Prevalent role of homologous recombination in the repair of specific double-strand breaks in Rhizobium etli.

Double-strand breaks (DSBs) are the most dangerous injuries for a genome. When unrepaired, death quickly ensues. In most bacterial systems, DSBs are repaired through homologous recombination. Nearly one-quarter of bacterial species harbor a second system, allowing direct ligation of broken ends, known as Non-Homologous End Joining (NHEJ). The relative role of both systems in DSBs repair in bacteria has been explored only in a few cases. To evaluate this in the bacterium Rhizobium etli, we used a modified version of the symbiotic plasmid (264 kb), containing a single copy of the nifH gene. In this plasmid, we inserted an integrative plasmid harboring a modified nifH gene fragment containing an I-SceI site. DSBs were easily inflicted in vivo by conjugating a small, replicative plasmid that expresses the I-SceI nuclease into the appropriate strains. Repair of a DSB may be achieved through homologous recombination (either between adjacent or distant repeats) or NHEJ. Characterization of the derivatives that repaired DSB in different configurations, revealed that in most cases (74%), homologous recombination was the prevalent mechanism responsible for repair, with a relatively minor contribution of NHEJ (23%). Inactivation of the I-SceI gene was detected in 3% of the cases. Sequence analysis of repaired derivatives showed the operation of NHEJ. To enhance the number of derivatives repaired through NHEJ, we repeated these experiments in a recA mutant background. Derivatives showing NHEJ were readily obtained when the DSB occurred on a small, artificial plasmid in a recA mutant. However, attempts to deliver a DSB on the symbiotic plasmid in a recA background failed, due to the accumulation of mutations that inactivated the I-SceI gene. This result, coupled with the absence of derivatives that lost the nonessential symbiotic plasmid, may be due to an unusual stability of the symbiotic plasmid, possibly caused by the presence of multiple toxin-antitoxin modules.

Draft genome sequence of the highly copper-tolerant Methylobacterium radiotolerans MLP1 isolated from the rhizosphere of grasses adjacent to mine tailings.

We present the genome of a highly copper-tolerant pink-pigmented facultative methylotroph isolated from the rhizosphere of grasses growing close to mine tailings. Based on whole-genome taxonomic analyses, this isolate was named Methylobacterium radiotolerans MLP1. Studies are in progress to infer its genome-based copper resistome.

Five copper homeostasis gene clusters encode the Cu-efflux resistome of the highly copper-tolerant Methylorubrum extorquens AM1.

Background: In the last decade, the use of copper has reemerged as a potential strategy to limit healthcare-associated infections and to control the spread of multidrug-resistant pathogens. Numerous environmental studies have proposed that most opportunistic pathogens have acquired antimicrobial resistance in their nonclinical primary habitat. Thus, it can be presumed that copper-resistant bacteria inhabiting a primary commensal niche might potentially colonize clinical environments and negatively affect the bactericidal efficacy of Cu-based treatments. The use of copper in agricultural fields is one of the most important sources of Cu pollution that may exert selection pressure for the increase of copper resistance in soil and plant-associated bacteria. To assess the emergence of copper-resistant bacteria in natural habitats, we surveyed a laboratory collection of bacterial strains belonging to the order Rhizobiales. This study proposes that Methylorubrum extorquens AM1 is an environmental isolate well adapted to thrive in copper-rich environments that could act as a reservoir of copper resistance genes. Methods: The minimal inhibitory concentrations (MICs) of CuCl2 were used to estimate the copper tolerance of eight plant-associated facultative diazotrophs (PAFD) and five pink-pigmented facultative methylotrophs (PPFM) belonging to the order Rhizobiales presumed to come from nonclinical and nonmetal-polluted natural habitats based on their reported source of isolation. Their sequenced genomes were used to infer the occurrence and diversity of Cu-ATPases and the copper efflux resistome of Mr. extorquens AM1. Results: These bacteria exhibited minimal inhibitory concentrations (MICs) of CuCl2 ranging between 0.020 and 1.9 mM. The presence of multiple and quite divergent Cu-ATPases per genome was a prevalent characteristic. The highest copper tolerance exhibited by Mr. extorquens AM1 (highest MIC of 1.9 mM) was similar to that found in the multimetal-resistant model bacterium Cupriavidus metallidurans CH34 and in clinical isolates of Acinetobacter baumannii. The genome-predicted copper efflux resistome of Mr. extorquens AM1 consists of five large (6.7 to 25.7 kb) Cu homeostasis gene clusters, three clusters share genes encoding Cu-ATPases, CusAB transporters, numerous CopZ chaperones, and enzymes involved in DNA transfer and persistence. The high copper tolerance and the presence of a complex Cu efflux resistome suggest the presence of relatively high copper tolerance in environmental isolates of Mr. extorquens.

RdsA Is a Global Regulator That Controls Cell Shape and Division in Rhizobium etli.

Unlike other bacteria, cell growth in rhizobiales is unipolar and asymmetric. The regulation of cell division, and its coordination with metabolic processes is an active field of research. In Rhizobium etli, gene RHE_PE00024, located in a secondary chromosome, is essential for growth. This gene encodes a predicted hybrid histidine kinase sensor protein, participating in a, as yet undescribed, two-component signaling system. In this work, we show that a conditional knockdown mutant (cKD24) in RHE_PE00024 (hereby referred as rdsA, after rhizobium division and shape) generates a striking phenotype, where nearly 64% of the cells present a round shape, with stochastic and uncoordinated cell division. For rod-shaped cells, a large fraction (12 to 29%, depending on their origin) present growth from the old pole, a sector that is normally inactive for growth in a wild-type cell. A fraction of the cells (1 to 3%) showed also multiple ectopic polar growths. Homodimerization of RdsA appears to be required for normal function. RNAseq analysis of mutant cKD24 reveals global changes, with downregulated genes in at least five biological processes: cell division, wall biogenesis, respiration, translation, and motility. These modifications may affect proper structuring of the divisome, as well as peptidoglycan synthesis. Together, these results indicate that the hybrid histidine kinase RdsA is an essential global regulator influencing cell division and cell shape in R. etli.

A novel way to synthesize pantothenate in bacteria involves ß-alanine synthase present in uracil degradation pathway.

Pantothenate is an indispensable vitamin precursor of the synthesis of coenzyme A (CoA), a key metabolite required in over 100 metabolic reactions. ß-Alanine (ß-ala) is an indispensable component of pantothenate. Due to the metabolic relevance of this pathway, we assumed that orthologous genes for ß-alanine synthesis would be present in the genomes of bacteria, archaea, and eukaryotes. However, comparative genomic studies revealed that orthologous gene replacement and loss of synteny occur at high frequency in panD genes. We have previously reported the atypical plasmid-encoded location of the pantothenate pathway genes panC and panB (two copies) in R. etli CFN42. This study also revealed the unexpected absence of a panD gene encoding the aspartate decarboxylase enzyme (ADC), required for the synthesis of ß-ala. The aim of this study was to identify the source of ß-alanine in Rhizobium etli CFN42. In this study, we present a bioinformatic analysis and an experimental validation demonstrating that the source of ß-ala in this R. etli comes from ß-alanine synthase, the last enzyme of the uracil degradation pathway.

Rhizobium tropici CIAT 899 copA gene plays a fundamental role in copper tolerance in both free life and symbiosis with Phaseolus vulgaris.

Rhizobium tropici CIAT 899 is a facultative symbiotic diazotroph able to deal with stressful concentrations of metals. Nevertheless the molecular mechanisms involved in metal tolerance have not been elucidated. Copper (Cu2+) is a metal component essential for the heme-copper respiratory oxidases and enzymes that catalyse redox reactions, however, it is highly toxic when intracellular trace concentrations are surpassed. In this study, we report that R. tropici CIAT 899 is more tolerant to Cu2+ than other Rhizobium and Sinorhizobium species. Through Tn5 random mutagenesis we identify a R. tropici mutant strain with a severe reduction in Cu2+ tolerance. The Tn5 insertion disrupted the gene RTCIAT899_CH17575, encoding a putative heavy metal efflux P1B-1-type ATPase designated as copA. Phaseolus vulgaris plants inoculated with the copA::Tn5 mutant in the presence of toxic Cu2+ concentrations showed a drastic reduction in plant and nodule dry weight, as well as nitrogenase activity. Nodules induced by the copA::Tn5 mutant present an increase in H2O2 concentration, lipoperoxidation and accumulate 40-fold more Cu2+ than nodules formed by the wild-type strain. The copA::Tn5 mutant complemented with the copA gene recovered the wild-type symbiotic phenotypes. Therefore, the copA gene is essential for R. tropici CIAT 899 to survive in copper-rich environments in both free life and symbiosis with P. vulgaris plants.

Analysis of genome sequence and symbiotic ability of rhizobial strains isolated from seeds of common bean (Phaseolus vulgaris).

BACKGROUND: Rhizobia are alpha-proteobacteria commonly found in soil and root nodules of legumes. It was recently reported that nitrogen-fixing rhizobia also inhabit legume seeds. In this study, we examined whole-genome sequences of seven strains of rhizobia isolated from seeds of common bean (Phaseolus vulgaris). RESULTS: Rhizobial strains included in this study belonged to three different species, including Rhizobium phaseoli, R. leguminosarum, and R. grahamii. Genome sequence analyses revealed that six of the strains formed three pairs of highly related strains. Both strains comprising a pair shared all but one plasmid. In two out of three pairs, one of the member strains was effective in nodulation and nitrogen fixation, whereas the other was ineffective. The genome of the ineffective strain in each pair lacked several genes responsible for symbiosis, including nod, nif, and fix genes, whereas that of the effective strain harbored the corresponding genes in clusters, suggesting that recombination events provoked gene loss in ineffective strains. Comparisons of genomic sequences between seed strains and nodule strains of the same species showed high conservation of chromosomal sequences and lower conservation of plasmid sequences. Approximately 70% of all genes were shared among the strains of each species. However, paralogs were more abundant in seed strains than in nodule strains. Functional analysis showed that seed strains were particularly enriched in genes involved in the transport and metabolism of amino acids and carbohydrates, biosynthesis of cofactors and in transposons and prophages. Genomes of seed strains harbored several intact prophages, one of which was inserted at exactly the same genomic position in three strains of R. phaseoli and R. leguminosarum. The R. grahamii strain carried a prophage similar to a gene transfer agent (GTA); this represents the first GTA reported for this genus. CONCLUSIONS: Seeds represent a niche for bacteria; their access by rhizobia possibly triggered the infection of phages, recombination, loss or gain of plasmids, and loss of symbiosis genes. This process probably represents ongoing evolution that will eventually convert these strains into obligate endophytes.

The ropAe gene encodes a porin-like protein involved in copper transit in Rhizobium etli CFN42.

Copper (Cu) is an essential micronutrient for all aerobic forms of life. Its oxidation states (Cu+ /Cu2+ ) make this metal an important cofactor of enzymes catalyzing redox reactions in essential biological processes. In gram-negative bacteria, Cu uptake is an unexplored component of a finely regulated trafficking network, mediated by protein-protein interactions that deliver Cu to target proteins and efflux surplus metal to avoid toxicity. Rhizobium etliCFN42 is a facultative symbiotic diazotroph that must ensure its appropriate Cu supply for living either free in the soil or as an intracellular symbiont of leguminous plants. In crop fields, rhizobia have to contend with copper-based fungicides. A detailed deletion analysis of the pRet42e (505 kb) plasmid from an R. etli mutant with enhanced CuCl2 tolerance led us to the identification of the ropAe gene, predicted to encode an outer membrane protein (OMP) with a ß-barrel channel structure that may be involved in Cu transport. In support of this hypothesis, the functional characterization of ropAe revealed that: (I) gene disruption increased copper tolerance of the mutant, and its complementation with the wild-type gene restored its wild-type copper sensitivity; (II) the ropAe gene maintains a low basal transcription level in copper overload, but is upregulated when copper is scarce; (III) disruption of ropAe in an actP (copA) mutant background, defective in copper efflux, partially reduced its copper sensitivity phenotype. Finally, BLASTP comparisons and a maximum likelihood phylogenetic analysis highlight the diversification of four RopA paralogs in members of the Rhizobiaceae family. Orthologs of RopAe are highly conserved in the Rhizobiales order, poorly conserved in other alpha proteobacteria and phylogenetically unrelated to characterized porins involved in Cu or Mn uptake.

The cation diffusion facilitator protein EmfA of Rhizobium etli belongs to a novel subfamily of Mn(2+)/Fe(2+) transporters conserved in α-proteobacteria.

Manganese (Mn(2+)) plays a key role in important cellular functions such as oxidative stress response and bacterial virulence. The mechanisms of Mn(2+) homeostasis are not fully understood, there are few data regarding the functional and taxonomic diversity of Mn(2+) exporters. Our recent phylogeny of the cation diffusion facilitator (CDF) family of transporters classified the bacterial Mn(2+)-CDF transporters characterized to date, Streptococcus pneumoniae MntE and Deinococcus radiodurans DR1236, into two monophyletic groups. DR1236 was shown to belong to the highly-diverse metal specificity clade VI, together with TtCzrB, a Zn(2+)/Cd(2+) transporter from Thermus thermophilus, the Fe(2+) transporter Sll1263 from Synechocystis sp and eight uncharacterized homologs whose potential Mn(2+)/Zn(2+)/Cd(2+)/Fe(2+) specificities could not be accurately inferred because only eleven proteins were grouped in this clade. A new phylogeny inferred from the alignment of 197 clade VI homologs revealed three novel subfamilies of uncharacterized proteins. Remarkably, one of them contained 91 uncharacterized α-proteobacteria transporters (46% of the protein data set) grouped into a single subfamily. The Mn(2+)/Fe(2+) specificity of this subfamily was proposed through the functional characterization of the Rhizobium etli RHE_CH03072 gene. This gene was upregulated by Mn(2+), Zn(2+), Cd(2+) and Fe(2+) but conferred only Mn(2+) resistance to R. etli. The expression of the RHE_CH03072 gene in an E. coli mntP/zitB/zntA mutant did not relieve either Zn(2+) or Mn(2+) stress but slightly increased its Fe(2+) resistance. These results indicate that the RHE_CH03072 gene, now designated as emfA, encodes for a bacterial Mn(2+)/Fe(2+) resistance CDF protein, having orthologs in more than 60 α-proteobacterial species.

Housekeeping genes essential for pantothenate biosynthesis are plasmid-encoded in Rhizobium etli and Rhizobium leguminosarum.

BACKGROUND: A traditional concept in bacterial genetics states that housekeeping genes, those involved in basic metabolic functions needed for maintenance of the cell, are encoded in the chromosome, whereas genes required for dealing with challenging environmental conditions are located in plasmids. Exceptions to this rule have emerged from genomic sequence data of bacteria with multipartite genomes. The genome sequence of R. etli CFN42 predicts the presence of panC and panB genes clustered together on the 642 kb plasmid p42f and a second copy of panB on plasmid p42e. They encode putative pantothenate biosynthesis enzymes (pantoate-ß-alanine ligase and 3-methyl-2-oxobutanoate hydroxymethyltransferase, respectively). Due to their ubiquitous distribution and relevance in the central metabolism of the cell, these genes are considered part of the core genome; thus, their occurrence in a plasmid is noteworthy. In this study we investigate the contribution of these genes to pantothenate biosynthesis, examine whether their presence in plasmids is a prevalent characteristic of the Rhizobiales with multipartite genomes, and assess the possibility that the panCB genes may have reached plasmids by horizontal gene transfer. RESULTS: Analysis of mutants confirmed that the panC and panB genes located on plasmid p42f are indispensable for the synthesis of pantothenate. A screening of the location of panCB genes among members of the Rhizobiales showed that only R. etli and R. leguminosarum strains carry panCB genes in plasmids. The panCB phylogeny attested a common origin for chromosomal and plasmid-borne panCB sequences, suggesting that the R. etli and R. leguminosarum panCB genes are orthologs rather than xenologs. The panCB genes could not totally restore the ability of a strain cured of plasmid p42f to grow in minimal medium. CONCLUSIONS: This study shows experimental evidence that core panCB genes located in plasmids of R. etli and R. leguminosarum are indispensable for the synthesis of pantothenate. The unusual presence of panCB genes in plasmids of Rhizobiales may be due to an intragenomic transfer from chromosome to plasmid. Plasmid p42f encodes other functions required for growth in minimal medium. Our results support the hypothesis of cooperation among different replicons for basic cellular functions in multipartite rhizobia genomes.

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.

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

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 (H(2)O(2)). Pre-exposure to a sublethal concentration of H(2)O(2) allowed R. etli to adapt and survive subsequent exposure to higher concentrations of H(2)O(2). 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 H(2)O(2) 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.

AniA regulates reserve polymer accumulation and global protein expression in Rhizobium etli.

Previously, it was reported that the oxidative capacity and ability to grow on carbon sources such as pyruvate and glucose were severely diminished in the Rhizobium etli phaC::OmegaSm(r)/Sp(r) mutant CAR1, which is unable to synthesize poly-beta-hydroxybutyric acid (PHB) (M. A. Cevallos, S. Encarnación, A. Leija, Y. Mora, and J. Mora, J. Bacteriol. 178:1646-1654, 1996). By random Tn5 mutagenesis of the phaC strain, we isolated the mutants VEM57 and VEM58, both of which contained single Tn5 insertions and had recovered the ability to grow on pyruvate or glucose. Nucleotide sequencing of the region surrounding the Tn5 insertions showed that they had interrupted an open reading frame designated aniA based on its high deduced amino acid sequence identity to the aniA gene product of Sinorhizobium meliloti. R. etli aniA was located adjacent to and divergently transcribed from genes encoding the PHB biosynthetic enzymes beta-ketothiolase (PhaA) and acetoacetyl coenzyme A reductase (PhaB). An aniA::Tn5 mutant (VEM5854) was constructed and found to synthesize only 40% of the wild type level of PHB. Both VEM58 and VEM5854 produced significantly more extracellular polysaccharide than the wild type. Organic acid excretion and levels of intracellular reduced nucleotides were lowered to wild-type levels in VEM58 and VEM5854, in contrast to those of strain CAR1, which were significantly elevated. Proteome analysis of VEM58 showed a drastic alteration of protein expression, including the absence of a protein identified as PhaB. We propose that the aniA gene product plays an important role in directing carbon flow in R. etli.