suggest that exopolysaccharides could act as suppressors of host-plant defence responses. This may explain the normal nodulation and nitrogen fixation in the VEM1673 mutant strain (Baron & Zambryski, 1995). A detailed proteome analysis of this mutant will be needed to identify the components of this compensatory or alternative mechanism required for protection during symbiosis.


   ACKNOWLEDGEMENTS
 
This work was supported by DGAPA-UNAM grants IN227598 and IN206800, and CONACYT grant 34903-N. We thank Magdalena Hernández and Aurora Arroyo Mora for technical assistance, Antonia Jaimes and Andrés Barrera for plant experiments, José Luis Zitlalpopoca for greenhouse support and Michael Dunn for helpful discussions and for critically reviewing the manuscript.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 26 July 2002; revised 30 January 2003; accepted 5 February 2003.



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