Araceli Dávalos

Differential regulation of fixN-reiterated genes in Rhizobium etli by a novel fixL-fixK cascade.

Among the complexities in the regulation of nitrogen fixation in the Rhizobiaceae are reiteration of regulatory components as well as variant roles for each component between species. For Rhizobium etli CFN42, we reported that the symbiotic plasmid (pCFN42d) contains a key regulatory gene (fixKd) and genes for a symbiotic cytochrome oxidase (fixNOQPd). Here we discuss the occurrence of reiteration of these genes (fixKf and fixNOQPf) and the finding of an unusual fixL homolog on a plasmid previously considered cryptic (pCFN42f). The structure of the deduced FixL polypeptide is suggestive of a fusion of the receiver and transmitter modules of a two-component regulatory system as described in R. leguminosarum bv. viciae VF39. Gene fusion analysis, coupled with mutation of each regulatory element, revealed that free-living expression of FixKf was dependent fully on FixL. In contrast, synthesis of FixKd was not detected under the conditions tested. The FixKf protein is needed for microaerobic expression of both fixN reiterations, whereas the FixKd protein appears to be dispensable. Interestingly, expression of the fixN reiterations exhibits a differential dependence for FixL, where transcription of fixNf was suppressed in the absence of FixL but expression of fixNd still showed significant levels. This suggests the existence of a FixL-independent mechanism for expression of the fixNd reiteration. Surprisingly, mutations in fixL, fixKd, or fixKf (either singly or in combination) did not alter symbiotic effectiveness. A mutation in fixNd (but not in fixNf) was, however, severely affected, indicating a differential role for these reiterations in nitrogen fixation.

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.

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.

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

BackgroundA 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-β-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.ResultsAnalysis 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.ConclusionsThis 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.

Characterization of Rhizobium phaseoli Sym plasmid regions involved in nodule morphogenesis and host‐range specificity

Two nodulation regions from the symbiotic plasmid (pSym) of Rhizobium phaseoil CE‐3 were identified. The two regions were contained in overlapping cosmids pSM927 and pSM991. These cosmids, in a R phaseoli pSym‐cured strain background, induced ineffective nodules on Phaseolus vulgaris roots.

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 β–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.

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

BackgroundRhizobia 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).ResultsRhizobial 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.ConclusionsSeeds 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.

Role of the Pyruvate Dehydrogenase (PDH) and Pyruvate Formate Lyase (PFL) in Rhizobium elti Symbiosis

We have shown that R. etli CE3 develops a fermentation-like response (Encarnacion et al. 1995). Because of biotin or thiamine added to minimal medium (MM) favors an aerobic metabolism, possibly there is a down regulation of the synthesis of these vitamins that affects PYC and PDH activities, driving the bacteria to fermentative metabolism. In E. coli, the production of acetylCoA in aerobic conditions is through the PDH enzyme, however, during anaerobiosis pyruvate formate lyase (PFL) provides acetylCoA.

Characterization of R. etli Mutants in the Purine-Thiamin Metabolism Suggest That 5-Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) is a Negative Effector of Symbiotic Cytochrome Terminal Oxidase cbb3 Production

Two Rhizobium etli Tn5mob -induced mutants (CFN035 and CFN037) exhibited enhanced capacity to oxidize N,N,N′,N′,tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase. Sequence of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl-transferase enzyme (purF), catalyzing the first step of the purine biosynthetic pathway (1). In CFN037 the Tn5mob insertion was located in the promoter region of thethiCOGE gene cluster and promotes a constitutive expression of thiC (thiC C mutant). 4-methyl-5-(s-hydroxyethyl)thiazole monophosphate (THZ-P) and 4-amino-5-hydroxymethylpyrimidine pyrophosphate (HMP-P), are coupled to form thiamin monophosphate, which is phosphorylated to make thiamin pyrophosphate. ThiC from R. etli shared significant homology with thiC from E. coli which is involved in the synthesis of HMP from the purine intermediate 5-Aminoimidazole-ribonucleotide. The second ORF of 327 residues is the product of a novel gene which is denoted as thiO. Analysis of the protein sequence suggests that ThiO catalyzes the oxidative deamination of some intermediate of thiamin biosynthesis. ThiG and ThiE from R. etli shared significant homology with ThiG and thiE from E. coli which are involved in the synthesis of THZ and in the condensation of HMP-P with THZ-P respectively. CFN035 and CFN037 produced the cbb 3 terminal oxidase as did the wild-type R. etli strain expressing the B. japonicum fixNOQP genes, which code for the symbiotic cbb 3 terminal oxidase. A blockade in the first step of the purine biosynthetic pathway and the constitutive expression of thiC would lower the concentration of several metabolites of the purine biosynthetic pathway. In order to identify the possible metabolic effector involved in cbb 3, production, the expression of a R. etli fixN-lacZ gene fusion was measured in several mutants affected in different steps of the purine biosynthetic pathway.

Cloning, Sequencing and Regulation of Catalase-Peroxidase of Rhizobium etli

The growth of R. etli is impaired when subcultured from rich to minimal medium (MM) and this unbalanced growth is involved with a change from aerobic to fermentative metabolism and is characterized by poly-β-hydroxybutyrate accumulation, cell aggregation and a drastic reduction in the activity of several enzymes of the TCA cycle (Encarnacion et al., 1995 J. Bacteriol. 177:3058–3066). Cell agregation, in addition to imposing a microaerobic enviroment and fermentative state, is also responsible for affording protection agains hidrogen peroxide (H2O2) to R. etli.