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Applied and Environmental Microbiology, April 2008, p. 2398-2403, Vol. 74, No. 8
0099-2240/08/$08.00+0     doi:10.1128/AEM.02457-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Requirement of a Plasmid-Encoded Catalase for Survival of Rhizobium etli CFN42 in a Polyphenol-Rich Environment

Alejandro García-de los Santos,^1* Erika López,¹ Ciro A. Cubillas,¹ K. Dale Noel,² Susana Brom,¹ and David Romero¹

Programa de Ingeniería Genómica, Centro de Ciencias Genómicas, UNAM, Cuernavaca, Morelos, Mexico,¹ Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin²

Received 31 October 2007/ Accepted 15 February 2008

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Nitrogen-fixing bacteria collectively called rhizobia are adapted to live in polyphenol-rich environments. The mechanisms that allow these bacteria to overcome toxic concentrations of plant polyphenols have not been clearly elucidated. We used a crude extract of polyphenols released from the seed coat of the black bean to simulate a polyphenol-rich environment and analyze the response of the bean-nodulating strain Rhizobium etli CFN42. Our results showed that the viability of the wild type as well as that of derivative strains cured of plasmids p42a, p42b, p42c, and p42d or lacking 200 kb of plasmid p42e was not affected in this environment. In contrast, survival of the mutant lacking plasmid p42f was severely diminished. Complementation analysis revealed that the katG gene located on this plasmid, encoding the only catalase present in this bacterium, restored full resistance to testa polyphenols. Our results indicate that oxidation of polyphenols due to interaction with bacterial cells results in the production of a high quantity of H₂O₂, whose removal by the katG-encoded catalase plays a key role for cell survival in a polyphenol-rich environment.

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Flavonoids are a widespread group of polyphenolic compounds in the plant kingdom. Their basic chemical structure consists of two benzene (A and B) rings linked through a heterocyclic pyran or pyrone (C) ring. Substitutions in the C ring give rise to anthocyanidins, flavanols, flavonols, flavones, flavanones, chalcones, and isoflavonoids. Many are esterified at hydroxyl groups with different sugars, commonly glucose, galactose, or rhamnose, to produce glycosides. They are distributed throughout the plant, including in the fruit and seeds.

Several groups of bacteria are adapted to live in environments rich in polyphenols, such as the gastrointestinal tracts of ruminants or the rhizosphere, the soil region surrounding the plant root (6, 45). Nitrogen-fixing bacteria belonging to the genera Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, and Azorhizobium (collectively called rhizobia) live surrounded by a wide variety of organic substances released by germinating seeds and plant roots, including polyphenolic compounds. Some of the flavonoids exuded by legume seeds and roots induce transcription of rhizobial nodulation (nod, noe, and nol) genes, which allow these bacteria to establish a symbiotic association with their host plant (9, 27). In addition, flavonoids enhance the growth rates of bacterial cells and promote bacterial movement toward the plant (12, 22, 42). Since root exudation is a highly dynamic process influenced by multiple biotic and abiotic factors, it is very likely that under field growth conditions, rhizobia are constantly exposed to large amounts and wide varieties of polyphenols in addition to the specific nod gene-inducing flavonoids (5). For instance, the presence of multiple microorganisms, including plant pathogens, may influence the quality and quantity of flavonoids produced by the roots (43, 47). It has also been shown that diverse environmental stress factors increase the synthesis of flavonoids (13, 31). The use of mixed plant cultures involving legumes, cereals, vegetables, and tuber crops is a common practice in agricultural systems, probably resulting in a large diversity of polyphenolic compounds in the rhizosphere ecosystem (11). Moreover, flavonoids leaching from decomposing plant litter may also increase the diversity and concentration of these molecules in the rhizosphere (11, 23).

There are few data regarding the mechanisms that allow rhizobia to live in a flavonoid-rich environment. Studies with Rhizobium etli strain CFN42, a symbiont of Phaseolus vulgaris, revealed that mutants defective in both exopolysaccharides and lipopolysaccharide (LPS) were more sensitive to coumestrol, genistein, and daidzein than mutants deficient only in one type of polysaccharide (17). In this same strain, genes encoding a putative multidrug efflux pump were shown to be upregulated by root exudates of P. vulgaris and flavonoids. Mutants of these genes showed increased sensitivity to phytoalexins, flavonoids, and salicylic acid in comparison to the wild-type strain (21). Isoflavonoid-inducible resistance to the soybean phytoalexin glyceollin was also reported to occur in Bradyrhizobium japonicum and Sinorhizobium fredii, but the molecular mechanism involved remains unknown (32, 39). On the other hand, it is known that rhizobia catabolize a wide range of flavonoids (34). This metabolic capacity might also be implicated in the adaptation of rhizobia to survive under toxic levels of flavonoids.

We have focused on the role of plasmids in the adaptation of R. etli CFN42 to rhizosphere and nodule environments. The genome of CFN42 consists of one circular chromosome and six plasmids, designated p42a to p42f, whose sizes range from 184 to 642 kb (20). The isolation and characterization of plasmid-cured strains have allowed the identification of plasmid-associated traits for nodulation competitivity and cellular growth as well as genes required for LPS synthesis and response to oxidative stress (7, 8, 19, 48).

In this paper, we report that R. etli CFN42 is highly resistant to a crude extract of polyphenols (CEP) released from the testae of black bean seeds and that this phenotype depends on the single catalase enzyme encoded by the 642-kb plasmid p42f, which is present in the genome of this bacterium.

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Bacterial strains, plasmids, and growth conditions.
The strains used in this work are listed in Table 1. Rhizobium strains were grown at 30°C in PY medium (38) or TY medium (0.5% tryptone, 0.3% yeast extract, 10 mM CaCl₂). Escherichia coli strains were grown in Luria-Bertani medium. When required, antibiotics were added at the following concentrations: nalidixic acid, 20 µg ml^–1; neomycin, 60 µg ml^–1; and tetracycline, 3 µg ml^–1 for Rhizobium and 10 µg ml^–1 for E. coli.

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TABLE 1. Bacterial strains and plasmids

Genetic manipulations.
Cosmid clones carrying DNA fragments belonging to plasmid p42f were used to complement the p42f-cured derivative CFNX186. Conjugative transfer of cosmids from E. coli to R. etli was done in triparental matings, using pRK2013 as a helper plasmid. Transconjugants were first selected for tetracycline resistance on PY plates and then tested for survival in the presence of CEP in a liquid assay. EcoRI fragments of cosmid pCos24 were first cloned into the pBluescript II SK(+) phagemid vector (Stratagene, La Jolla, CA) and then subcloned in the broad-host-range conjugative plasmid pRK7813. The resulting plasmids were introduced by conjugation into CFNX186 and transconjugants selected as mentioned above. The complementing 2.4-kb HindIII fragment was sequenced on both strands by using the pBluescript primers T7 and T3.

Growth inhibition by testa exudate spots on PY agar plates.
Surface-sterilized bean seeds of Phaseolus vulgaris cv. Black Jamapa were imbibed on PY soft agar plates for 16 h at 30°C. At this time, the polyphenols released from the seed coat produced a black spot under the imbibed seeds. The seeds were removed, and bacterial cultures (15 µl of a 10^–5-diluted culture grown overnight in liquid PY) were inoculated on the black spot of polyphenols left by the seed coat. As controls, the same volume of diluted culture was inoculated on the plate outside the polyphenol spot. Bacterial growth was monitored after 48 h of incubation at 30°C.

CEP from seed coats.
The CEP used in survival assays in the present study was extracted from lyophilized seed coats of P. vulgaris cv. Black Jamapa, and the sample of lyophilized seed coats was kindly provided by G. Loarca-Piña from Research and Graduate Studies in Food Science, School of Chemistry, Autonomous University of Queretaro, Mexico. Each gram of seed coat contains 10.5 mM of proanthocyanidins [expressed in mmol liter^–1 of (+)-catechin equivalents] and 2.4 mM of anthocyanins (expressed in mmol liter^–1 of cyanidin 3-glucoside equivalents) (3). Aparicio-Fernandez et al. (2) reported that the main flavonoids present in this testa sample (detected by high-performance liquid chromatography-mass spectrometry after silica gel fractionation) were proanthocyanidins (monomers to hexamers), anthocyanins (delphinidin, petunidin, and malvidin glycosides), and flavonols (kaempferol, quercetin, and myricetin glycosides). The CEP used in survival assays in the present study was collected from 4 g of lyophilized seed coats soaked in 20 ml of deionized water at 30°C for 16 h without shaking. The testa rinse obtained was sterilized by filtration through a 0.22-µm syringe filter. The total volume was adjusted to 20 ml with sterile deionized water. This sample of CEP contained 41.3 mM of proanthocyanidins [expressed in mmol liter^–1 of (+)-catechin equivalents] and 9.6 mM of anthocyanins (expressed in mmol liter^–1 of cyanidin 3-glucoside equivalents).

Determination of cell survival in the presence of CEP.
Overnight cultures grown in PY were adjusted to an optical density at 600 nm of 0.5. Cells were washed twice with sterile deionized water. The pellet of 1 milliliter of washed cells (approximately 1 x 10⁹ viable cells) was suspended in 1 milliliter of CEP. After incubation at 30°C for 16 h, cell suspensions were centrifuged, washed twice with sterile deionized water, and plated on PY with appropriate antibiotics to determine the number of viable cells. In experiments designed to determine whether metal ions are involved in the bactericidal effect of CEP on katG mutant strains, samples with 1 milliliter of washed cells were suspended in 1 milliliter of CEP containing 10 mM EDTA, incubated at 30°C for 16 h, and used to determine the number of viable cells.

Crude anthocyanidin extract from seeds.
Black P. vulgaris (cv. Midnight Black Turtle Soup) (Idaho Seed Bean, Twin Falls, ID) seeds were extracted under acid conditions (0.1% HCl) as described previously (15, 37). This extract was then boiled in 2 N HCl for 40 min, chilled, and extracted in pentanol to enrich for anthocyanidins (15). The concentration of anthocyanidins was estimated from the A₅₅₅ values of aliquots diluted in 0.01% HCl in methanol (using 30,900 as the molar extinction coefficient).

Agar diffusion assay of sensitivity to anthocyanidins.
The strain to be tested was grown overnight to stationary phase in TY broth. To 3.3 ml of TY in 0.75% agar at 45°C, 0.10 ml of this overnight culture was added, and the agar was mixed rapidly and poured evenly over a TY agar (1.5%) plate. Once the top agar was solidified, a sterile paper disk (6-mm diameter [Becton Dickinson and Co.]) was laid upon the center and 10 µl of 21 mM P. vulgaris anthocyanidins was added. Plates were incubated for 4 days at 30°C and monitored for the presence of halos of inhibition.

Qualitative assay for catalase activity.
The catalase activity was tested by the production of O₂ gas (bubbling) upon addition of 100 µl of 3% hydrogen peroxide to 1 ml of liquid bacterial culture grown overnight.

Quantification of hydrogen peroxide.
The amounts of hydrogen peroxide (H₂O₂) present in CEP samples with or without bacterial cells were determined with a PeroXOquant quantitative peroxide assay kit (Pierce, Rockford, IL), following the manufacturer's instructions.

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Effect of a CEP on the survival of R. etli CFN42 and its plasmid-cured derivatives.
The relative sensitivities of R. etli CFN42 and its plasmid-cured derivatives to testa polyphenols were initially determined with PY soft agar plates. Bacterial cells were drop inoculated over the spot of testa exudates left by bean seeds imbibed on PY soft agar plates (Fig. 1A and B). As controls, the strains were also inoculated on the same plate outside the testa exudates. After 48 h of incubation, full growth inside and outside the spot of testa exudates was observed for the wild type (Fig. 1C). In contrast, strain CFNX186 (cured of plasmid p42f) did not grow on the spot of testa exudates but grew very well outside the spot (Fig. 1D). All the other derivatives cured of plasmids p42a, p42b, p42c, and p42d and a strain carrying a deletion of plasmid p42e grew very well inside and outside the spot of testa exudates, similar to the wild-type strain (data not shown).

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FIG. 1. Effect of the compounds released from P. vulgaris seeds on the growth of wild-type R. etli CFN42 and its p42f-cured derivative CFNX186. (A) Surface-sterilized seeds were imbibed on PY soft agar plates at 30°C in the dark for 16 h. (B) Polyphenol spot appearance after seed removal from the agar plates. (C) Full growth of the CFN42 strain inside and outside the polyphenol spot. (D) Growth inhibition of CFNX186 by the polyphenol spot.

The sensitivity to testa exudates shown by the mutant CFNX186 was quantitatively analyzed through survival assays performed with a CEP obtained from detached seed coats of P. vulgaris. Different dilutions of CEP in water (25%, 50%, and 75%, vol/vol), the undiluted sample (100%), and a control without CEP (0%) were inoculated with 1 x 10⁹ viable cells. The millimolar concentrations of proanthocyanidins [expressed in mmol liter^–1 of (+)-catechin equivalents] and anthocyanins (expressed in mmol liter^–1 of cyanidin 3-glucoside equivalents) in the full-strength extract (undiluted sample) were 41.3 and 9.6, respectively. After 16 h of incubation, the viability of the wild-type strain was not affected by the presence of CEP, while strain CFNX186 showed strong decreases in viability in the full range of concentrations tested (Fig. 2).

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FIG. 2. Effect of the CEP from the P. vulgaris seed coat on the survival of wild-type R. etli CFN42 and its p42f-cured derivative CFNX186. Washed cells (1 x 109 cells/ml) were exposed for 16 h to different concentrations of CEP diluted in water. After this time, viable-cell numbers were determined (means ± standard deviations; n = 3).

The absence of katG is responsible for the CEP sensitivity of CFNX186.
To identify the gene(s) involved in resistance to testa polyphenols, cosmid clones carrying DNA fragments specific for plasmid p42f were introduced by conjugation into the p42f-cured strain CFNX186. One of the cosmids (pCos24), harboring a fragment of approximately 20 kb from plasmid p42f, was chosen for subcloning and further analysis. Four subclones, carrying EcoRI fragments of 4.3 kb, 4.0 kb, 3.1 kb, and 2.8 kb, were constructed and analyzed for complementation of CFNX186. Only the subclone pAGS1 (4.3 kb) was able to complement mutant CFNX186 for CEP resistance (Fig. 3). A HindIII-BamHI fragment of pAGS1 was used to construct pAGS2, and a 2.4-kb HindIII fragment was used to construct pAGS3. Only pAGS3 restored the resistance to CEP (Fig. 3). To identify the genes in this fragment, both ends were sequenced and compared with the complete DNA sequence of plasmid p42f. The comparison revealed that plasmid pAGS3 contains the katG gene (2,187 bp), which encodes the unique catalase enzyme present in this bacterium (GenBank accession number AF486647) (Fig. 3). This fragment also contains 43 bp of the 5' end of an open reading frame encoding the putative transcriptional regulator protein OxyR (Fig. 3). In order to further confirm that the survival of R. etli CFN42 on testa polyphenols depends only on the presence of katG, an isogenic Tn5 insertional mutant defective in catalase activity (VEM1673) was assayed for survival on CEP. This mutant showed the same level of sensitivity to CEP as strain CFNX186 (data not shown). After complementation with plasmid pAGS3, VEM1673 recovered the polyphenol resistance phenotype (Fig. 3). These results clearly indicate that the survival of R. etli on CEP fully depends on the catalase activity encoded by plasmid p42f.

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FIG. 3. Physical and genetic map of the 4,318-bp EcoRI fragment of plasmids pAGS1, pAGS2, and pAGS3 and ability to complement p42f-cured strain CFNX186 and katG mutant VEM1673. The entire katG gene, encoding the catalase enzyme, and 42 bp of the oxyR gene contained in the subclone pAGS3 are denoted by arrow-shaped boxes. Positive complementation (+) indicates that the numbers of CFU of transconjugants harboring plasmids pAGS1 and pAGS3 remained steady after 16 h of treatment with CEP (109 CFU). Negative complementation (–) indicates that the numbers of CFU of transconjugants harboring plasmid pAGS2 diminished 5 orders of magnitude after 16 h of treatment with CEP (109 to 104 CFU). Abbreviations: E, EcoRI; H, HindIII; B, BamHI.

Quantification of hydrogen peroxide in the CEP.
The above data suggest that a bactericidal concentration of H₂O₂ should be present in the CEP or is generated during the treatment of bacterial cells with CEP. It has been demonstrated that the auto-oxidation of polyphenols produces a substantial amount of H₂O₂ (1). This suggests that hydrogen peroxide generated during auto-oxidation of testa polyphenols might be responsible for the bactericidal effect on mutants lacking catalase. However, another possibility might be that endogenous hydrogen peroxide increases during the catabolism of testa polyphenols. In order to determine if the bactericidal effect of testa compounds was due to the production of intra- or extracellular H₂O₂, 300 units of catalase from Aspergillus niger were added to 1 ml of 50% CEP inoculated with strain VEM1673 (10⁹ CFU/ml). After 16 h of incubation, the viable count remained steady. In contrast, the viable count of VEM1673 inoculated on CEP with heat-inactivated catalase showed a 5-log-unit reduction in CFU (10⁹ to 10⁴ CFU/ml) (data not shown). This experiment supports the hypothesis that the extracellular H₂O₂ present in the CEP contributes to the loss of viability of VEM1673.

We determined the amount of H₂O₂ present in a fresh sample of CEP as well as that of the H₂O₂ generated during the incubation of CEP (50%, vol/vol) at 30°C for 16 h in the presence or absence of bacterial cells. The initial concentration of H₂O₂ in a sample of fresh CEP was 19.61 ± 4.1 µM. After 16 h of incubation, the same sample showed an increase of 28.5-fold in its concentration of H₂O₂ (559 ± 46.3 µM). This indicates that auto-oxidation of testa compounds plays an important role in the generation of H₂O₂. In contrast, a sample of fresh CEP inoculated with the wild-type strain (10⁹ cell/ml) did not show a significant increase of H₂O₂ after 16 h of incubation (21.8 ± 5.6 µM), indicating that the catalase activity substantially reduces the accumulation of H₂O₂ produced by auto-oxidation. Accordingly, mutant VEM1673 complemented with plasmid pAGS3 (VEM1673/katG) showed a reduction in H₂O₂ accumulation similar to that for the wild type. A sample of fresh CEP inoculated with the catalase-defective mutant VEM1673 (10⁹ cell/ml) showed a 66-fold increase in the concentration of H₂O₂ after 16 h of incubation. This amount of H₂O₂ (1,300.36 ± 175.8 µM) was 2.3-fold higher than that of the H₂O₂ (559 ± 46.3 µM) produced solely by auto-oxidation in the uninoculated sample of CEP, indicating that the presence of bacterial cells actively increased the generation of H₂O₂ (the unpaired t test revealed a significant difference [P < 0.01] between both samples).

EDTA eliminates the bactericidal effect of CEP on the katG mutant VEM1673.
It has been reported that the H₂O₂-generating property of polyphenolic compounds depends on their prooxidant activity, which is defined as the capacity of some polyphenols to generate active oxygen species (superoxide anion, hydrogen peroxide, and hydroxyl radicals) in the presence of oxygen and a transition metal ion, such as Fe²⁺ or Cu²⁺ (1, 18). Since significant amounts of iron (Fe) have been found in the seed coats of black beans (35), we analyzed the influence of metal ions on the bactericidal activity of CEP by the addition of the chelating agent EDTA. This experiment showed that the bactericidal effect of CEP on the mutant VEM1673 was totally abolished by the addition of 10 mM of EDTA.

Mutants sensitive to P. vulgaris seed anthocyanidins generally lack catalase activity.
Over the years, a large number of Lps mutants have derived from R. etli CFN42 by various means. In this collection of mutants, several had been observed to be much more sensitive to anthocyanidins than the wild type or other Lps mutants. The differences in sensitivity did not seem to correlate with LPS structural differences (data not shown). The above-mentioned experiments suggested that, instead, there might be a correlation between high-frequency loss of p42f, a consequent loss of katG, and sensitivity to anthocyanidins. Hence, a random set of strains in the collection was tested for catalase activity by a traditional qualitative assay with glass slides (48) and anthocyanidin sensitivity was tested using an agar diffusion assay. For the latter assay, the anthocyanidins had been extracted by a procedure involving hydrolysis with strong acid. A previous report indicates that the main anthocyanidins in the preparation are delphinidin, cyanidin, petunidin, and malvidin (16). The strains fell into three groups: (i) the wild type and the majority of the mutant strains were vigorously positive in the catalase assay and exhibited no zone of inhibition beyond the disc (Fig. 4A); (ii) several strains, including katG mutant CE457, had no catalase activity, and all exhibited equally large zones of inhibition (14 mm in diameter) (Fig. 4C); and (iii) one uncharacterized mutant with a reduced catalase activity (CE518) showed a zone of inhibition larger than that of the wild type but smaller than that of CE457 (Fig. 4B).

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FIG. 4. Agar diffusion assay of sensitivity to anthocyanidins. (A) R. etli CFN42; (B) mutant strain CE518, with reduced catalase activity; (C) mutant strain CE457, with no detectable catalase activity.

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It has been assumed that the main polyphenols that rhizobia face are those exuded by the roots of their legume hosts. However, in their natural environment, rhizobia have to deal with a great variety of polyphenolic compounds entering the rhizosphere from different sources. For instance, R. etli, the symbiont of P. vulgaris, lives surrounded by a large diversity of polyphenolic compounds derived from the crop association among beans, maize, squash, and chili peppers, which have been planted together for thousands of years in Mesoamerica (41, 46). Legume seed coats are also a rich source of flavonoids, some of which differ from those present in root exudates (22, 27, 28, 36). It has been shown that R. etli bacteria are commonly exposed to testa flavonoids, since P. vulgaris seeds carry Rhizobium etli strains on their testae, presumably in a dormant state (40).

In this study, we used the water-soluble polyphenols released from the seed coat of the black bean to simulate a polyphenol-rich environment for R. etli CFN42. We demonstrated that the viability of this bacterium was not affected by the complex mix of polyphenols present in this environment. Interestingly, the polyphenol-rich environment had a bactericidal effect on a mutant cured of plasmid p42f. Analysis of the mutant strain revealed that the katG-encoded catalase was the key enzyme for survival of R. etli CFN42 under this environmental condition, due to its ability to reduce the accumulation of bactericidal levels of hydrogen peroxide. It is important to emphasize that the R. etli CFN42 genome contains a single catalase-peroxidase-encoding gene, localized extrachromosomally (48). In addition to survival assays performed with CEP, we also demonstrated that a crude extract enriched in anthocyanidins was toxic for mutants defective in catalase, suggesting that oxidation of delphinidin, cyanidin, petunidin, and malvidin, the major anthocyanins present in the testae of different black bean cultivars (2, 16, 27), may contribute to generate H₂O₂.

Previous studies of rhizobia catalases have been centered on their role in the oxidative stress faced inside the nodule (14, 29, 48). The data presented in this work show that under free-living conditions the oxidation of polyphenols represents an important source of H₂O₂ for R. etli and highlight the vital participation of the R. etli katG gene for bacterial persistence in a polyphenol-rich environment.

Phenolics are readily oxidized in the environment by enzymes and abiotic factors (pH, redox potential, and metal catalysts) in soil, sediments, water, and the digestive tracts of herbivores (4). Some polyphenols have the capacity to generate active oxygen species (superoxide anion, hydrogen peroxide, and hydroxyl radicals) in the presence of oxygen and a transition metal ion, such as Fe³⁺ or Cu²⁺ (1, 10, 18). This prooxidant activity has been proven for quercetin, myricetin, and kaempferol (10). Since these flavonols are abundant in the seed coat of the black jamapa bean, they may also contribute to the generation of hydrogen peroxide (2). Studies with E. coli have shown that the flavonoids catechin, epigallocatechin, and epichatechin in the presence of copper(II) produce bactericidal concentrations of H₂O₂ (25, 26, 33). According to Hoshino et al. (26), catechin-Cu(II) complexes where Cu(II) is reduced to Cu(I) are formed, and the reoxidation of Cu(I) to Cu(II) by molecular oxygen generates H₂O₂. In the present study, we showed that the addition of EDTA to the CEP eliminated the bactericidal effect on the katG mutant strain. Thus, our data are consistent with the model proposed for E. coli.

We found that the CEP inoculated with the katG mutant strain generated 1,300 µM of H₂O₂ after 16 h of incubation. This amount was 2.3-fold higher than that of the H₂O₂ produced by auto-oxidation of the uninoculated sample (559 µM), suggesting that R. etli cells actively contribute to the oxidation of the polyphenols. Polyphenol oxidases (PPOs), a group of enzymes able to catalyze the oxidation of aromatic compounds, have been found in several bacterial species able to interact with plants (24). Two putative PPOs, which show 50% similarity with PPOs of Ralstonia solanacearum, have been identified in the genome sequence of R. etli CFN42 (20). The above data suggest that in addition to auto-oxidation, bacterial enzymes also participate in oxidation of polyphenols, contributing to a variable pool of H₂O₂. A strategy used by soil bacteria for contending with this condition seems to rely on catalase activity. It will be interesting to determine if the extrachromosomal localization of R. etli katG represents an advantage for the population, regarding distribution among cells, through horizontal gene transfer.

 
We thank G. Loarca-Piña for providing the sample of seed coat used in this study and for helpful suggestions. We are grateful to Laura Cervantes, Javier Rivera, Jodie Box, and Mitchell Klement for their excellent technical assistance and to Rosa Isela Santamaría for DNA sequencing.

This work was supported by DGAPA-UNAM grant IN201206.

 
* Corresponding author. Mailing address: Programa de Ingeniería Genómica, Centro de Ciencias Genómicas, UNAM. Ap. Postal 565-A, Cuernavaca, Morelos, Mexico. Phone: (52) (777) 329 16 91. Fax: (52) (777) 317 55 81. E-mail: alex{at}ccg.unam.mx

Published ahead of print on 29 February 2008.

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Applied and Environmental Microbiology, April 2008, p. 2398-2403, Vol. 74, No. 8
0099-2240/08/$08.00+0     doi:10.1128/AEM.02457-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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