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Cellular metabolism of molecular oxygen produces reactive and potentially toxic oxygen species such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals (16). For defense against these reactive oxygen species, organisms contain antioxidants and enzymes that repair oxidative damage. Catalases (H₂O₂:H₂O₂ oxidoreductase; EC 1.11.1.6) are heme-containing enzymes involved in the dismutation of H₂O₂ in O₂ and H₂O. These enzymes play an important role in reducing the formation of the highly reactive hydroxyl radical which arises from H₂O₂ degradation via the Fenton reaction (16). The response of bacteria to oxidative stress has been most extensively studied in the enteric bacterium Escherichia coli (reviewed in reference 7) which synthesizes two types of catalase enzyme, a bifunctional catalase/peroxidase (HPI) encoded by katG (32) and a monofunctional catalase (HPII) encoded by katE (34). These two kat genes are regulated differently in terms of growth phase and response to oxidative stress (reviewed in reference 24).

Sinorhizobium meliloti is a soil bacterium able to establish a symbiosis with alfalfa (Medicago sativa). This symbiosis leads to the formation of nodules on the alfalfa roots following a flow of signals transmitted between plant and bacteria (13). In several aspects, this symbiosis can be considered a controlled incompatible reaction (29). Most infections can be aborted by a hypersensitivity-like response mediated by an H₂O₂ oxidative burst which results in the plant exhibiting control over the number of nodules formed (33). Inside the nodules, the bacteria differentiate into their endosymbiotic, bacteroid forms. The microsymbiont is able to reduce nitrogen to ammonia, which can subsequently be metabolized by the plant. Nitrogenase, the key enzyme in this fixation of atmospheric nitrogen, is quickly and irreversibly inactivated by oxygen, but the energy for N₂ fixation requires a high bacteroid respiration rate. To solve this seeming paradox, a variable diffusion barrier controls the entry of O₂ into the infected region (36), and a large amount of the O₂-carrying protein leghemoglobin facilitates the supply of O₂ to the microsymbiont (1). Additionally, an important antioxidant defense occurs in the peripheral cell layers of legume root nodules (5). Bacteroids produce a high concentration of reactive oxygen species because of the stringent conditions required to reduce N₂, the potential of nitrogenase to directly reduce O₂ (4), and the high rates of bacteroid respiration; it has been suggested that these oxygen-derived species have a role in the inactivation of nitrogenase (28).

To elucidate the role of catalases in the establishment and/or maintenance of Medicago-Sinorhizobium functional nodules, we have previously cloned the S. meliloti katA gene, which encodes the H₂O₂-inducible catalase KatA (18). We showed that free-living S. meliloti bacteria in stationary phase on rich medium produce two catalases, namely a monofunctional catalase (KatA) and a bifunctional catalase/peroxidase (KatB). A katA::Tn5 mutant showed a drastic sensitivity to H₂O₂, and KatA appeared to be the major component of an H₂O₂-adaptative response. Neither nodulating capacity nor nitrogen fixing activity were impaired in the katA mutant, suggesting that KatA is not essential for the nodulation and nitrogen fixation processes.

Cloning and analysis of the katA-homologous gene in S. meliloti. We previously took advantage of the high homology between regions of E. coli HPII and several catalases from various phyla to clone the katA gene of S. meliloti by nested PCR (18). Southern analysis of genomic DNA, digested with different restriction enzymes, showed a pattern of two bands (7.1- and 14-kb ApaI-ApaI fragments), suggesting the presence of a second, katA-homologous, catalase gene. The 1.4-kb ApaI-EcoRI fragment corresponding to the katA coding region was radiolabeled by using the Prime-a-Gene labeling system (Promega, Charbonnières, France) and used as a probe to hybridize with an S. meliloti genomic cosmid library (12) under low stringency (55°C). Restriction analysis of five positive clones carrying a DNA insert of 22 kb indicated that three cosmids represented the same genomic region but were different from katA. Restriction enzyme mapping, Southern blotting, and deletion analysis of one of these three cosmids, pLRK2, were performed to localize the katA-homologous gene. A 3.3-kb EcoRI-PstI katA-hybridizing fragment from pLRK2 was subcloned into a pBluescript vector (pBSKC1) and fully sequenced by the dideoxy chain termination method in accordance with the U.S. Biochemicals protocol for the Sequenase 2.0 enzyme with -³⁵S-dATP (ICN, Orsay, France). Analysis of the sequence revealed one major open reading frame encoding 687 amino acid residues, corresponding to a protein with an M_r of 76,000 and a pI of 6.5. The ATG was preceded by a potential ribosome binding site (AAGGAG) located 7 bp upstream. Inverted repeat sequences are present downstream of the stop codon and might serve as a transcription terminator. Southern analysis of digested genomic DNA with a radiolabeled 2.2-kb EcoRI-ApaI fragment of this katA-homologous gene, kat2, confirmed that this probe hybridized with the 14-kb ApaI-ApaI fragment of the S. meliloti chromosome previously detected (data not shown). As expected, a search of the current nonredundant DNA and protein databases with the BLAST algorithm (Beckman Center for Molecular and Genetic Medicine, Stanford, Calif.) revealed that the deduced amino acid sequence of kat2 had regions of high homology with monofunctional catalases from mammals, plants, and bacteria but not with bifunctional catalases. A multiple alignment of KatA (U59271) and Kat2 amino acid sequences from S. meliloti was performed with Xanthomonas oryzae KatX (X97673), E. coli hyperoxidase II (M55161), and Rhizobium sp. strain SNU003 (U56239), using the Genetics Computer Group programs PILEUP and PRETTY (data not shown). Considering both identical and conservative replacement of amino acids, Kat2 showed a high degree of identity with X. oryzae KatX (identity of 56.9%) and E. coli HPII (identity of 48.9%). Surprisingly, the Kat2 sequence showed very low amino acid identity with the S. meliloti KatA (28.2%) and with the Rhizobium sp. strain SNU003 catalase (28%), since a large divergence in the C-terminal region was observed between Kat2 and these two catalases. However, the amino acid residues thought to be involved in the active-site and the proximal- and distal-heme-site ligands (23) were highly conserved in the five sequences.

Induction of a new catalase during stationary phase in minimum medium. Strains and plasmids used in this study are listed in Table 1. Previously, we detected only KatA and KatB in protein extracts of stationary-phase cells grown at 30°C in rich medium (Luria broth [LB]-MC: yeast extract, 5 g/liter; tryptone, 10 g/liter; NaCl, 10 g/liter; 2.5 mM MgSO₄; and 2.5 mM CaCl₂), (18). Sequence analysis indicates that the kat2 product could not correspond to a bifunctional, HPI-like catalase/peroxidase. To test this, a recombinant Rm5000 strain carrying an interposon () on the kat2 gene (MK5002) was constructed. The 1.5-kb SacI-ApaI subclone (pBSKC2) containing the 5' end of kat2 was cleaved at the neighboring SmaI sites within the coding region, creating a deletion. The kat2 was disrupted by ligation within an -interposon cassette containing a Sp^r/Sm^r element flanked by transcriptional terminators translational stops in all three reading frames (8). The SacI-KpnI fragment containing this null allele was subcloned into pRK415. Recombinant plasmids were transferred to the rifampin-resistant SU47 derivative Rm5000 by triparental mating with E. coli MT616 as a helper in accordance with the protocol previously described (14). The recipient strains containing the recombinant plasmid were selected by growth on medium complemented with rifampin (20 µg/ml), spectinomycin (100 µg/ml), and tetracycline (10 µg/ml). The plasmid incompatibility technique (30) was then used to detect strains in which the insertion had recombined from the plasmid to the S. meliloti genome. Recombinants carrying  on the kat2 gene (MK5002) were selected for the gentamicin-resistant plasmid pGM2, on LB-MC medium containing gentamicin (70 µg/ml), spectinomycin, and rifampin. Southern analysis of the MK5002 genomic DNA confirmed the insertion of the -spectinomycin cassette in the coding region of kat2. Analysis of the catalase pattern of the kat2 mutant revealed no difference between it and the wild-type profile, confirming that this gene does not encode KatB. These results suggested the presence of at least three catalase genes in S. meliloti. Three catalases have been detected in Rhizobium leguminosarum bv. phaseoli free-living bacteria grown in YEM medium (3). Analysis of the transcription of katE in E. coli by using lacZ fusion clearly showed that the katE promoter gave rise to low expression during growth in rich medium but elevated expression during growth in poor medium (27).

View larger version (71K): [in this window] [in a new window]   FIG. 1.   Expression of KatA, KatB, and KatC during growth of free-living S. meliloti on M9 minimal medium and catalase profile in 5-week-old bacteroids. Catalase activity was detected in samples (25 µg of proteins) after electrophoresis on native polyacrylamide gels.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Effect of a katA or katC mutation on total catalase activity during growth on M9 medium. Stationary cultures of RM5000, MK5001, and MK5002 were inoculated into fresh M9 medium to an initial optical density at 600 nm (OD600) of 0.05. Samples were taken from cultures at the indicated times. Bacterial growth was monitored as OD600 (A), and total catalase activity was measured (B). Values represent averages of at least two experiments; error bars show standard deviations.

Regulation of the katC expression. To determine if KatC induction during stationary phase is controlled at the transcriptional level, a katC::lacZ transcriptional fusion was constructed. Plasmid pBSKC3 carries a 2,190-bp EcoRI-ApaI fragment with a segment of katC purified from pBSKC1 and subcloned into pBluescript KS(+). pSupKC carries a 2.2-kb PstI-PstI fragment purified from pBSKC3 and subcloned at the PstI site of the vector pSUP202. To construct a transcriptional lacZ fusion to the katC promoter, a BamHI-BamHI lacZ-Km^r cartridge purified from plasmid pKOK5 was inserted in BglII sites of pSupKC. After transfer by conjugation to S. meliloti Rm5000, a simple recombinant clone, designated RKCZ01, was isolated on LB-MC medium containing rifampin and neomycin (200 µg/ml). Recombination at the correct location was checked by hybridization with the specific DNA fragment from pBSKC1. The bacterial growth in M9 medium and the -galactosidase activity of the katC::lacZ recombinant strain RKCZ01 and wild-type strain RM5000 as a control are shown in Fig. 3. -Galactosidase activity was determined by measuring the hydrolysis of o-nitrophenyl--D-galactoside as described by Miller (26), and protein concentration was determined as described previously (18). The growth curves of the two strains were very similar (Fig. 3A). However, a threefold increase of the -galactosidase activity was observed during the stationary phase for strain RKCZ01 (Fig. 3B), which could be correlated with the induction of KatC (Fig. 1). These data suggest that growth-dependent KatC regulation is mainly or solely transcriptional.

View larger version (15K): [in this window] [in a new window]   FIG. 3.   Growth stage induction of katC::lacZ fusion. Stationary cultures of Rm5000 and RCZ01 (katC::lacZ transcriptional fusion) were inoculated in fresh M9 medium to an initial optical density at 600 nm (OD600) of 0.05. Bacterial growth was monitored as OD600 (A). At the indicated times, samples were taken to assay -galactosidase activity (B). Values represent averages of at least two experiments; error bars show standard deviations.

View this table: [in this window] [in a new window]   TABLE 2.   Effect of different stresses on the regulation of katC expression determined by measuring -galactosidase activity

Catalase expression pattern in bacteroids. To further investigate the role of catalases in the development of functional nodules, M. sativa plants were inoculated with the wild-type Rm5000 strain, and bacteroids were isolated from 5-week-old nodules. Around 1.5 g of freshly harvested nodules was crushed in a mortar at 4°C in 3 ml of homogenization medium containing 50 mM phosphate buffer (pH 7.4) and insoluble polyvinylpyrrolidone. The homogenate was filtered through a 200-µm nylon mesh. A first centrifugation (1,500 × g, 5 min, 4°C) eliminated cell debris and polyvinylpyrrolidone residues. The bacteroids were sedimented during a second centrifugation (8,000 × 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. The pellet was resuspended in 1 ml of extraction buffer containing 50 mM phosphate buffer (pH 7) and 1 mM EDTA. The bacteroids were then sonicated, and membrane debris was eliminated by centrifugation (8,000 × g, 10 min, 4°C). Analysis of bacteroid protein extracts from five independent experiments showed that a higher catalase activity (sevenfold) was detected in bacteroids (214.9 ± 38.6 U/mg of protein) than in free-living bacteria (30.3 ± 5.3 U/mg of protein), suggesting that a large amount of H₂O₂ was directly or indirectly generated in the microsymbiont.

Reduction of nitrogen fixation capacity in a katA katC double mutant. To test the effect of a katA and/or katC mutation on nodulation and nitrogen fixation, a katA katC double mutant (MK5003) was constructed by transduction. The katA::Tn5 mutation from strain MK5001 was transferred to MK5002 by general transduction by using the M12 phage, in accordance with a standard protocol (14). Transductants (MK5003) were selected on LB-MC medium containing kanamycin (50 µg/ml) and spectinomycin (100 µg/ml). M. sativa host plants were inoculated with MK5001, MK5002, and MK5003 mutant strains and with the wild-type Rm5000 strain as a control. Seventy-two plants were grown in sterile tubes (three plantlets per tube) containing 20 ml of a nitrogen-free nutrient medium with 0.8% agarose prepared as a slant. Plants were inoculated with the appropriate S. meliloti strains 1 week after germination. Nitrogen fixation activity was determined by C₂H₂ reduction by using a gas chromatograph (ATI-Unicam, model 610) equipped with a column of Porapak T (80/100 mesh) as described previously (18). Incubations of these nodulated plantlets were made at 25°C, in rubber-cap tubes containing O₂ (20 kPa) and C₂H₂ (10 kPa) in argon. The fresh weight of nodules per tube was measured, and the phenotypes of the bacteria recovered from the nodules were checked on the appropriate media. Plants were visually screened for nodule formation by observing the root system 5 weeks after Sinorhizobium inoculation. A high efficiency of nodulation (87 to 97%) was observed for the plants inoculated with the single mutants MK5001 and MK5002 and with Rm5000. In contrast, only 75% of the plants inoculated with the double-mutant katA katC MK5003 strain showed a nodulating phenotype. Acetylene reduction activity was assayed for M. sativa nodulated with the different strains at 5 and 9 weeks after bacterial infection (Fig. 4). The level of C₂H₂ reduction observed in MK5001-nodulated plants was not significantly different from that of Rm5000-nodulated plants, confirming our previous results (18). There was also no significant reduction of nitrogen fixation in MK5002-nodulated plants compared to Rm5000-nodulated plants, which is consistent with the nondetection of katC in bacteroids. However, a drastic decrease in C₂H₂ reduction, especially in 9-week-old nodules, was observed for MK5003-nodulated plants.

View larger version (39K): [in this window] [in a new window]   FIG. 4.   Effect of catalase mutations on nitrogen fixation. C2H2 reduction activity was measured in a tube containing three plants at 5 weeks and 9 weeks after inoculation with the wild-type Rm5000 strain or the MK5001, MK5002, or MK5003 mutant strain. Values are means ± standard errors (n = 24). Experimental data were assessed for statistical significance by means of Student's t test. FW, fresh weight.

Nucleotide sequence accession number. The 3.3-kb EcoRI-PstI katA-hybridizing fragment from pLRK2 has been assigned GenBank accession no. AF121348.