The gap gene of Rhizobium etli is required for both free life and symbiosis with common beans.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH or Gap) is a ubiquitous enzyme essential for carbon and energy metabolism in most organisms. Despite its primary role in sugar metabolism, GAPDH is recognized for its involvement in diverse cellular processes, being considered a paradigm among multifunctional/moonlighting proteins. Besides its canonical cytoplasmic location, GAPDH has been detected on cell surfaces or as a secreted protein in prokaryotes, yet little is known about its possible roles in plant symbiotic bacteria. Here we report that Rhizobium etli, a nitrogen-fixing symbiont of common beans, carries a single gap gene responsible for both GAPDH glycolytic and gluconeogenic activities. An active Gap protein is required throughout all stages of the symbiosis between R. etli and its host plant Phaseolus vulgaris. Both glycolytic and gluconeogenic Gap metabolic activities likely contribute to bacterial fitness during early and intermediate stages of the interaction, whereas GAPDH gluconeogenic activity seems critical for nodule invasion and nitrogen fixation. Although the R. etli Gap protein is secreted in a c-di-GMP related manner, no involvement of the R. etli gap gene in c-di-GMP related phenotypes, such as flocculation, biofilm formation or EPS production, was observed. Notably, the R. etli gap gene fully complemented a double gap1/gap2 mutant of Pseudomonas syringae for free life growth, albeit only partially in planta, suggesting potential specific roles for each type of Gap protein. Nevertheless, further research is required to unravel additional functions of the R. etli Gap protein beyond its essential metabolic roles.

Phenotypic plasticity and a new small molecule are involved in a fungal-bacterial interaction.

Nitrogen-fixing bacteria have been extensively studied in the context of interactions with their host plants; however, little is known about the phenotypic plasticity of these microorganisms in nonmutualistic interactions with other eukaryotes. A dual-species coculture model was developed by using the plant symbiotic bacterium Rhizobium etli and the well-studied eukaryote Saccharomyces cerevisiae as a tractable system to explore the molecular mechanisms used by R. etli in nonmutual interactions. Here, we show that the fungus promotes the growth of the bacterium and that together, these organisms form a mixed biofilm whose biomass is ~ 3 times greater and is more structured than that of either single-species biofilm. We found that these biofilm traits are dependent on a symbiotic plasmid encoding elements involved in the phenotypic plasticity of the bacterium, mitochondrial function and in the production of a yeast-secreted sophoroside. Interestingly, the promoters of 3 genes that are key in plant bacteria-interaction (nifH, fixA and nodA) were induced when R. etli coexists with yeast. These results show that investigating interactions between species that do not naturally coexist is a new approach to discover gene functions and specialized metabolites in model organisms.

Proteins in the periplasmic space and outer membrane vesicles of Rhizobium etli CE3 grown in minimal medium are largely distinct and change with growth phase.

Rhizobium etli CE3 grown in succinate-ammonium minimal medium (MM) excreted outer membrane vesicles (OMVs) with diameters of 40 to 100 nm. Proteins from the OMVs and the periplasmic space were isolated from 6 and 24 h cultures and identified by proteome analysis. A total of 770 proteins were identified: 73.8 and 21.3 % of these occurred only in the periplasm and OMVs, respectively, and only 4.9 % were found in both locations. The majority of proteins found in either location were present only at 6 or 24 h: in the periplasm and OMVs, only 24 and 9 % of proteins, respectively, were present at both sampling times, indicating a time-dependent differential sorting of proteins into the two compartments. The OMVs contained proteins with physiologically varied roles, including Rhizobium adhering proteins (Rap), polysaccharidases, polysaccharide export proteins, auto-aggregation and adherence proteins, glycosyl transferases, peptidoglycan binding and cross-linking enzymes, potential cell wall-modifying enzymes, porins, multidrug efflux RND family proteins, ABC transporter proteins and heat shock proteins. As expected, proteins with known periplasmic localizations (phosphatases, phosphodiesterases, pyrophosphatases) were found only in the periplasm, along with numerous proteins involved in amino acid and carbohydrate metabolism and transport. Nearly one-quarter of the proteins present in the OMVs were also found in our previous analysis of the R. etli total exproteome of MM-grown cells, indicating that these nanoparticles are an important mechanism for protein excretion in this species.

Soil commensal rhizobia promote Rhizobium etli nodulation efficiency through CinR-mediated quorum sensing.

The rhizosphere microbiome is composed of diverse microorganisms directly interacting with plants and each other. We sought to achieve a better understanding of how rhizobia interact with other soil bacteria during the initial symbiosis period. In this study, we investigated how soil commensals, particularly other rhizobia, affect Rhizobium etli-Phaseolus vulgaris interactions. We found that R. etli formed significantly more nodules on beans grown in unsterilized soil than those in sterilized soil. Furthermore, a strain identified as Rhizobium fabae, isolated from unsterilized soil, was found to affect R. etli nodulation. Interestingly, we found that the key quorum sensing regulator CinR is important for R. etli nodulation efficiency when it is co-inoculated with R. fabae. Moreover, we found that quorum sensing signals produced by R. fabae promoted CinR-mediated gene expression in R. etli. These data suggest that the effects of R. fabae on R. etli symbiosis may act through multispecies bacterial cell-cell communication.

A quinol oxidase, encoded by cyoABCD, is utilized to adapt to lower O2 concentrations in Rhizobium etli CFN42.

Bacteria have branched aerobic respiratory chains that terminate at different terminal oxidases. These terminal oxidases have varying properties such as their affinity for oxygen, transcriptional regulation and proton pumping ability. The focus of this study was a quinol oxidase encoded by cyoABCD. Although this oxidase (Cyo) is widespread among bacteria, not much is known about its role in the cell, particularly in bacteria that contain both cytochrome c oxidases and quinol oxidases. Using Rhizobium etli CFN42 as a model organism, a cyo mutant was analysed for its ability to grow in batch cultures at high (21 % O2) and low (1 and 0.1 % O2) ambient oxygen concentrations. In comparison with other oxidase mutants, the cyo mutant had a significantly longer lag phase under low-oxygen conditions. Using a cyo :: lacZ transcriptional fusion, it was shown that cyo expression in the wild type peaks between 1 and 2.5 % O2. In addition, it was shown with quantitative reverse transcriptase PCR that cyoB is upregulated approximately fivefold in 1 % O2 compared with fully aerobic (21 % O2) conditions. Analysis of the cyo mutant during symbiosis with Phaseolous vulgaris indicated that Cyo is utilized during early development of the symbiosis. Although it is commonly thought that Cyo is utilized only at higher oxygen concentrations, the results from this study indicate that Cyo is important for adaptation to and sustained growth under low oxygen.

Down-regulation of PvTRE1 enhances nodule biomass and bacteroid number in the common bean.

Legume-rhizobium interactions have been widely studied and characterized, and the disaccharide trehalose has been commonly detected during this symbiotic interaction. It has been proposed that trehalose content in nodules during this symbiotic interaction might be regulated by trehalase. In the present study, we assessed the role of trehalose accumulation by down-regulating trehalase in the nodules of common bean plants. We performed gene expression analysis for trehalase (PvTRE1) during nodule development. PvTRE1 was knocked down by RNA interference (RNAi) in transgenic nodules of the common bean. PvTRE1 expression in nodulated roots is mainly restricted to nodules. Down-regulation of PvTRE1 led to increased trehalose content (78%) and bacteroid number (almost one order of magnitude). In addition, nodule biomass, nitrogenase activity, and GOGAT transcript accumulation were significantly enhanced too. The trehalose accumulation, triggered by PvTRE1 down-regulation, led to a positive impact on the legume-rhizobium symbiotic interaction. This could contribute to the agronomical enhancement of symbiotic nitrogen fixation.

Change in land use alters the diversity and composition of Bradyrhizobium communities and led to the introduction of Rhizobium etli into the tropical rain forest of Los Tuxtlas (Mexico).

Nitrogen-fixing bacteria of the Bradyrhizobium genus are major symbionts of legume plants in American tropical forests, but little is known about the effects of deforestation and change in land use on their diversity and community structure. Forest clearing is followed by cropping of bean (Phaseolus vulgaris) and maize as intercropped plants in Los Tuxtlas tropical forest of Mexico. The identity of bean-nodulating rhizobia in this area is not known. Using promiscuous trap plants, bradyrhizobia were isolated from soil samples collected in Los Tuxtlas undisturbed forest, and in areas where forest was cleared and land was used as crop fields or as pastures, or where secondary forests were established. Rhizobia were also trapped by using bean plants. Bradyrhizobium strains were classified into genospecies by dnaK sequence analysis supported by recA, glnII and 16S-23S rDNA IGS loci analyses. A total of 29 genospecies were identified, 24 of which did not correspond to any described taxa. A reduction in Bradyrhizobium diversity was observed when forest was turned to crop fields or pastures. Diversity seemed to recover to primary forest levels in secondary forests that derived from abandoned crop fields or pastures. The shifts in diversity were not related to soil characteristics but seemingly to the density of nodulating legumes present at each land use system (LUS). Bradyrhizobium community composition in soils was dependent on land use; however, similarities were observed between crop fields and pastures but not among forest and secondary forest. Most Bradyrhizobium genospecies present in forest were not recovered or become rare in the other LUS. Rhizobium etli was found as the dominant bean-nodulating rhizobia present in crop fields and pastures, and evidence was found that this species was introduced in Los Tuxtlas forest.

Glutathione is required by Rhizobium etli for glutamine utilization and symbiotic effectiveness.

Here, we provide genetic and biochemical evidence indicating that the ability of Rhizobium etli bacteria to efficiently catabolize glutamine depends on its ability to produce reduced glutathione (l-γ-glutamyl-l-cysteinylglycine [GSH]). We find that GSH-deficient strains, namely a gshB (GSH synthetase) and a gor (GSH reductase) mutant, can use different amino acids, including histidine, alanine, and asparagine but not glutamine, as sole source of carbon, energy, and nitrogen. Moreover, l-buthionine(S,R)-sulfoximine, a GSH synthesis inhibitor, or diamide that oxidizes GSH, induced the same phenotype in the wild-type strain. Among the steps required for its utilization, glutamine uptake, occurring through the two well-characterized carriers (Aap and Bra systems) but not glutamine degradation or respiration, was largely reduced in GSH-deficient strains. Furthermore, GSH-deficient mutants of R. etli showed a reduced symbiotic efficiency. Exogenous GSH was sufficient to rescue glutamine uptake or degradation ability, as well as the symbiotic effectiveness of GSH mutants. Our results suggest a previously unknown GSH-glutamine metabolic relationship in bacteria.

A comparative transcriptome analysis of Rhizobium etli bacteroids: specific gene expression during symbiotic nongrowth.

Rhizobium etli occurs either in a nitrogen-fixing symbiosis with its host plant, Phaseolus vulgaris, or free-living in the soil. During both conditions, the bacterium has been suggested to reside primarily in a nongrowing state. Using genome-wide transcriptome profiles, we here examine the molecular basis of the physiological adaptations of rhizobia to nongrowth inside and outside of the host. Compared with exponentially growing cells, we found an extensive overlap of downregulated growth-associated genes during both symbiosis and stationary phase, confirming the essentially nongrowing state of nitrogen-fixing bacteroids in determinate nodules that are not terminally differentiated. In contrast, the overlap of upregulated genes was limited. Generally, actively growing cells have hitherto been used as reference to analyze symbiosis-specific expression. However, this prevents the distinction between differential expression arising specifically from adaptation to a symbiotic lifestyle and features associated with nongrowth in general. Using stationary phase as the reference condition, we report a distinct transcriptome profile for bacteroids, containing 203 induced and 354 repressed genes. Certain previously described symbiosis-specific characteristics, such as the downregulation of amino acid metabolism genes, were no longer observed, indicating that these features are more likely due to the nongrowing state of bacteroids rather than representing bacteroid-specific physiological adaptations.

Stress response regulators identified through genome-wide transcriptome analysis of the (p)ppGpp-dependent response in Rhizobium etli.

BACKGROUND: The alarmone (p)ppGpp mediates a global reprogramming of gene expression upon nutrient limitation and other stresses to cope with these unfavorable conditions. Synthesis of (p)ppGpp is, in most bacteria, controlled by RelA/SpoT (Rsh) proteins. The role of (p)ppGpp has been characterized primarily in Escherichia coli and several Gram-positive bacteria. Here, we report the first in-depth analysis of the (p)ppGpp-regulon in an α-proteobacterium using a high-resolution tiling array to better understand the pleiotropic stress phenotype of a relA/rsh mutant. RESULTS: We compared gene expression of the Rhizobium etli wild type and rsh (previously rel) mutant during exponential and stationary phase, identifying numerous (p)ppGpp targets, including small non-coding RNAs. The majority of the 834 (p)ppGpp-dependent genes were detected during stationary phase. Unexpectedly, 223 genes were expressed (p)ppGpp-dependently during early exponential phase, indicating the hitherto unrecognized importance of (p)ppGpp during active growth. Furthermore, we identified two (p)ppGpp-dependent key regulators for survival during heat and oxidative stress and one regulator putatively involved in metabolic adaptation, namely extracytoplasmic function sigma factor EcfG2/PF00052, transcription factor CH00371, and serine protein kinase PrkA. CONCLUSIONS: The regulatory role of (p)ppGpp in R. etli stress adaptation is far-reaching in redirecting gene expression during all growth phases. Genome-wide transcriptome analysis of a strain deficient in a global regulator, and exhibiting a pleiotropic phenotype, enables the identification of more specific regulators that control genes associated with a subset of stress phenotypes. This work is an important step toward a full understanding of the regulatory network underlying stress responses in α-proteobacteria.

Plasmids with a chromosome-like role in rhizobia.

Replicon architecture in bacteria is commonly comprised of one indispensable chromosome and several dispensable plasmids. This view has been enriched by the discovery of additional chromosomes, identified mainly by localization of rRNA and/or tRNA genes, and also by experimental demonstration of their requirement for cell growth. The genome of Rhizobium etli CFN42 is constituted by one chromosome and six large plasmids, ranging in size from 184 to 642 kb. Five of the six plasmids are dispensable for cell viability, but plasmid p42e is unusually stable. One possibility to explain this stability would be that genes on p42e carry out essential functions, thus making it a candidate for a secondary chromosome. To ascertain this, we made an in-depth functional analysis of p42e, employing bioinformatic tools, insertional mutagenesis, and programmed deletions. Nearly 11% of the genes in p42e participate in primary metabolism, involving biosynthetic functions (cobalamin, cardiolipin, cytochrome o, NAD, and thiamine), degradation (asparagine and melibiose), and septum formation (minCDE). Synteny analysis and incompatibility studies revealed highly stable replicons equivalent to p42e in content and gene order in other Rhizobium species. A systematic deletion analysis of p42e allowed the identification of two genes (RHE_PE00001 and RHE_PE00024), encoding, respectively, a hypothetical protein with a probable winged helix-turn-helix motif and a probable two-component sensor histidine kinase/response regulator hybrid protein, which are essential for growth in rich medium. These data support the proposal that p42e and its homologous replicons (pA, pRL11, pRLG202, and pR132502) merit the status of secondary chromosomes.

Characterization of the NifA-RpoN regulon in Rhizobium etli in free life and in symbiosis with Phaseolus vulgaris.

The NifA-RpoN complex is a master regulator of the nitrogen fixation genes in alphaproteobacteria. Based on the complete Rhizobium etli genome sequence, we constructed an R. etli CFN42 oligonucleotide (70-mer) microarray and utilized this tool, reverse transcription (RT)-PCR analysis (transcriptomics), proteomics, and bioinformatics to decipher the NifA-RpoN regulon under microaerobic conditions (free life) and in symbiosis with bean plants. The R. etli NifA-RpoN regulon was determined to contain 78 genes, including the genes involved in nitrogen fixation, and the analyses revealed 42 new NifA-RpoN-dependent genes. More importantly, this study demonstrated that the NifA-RpoN regulon is composed of genes and proteins that have very diverse functions, that play fundamental and previously less appreciated roles in regulating the normal physiology of the cell, and that have important functions in providing adequate conditions for efficient nitrogen fixation in symbiosis. The R. etli NifA-RpoN regulon defined here has some components in common with other NifA-RpoN regulons described previously, but the vast majority of the components have been found only in the R. etli regulon, suggesting that they have a specific role in this bacterium and particular requirements during nitrogen fixation compared with other symbiotic bacterial models.

Sulphadimethoxine inhibits Phaseolus vulgaris root growth and development of N-fixing nodules.

Sulphonamides contamination of cultivated lands occurs through the recurrent spreading of animal wastes from intensive farming. The aim of this study was to test the effect(s) of sulphadimethoxine on the beneficial N-fixing Rhizobium etli-Phaseolus vulgaris symbiosis under laboratory conditions. The consequence of increasing concentrations of sulphadimethoxine on the growth ability of free-living R. etli bacteria, as well as on seed germination, seedling development and growth of common bean plants was examined. We have established that sulphadimethoxine inhibited the growth of both symbiotic partners in a dose-dependent manner. Bacterial invasion occurring in developing root nodules was visualized by fluorescence microscopy generating EGFP-marked R. etli bacteria. Our results proved that the development of symbiotic N-fixing root nodules is hampered by sulphadimethoxine thus identifying sulphonamides as toxic compounds for the Rhizobium-legume symbiosis: a low-input sustainable agricultural practice.

Role of the extracytoplasmic function sigma factor RpoE4 in oxidative and osmotic stress responses in Rhizobium etli.

The aims of this study were to functionally characterize and analyze the transcriptional regulation and transcriptome of the Rhizobium etli rpoE4 gene. An R. etli rpoE4 mutant was sensitive to oxidative, saline, and osmotic stresses. Using transcriptional fusions, we determined that RpoE4 controls its own transcription and that it is negatively regulated by rseF (regulator of sigma rpoE4; CH03274), which is cotranscribed with rpoE4. rpoE4 expression was induced not only after oxidative, saline, and osmotic shocks, but also under microaerobic and stationary-phase growth conditions. The transcriptome analyses of an rpoE4 mutant and an rpoE4-overexpressing strain revealed that the RpoE4 extracytoplasmic function sigma factor regulates about 98 genes; 50 of them have the rpoE4 promoter motifs in the upstream regulatory regions. Interestingly, 16 of 38 genes upregulated in the rpoE4-overexpressing strain encode unknown putative cell envelope proteins. Other genes controlled by RpoE4 include rpoH2, CH00462, CH02434, CH03474, and xthA1, which encode proteins involved in the stress response (a heat shock sigma factor, a putative Mn-catalase, an alkylation DNA repair protein, pyridoxine phosphate oxidase, and exonuclease III, respectively), as well as several genes, such as CH01253, CH03555, and PF00247, encoding putative proteins involved in cell envelope biogenesis (a putative peptidoglycan binding protein, a cell wall degradation protein, and phospholipase D, respectively). These results suggest that rpoE4 has a relevant function in cell envelope biogenesis and that it plays a role as a general regulator in the responses to several kinds of stress.

Pleiotropic effects of a rel mutation on stress survival of Rhizobium etli CNPAF512.

BACKGROUND: The rel gene of Rhizobium etli (relRet), the nodulating endosymbiont of the common bean plant, determines the cellular level of the alarmone (p)ppGpp and was previously shown to affect free-living growth and symbiosis. Here, we demonstrate its role in cellular adaptation and survival in response to various stresses. RESULTS: Growth of the R. etli relRet mutant was strongly reduced or abolished in the presence of elevated NaCl levels or at 37 degrees C, compared to the wild type. In addition, depending on the cell density, decreased survival of exponentially growing or stationary phase relRet mutant cells was obtained after H2O2, heat or NaCl shock compared to the wild-type strain. Survival of unstressed stationary phase cultures was differentially affected depending on the growth medium used. Colony forming units (CFU) of relRet mutant cultures continuously decreased in minimal medium supplemented with succinate, whereas wild-type cultures stabilised at higher CFU levels. Microscopic examination of stationary phase cells indicated that the relRet mutant was unable to reach the typical coccoid morphology of the wild type in stationary phase cultures. Assessment of stress resistance of re-isolated bacteroids showed increased sensitivity of the relRet mutant to H2O2 and a slightly increased resistance to elevated temperature (45 degrees C) or NaCl shock, compared to wild-type bacteroids. CONCLUSION: The relRet gene is an important factor in regulating rhizobial physiology, during free-living growth as well as in symbiotic conditions. Additionally, differential responses to several stresses applied to bacteroids and free-living exponential or stationary phase cells point to essential physiological differences between the different states.

Thiamine limitation determines the transition from aerobic to fermentative-like metabolism in Rhizobium etli CE3.

Both thiamine and biotin when added to minimal medium subcultures reversed the fermentative-like metabolism exhibited by Rhizobium etli CE3. Thiamine auxotrophs lacking thiCOGE genes were used to investigate the role of thiamine in this medium. A thiC1169::miniTn5lacZ1 thiamine auxotroph subjected to the above subcultures resulted in growth arrest, reduced pyruvate-dehydrogenase activity, and a smaller amount of poly-beta-hydroxybutyrate compared with the CE3 strain. Moreover, thiC and thiEb genes were overexpressed as result of thiamine limitation. The absence of classical thi genes suggests that thiamine is synthesized with low efficiency by an alternative pathway. Low levels of thiamine cause the CE3 strain to exhibit a fermentative-like metabolism.

Genetic determinants of swarming in Rhizobium etli.

Swarming motility is considered to be a social phenomenon that enables groups of bacteria to move coordinately atop solid surfaces. The differentiated swarmer cell population is embedded in an extracellular slime layer, and the phenomenon has previously been linked with biofilm formation and virulence. The gram-negative nitrogen-fixing soil bacterium Rhizobium etli CNPAF512 was previously shown to display swarming behavior on soft agar plates. In a search for novel genetic determinants of swarming, a detailed analysis of the swarming behavior of 700 miniTn5 mutants of R. etli was performed. Twenty-four mutants defective in swarming or displaying abnormal swarming patterns were identified and could be divided into three groups based on their swarming pattern. Fourteen mutants were completely swarming deficient, five mutants showed an atypical swarming pattern with no completely smooth edge and local extrusions, and five mutants displayed an intermediate swarming phenotype. Sequence analysis of the targeted genes indicated that the mutants were likely affected in quorum-sensing, polysaccharide composition or export, motility, and amino acid and polyamines metabolism. Several of the identified mutants displayed a reduced symbiotic nitrogen fixation activity.

Binding site determinants for the LysR-type transcriptional regulator PcaQ in the legume endosymbiont Sinorhizobium meliloti.

LysR-type transcriptional regulators represent one of the largest groups of prokaryotic regulators described to date. In the gram-negative legume endosymbiont Sinorhizobium meliloti, enzymes involved in the protocatechuate branch of the beta-ketoadipate pathway are encoded within the pcaDCHGB operon, which is subject to regulation by the LysR-type protein PcaQ. In this work, purified PcaQ was shown to bind strongly (equilibrium dissociation constant, 0.54 nM) to a region at positions -78 to -45 upstream of the pcaD transcriptional start site. Within this region, we defined a PcaQ binding site with dyad symmetry that is required for regulation of pcaD expression in vivo and for binding of PcaQ in vitro. We also demonstrated that PcaQ participates in negative autoregulation by monitoring expression of pcaQ via a transcriptional fusion to lacZ. Although pcaQ homologues are present in many alpha-proteobacteria, this work describes the first reported purification of this regulator, as well as characterization of its binding site, which is conserved in Agrobacterium tumefaciens, Rhizobium leguminosarum, Rhizobium etli, and Mesorhizobium loti.

The Rhizobium etli opt operon is required for symbiosis and stress resistance.

Rhizobium etli is a Gram-negative root-colonizing soil bacterium capable of fixing nitrogen while living in symbiosis with its leguminous host Phaseolus vulgaris. A genome-wide screening for R. etli symbiotic mutants revealed a R. etli operon encoding an oligopeptide ABC-transporter (Opt), two redA homologous genes and one redB gene. Expression analysis showed this opt operon to be transcribed both under free-living and symbiotic conditions and expression levels were demonstrated to be growth-phase-dependent. Plants nodulated by R. etli opt mutants showed a reduced symbiotic nitrogen fixation activity (approximately 50% reduction). Growth experiments with opt mutants in the presence of oligopeptides as the sole nitrogen source confirmed the involvement of the opt genes in oligopeptide uptake. Further phenotypic analysis of the opt mutants revealed them to display an enhanced resistance to the oligopeptide antibiotic bacitracin, an increased susceptibility to the beta-lactam antibiotic ampicillin and a decreased osmotolerance. In conclusion, our results demonstrate that the opt operon plays a crucial role during symbiosis and stress resistance.

Rhizobium etli USDA9032 engineered to produce a phenazine antibiotic inhibits the growth of fungal pathogens but is impaired in symbiotic performance.

Phenazine production was engineered in Rhizobium etli USDA9032 by the introduction of the phz locus of Pseudomonas chlororaphis O6. Phenazine-producing R. etli was able to inhibit the growth of Botrytis cinerea and Fusarium oxysporum in vitro. Black bean inoculated with phenazine-producing R. etli produced brownish Fix(-) nodules.