RESUMO
Biotin is a key cofactor of metabolic carboxylases, although many rhizobial strains are biotin auxotrophs. When some of these strains were serially subcultured in minimal medium, they showed diminished growth and increased excretion of metabolites. The addition of biotin, or genetic complementation with biotin synthesis genes resulted in full growth of Rhizobium etli CFN42 and Rhizobium phaseoli CIAT652 strains. Half of rhizobial genomes did not show genes for biotin biosynthesis, but three-quarters had genes for biotin transport. Some strains had genes for an avidin homologue (rhizavidin), a protein with high affinity for biotin but an unknown role in bacteria. A CFN42-derived rhizavidin mutant showed a sharper growth decrease in subcultures, revealing a role in biotin storage. In the search of biotin-independent growth of subcultures, CFN42 and CIAT652 strains with excess aeration showed optimal growth, as they also did, unexpectedly, with the addition of aspartic acid analogues α- and N-methyl aspartate. Aspartate analogues can be sensed by the chemotaxis aspartate receptor Tar. A tar homologue was identified and its mutants showed no growth recovery with aspartate analogues, indicating requirement of the Tar receptor in such a phenotype. Additionally, tar mutants did not recover full growth with excess aeration. A Rubisco-like protein was found to be necessary for growth as the corresponding mutants showed no recovery either with high aeration or aspartate analogues; also, diminished carboxylation was observed. Taken together, our results indicate a route of biotin-independent growth in rhizobial strains that included oxygen, a Tar receptor and a previously uncharacterized Rubisco-like protein.
Assuntos
Rhizobium etli , Rhizobium , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biotina/metabolismo , Receptores de Aminoácido , Rhizobium/genética , Rhizobium/metabolismo , Rhizobium etli/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
Flavonoids excreted by legume roots induce the expression of symbiotically essential nodulation (nod) genes in rhizobia, as well as that of specific protein export systems. In the bean microsymbiont Rhizobium etli CE3, nod genes are induced by the flavonoid naringenin. In this study, we identified 693 proteins in the exoproteome of strain CE3 grown in minimal medium with or without naringenin, with 101 and 100 exoproteins being exclusive to these conditions, respectively. Four hundred ninety-two (71%) of the extracellular proteins were found in both cultures. Of the total exoproteins identified, nearly 35% were also present in the intracellular proteome of R. etli bacteroids, 27% had N-terminal signal sequences and a significant number had previously demonstrated or possible novel roles in symbiosis, including bacterial cell surface modification, adhesins, proteins classified as MAMPs (microbe-associated molecular patterns), such as flagellin and EF-Tu, and several normally cytoplasmic proteins as Ndk and glycolytic enzymes, which are known to have extracellular "moonlighting" roles in bacteria that interact with eukaryotic cells. It is noteworthy that the transmembrane ß (1,2) glucan biosynthesis protein NdvB, an essential symbiotic protein in rhizobia, was found in the R. etli naringenin-induced exoproteome. In addition, potential binding sites for two nod-gene transcriptional regulators (NodD) occurred somewhat more frequently in the promoters of genes encoding naringenin-induced exoproteins in comparison to those ofexoproteins found in the control condition.
Assuntos
Proteínas de Bactérias/metabolismo , Flavanonas/farmacologia , Nodulação/genética , Proteoma/metabolismo , Rhizobium etli/genética , Rhizobium etli/metabolismo , Proteínas de Bactérias/genética , Fabaceae/microbiologia , Regulação da Expressão Gênica , Fixação de Nitrogênio/genética , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Proteoma/genética , Simbiose/genéticaRESUMO
Establishment of nitrogen-fixing symbiosis requires the recognition of rhizobial molecules to initiate the development of nodules. Using transcriptional profiling of roots inoculated with mutant strains defective in the synthesis of Nod Factor (NF), exopolysaccharide (EPS), or lipopolysaccharide (LPS), we identified 2,606 genes from common bean (Phaseolus vulgaris) that are differentially regulated at early stages of its interaction with Rhizobium etli. Many transcription factors from different families are modulated by NF, EPS, and LPS in different combinations, suggesting that the plant response depends on the integration of multiple signals. Some receptors identified as differentially expressed constitute excellent candidates to participate in signal perception of molecules derived from the bacteria. Several components of the ethylene signal response, a hormone that plays a negative role during early stages of the process, were down-regulated by NF and LPS. In addition, genes encoding proteins involved in small RNA-mediated gene regulation were regulated by these signal molecules, such as Argonaute7, a specific component of the trans-acting short interfering RNA3 pathway, an RNA-dependent RNA polymerase, and an XH/XP domain-containing protein, which is part of the RNA-directed DNA methylation. Interestingly, a number of genes encoding components of the circadian central oscillator were down-regulated by NF and LPS, suggesting that a root circadian clock is adjusted at early stages of symbiosis. Our results reveal a complex interaction of the responses triggered by NF, LPS, and EPS that integrates information of the signals present in the surface or secreted by rhizobia.
Assuntos
Phaseolus/genética , Phaseolus/microbiologia , Rhizobium etli/fisiologia , Transcriptoma , Relógios Circadianos/genética , Regulação da Expressão Gênica de Plantas , Lipopolissacarídeos/metabolismo , Mutação , Phaseolus/metabolismo , Interferência de RNA , Reprodutibilidade dos Testes , Rhizobium etli/genética , Rhizobium etli/metabolismo , Nódulos Radiculares de Plantas/metabolismo , Nódulos Radiculares de Plantas/microbiologia , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
Rhizobium etli CFN42 has a multipartite genome composed of one chromosome and six large plasmids with low copy numbers, all belonging to the repABC plasmid family. All elements essential for replication and segregation of these plasmids are encoded within the repABC operon. RepA and RepB direct plasmid segregation and are involved in the transcriptional regulation of the operon, and RepC is the initiator protein of the plasmid. Here we show that in addition to RepA (repressor) and RepB (corepressor), full transcriptional repression of the operon located in the symbiotic plasmid (pRetCFN42d) of this strain requires parS, the centromere-like sequence, and the operator sequence. However, the co-expression of RepA and RepB is sufficient to induce the displacement of the parental plasmid. RepA is a Walker-type ATPase that self associates in vivo and in vitro and binds specifically to the operator region in its RepA-ADP form. In contrast, RepA-ATP is capable of binding to non-specific DNA. RepA and RepB form high molecular weight DNA-protein complexes in the presence of ATP and ADP. RepA carrying ATP-pocket motif mutations induce full repression of the repABC operon without the participation of RepB and parS. These mutants specifically bind the operator sequence in their ATP or ADP bound forms. In addition, their expression in trans exerts plasmid incompatibility against the parental plasmid. RepA and RepB expressed in trans induce plasmid incompatibility because of their ability to repress the repABC operon and not only by their capacity to distort the plasmid segregation process.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Óperon , Plasmídeos , Rhizobium etli/genética , Difosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Dados de Sequência Molecular , Mutação , Rhizobium etli/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição GênicaRESUMO
Transcriptional control of the fixK gene in Rhizobium etli and R. leguminosarum bv. viciae is governed by a two-component signal transduction system that diverts from the conventional FixL-FixJ cascade that occurs in model rhizobia. Although a fixL gene, encoding a hybrid histidine kinase (hFixL), is present in R. etli, no fixJ, the cognate response regulator, has been identified. In this work, we present evidence that the pRet42f-located open reading frame RHE_PF00530 (fxkR) encodes a novel response regulator indispensable for fixKf activation under microaerobic growth. Moreover, results from complementation assays demonstrate that the activation of fixKf expression requires the presence of both hFixL and FxkR, and that the fxkR ortholog from R. leguminosarum bv. viciae is able to substitute for FxkR transcriptional control in R. etli. In addition, in these two organisms, hFixL- and FxkR-related proteins were identified in other bacteria, located in close proximity to a fixK-related gene. Using reporter fusions, site-directed mutagenesis, and electrophoretic mobility shift assays, we identified the FxkR binding site upstream from the transcriptional start site of fixKf. Similar to our previous observations for fixL and fixKf mutants, a null mutation in fxkR does not affect the symbiotic effectiveness of the strain. Thus, our findings reveal that FxkR is the long-standing missing key regulator that allows the transduction of the microaerobic signal for the activation of the FixKf regulon.
Assuntos
Proteínas de Bactérias/metabolismo , Rhizobium etli/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Mutagênese Sítio-Dirigida , Rhizobium etli/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Bacterial nitrogen fixation is the biological process by which atmospheric nitrogen is uptaken by bacteroids located in plant root nodules and converted into ammonium through the enzymatic activity of nitrogenase. In practice, this biological process serves as a natural form of fertilization and its optimization has significant implications in sustainable agricultural programs. Currently, the advent of high-throughput technology supplies with valuable data that contribute to understanding the metabolic activity during bacterial nitrogen fixation. This undertaking is not trivial, and the development of computational methods useful in accomplishing an integrative, descriptive and predictive framework is a crucial issue to decoding the principles that regulated the metabolic activity of this biological process. RESULTS: In this work we present a systems biology description of the metabolic activity in bacterial nitrogen fixation. This was accomplished by an integrative analysis involving high-throughput data and constraint-based modeling to characterize the metabolic activity in Rhizobium etli bacteroids located at the root nodules of Phaseolus vulgaris (bean plant). Proteome and transcriptome technologies led us to identify 415 proteins and 689 up-regulated genes that orchestrate this biological process. Taking into account these data, we: 1) extended the metabolic reconstruction reported for R. etli; 2) simulated the metabolic activity during symbiotic nitrogen fixation; and 3) evaluated the in silico results in terms of bacteria phenotype. Notably, constraint-based modeling simulated nitrogen fixation activity in such a way that 76.83% of the enzymes and 69.48% of the genes were experimentally justified. Finally, to further assess the predictive scope of the computational model, gene deletion analysis was carried out on nine metabolic enzymes. Our model concluded that an altered metabolic activity on these enzymes induced different effects in nitrogen fixation, all of these in qualitative agreement with observations made in R. etli and other Rhizobiaceas. CONCLUSIONS: In this work we present a genome scale study of the metabolic activity in bacterial nitrogen fixation. This approach leads us to construct a computational model that serves as a guide for 1) integrating high-throughput data, 2) describing and predicting metabolic activity, and 3) designing experiments to explore the genotype-phenotype relationship in bacterial nitrogen fixation.
Assuntos
Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Fixação de Nitrogênio/fisiologia , Phaseolus/microbiologia , Rhizobium etli/fisiologia , Nódulos Radiculares de Plantas/microbiologia , Biologia de Sistemas/métodos , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Espectrometria de Massas , Redes e Vias Metabólicas/genética , Análise em Microsséries , Fixação de Nitrogênio/genética , Proteômica/métodos , Rhizobium etli/genética , Rhizobium etli/metabolismoRESUMO
In this communication we reported the study of a cation channel present in the cytoplasmic membrane of the nitrogen fixing bacterium Rhizobium etli. Inner-membrane (IM) vesicles were purified and fused into planar lipid bilayers (PLBs), under voltage clamp conditions. We have found that fusion of IM-enriched vesicle fractions with these model membranes leads, mainly (>30% of 46 experiments), to the reconstitution of high-conductance channels. Following this strategy, the activity of a channel with main open conductance of 198 pS, in symmetrical 100 mM KCl, was recorded. The single-channel conductance increase to 653 pS in the presence of a 5:1 (cis to trans) gradient of KCl. The channel exhibits voltage dependency and a weak selectivity for cations showing a permeability ratios of P (Rb)/P (K) = 0.96, P (Na)/P (K) = 0.07, and a conductance ratio of gamma(Rb)/gamma(K) = 1.1. The channel here characterized represents a previously undescribed Rhizobium channel although its precise role in rhizobial physiology remains yet to be determined.
Assuntos
Proteínas de Bactérias/metabolismo , Canais Iônicos/metabolismo , Rhizobium etli/fisiologia , Cátions/metabolismo , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Rhizobium etli/metabolismo , Microbiologia do SoloRESUMO
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.
Assuntos
Proteínas de Bactérias/fisiologia , Pressão Osmótica/fisiologia , Estresse Oxidativo/genética , Rhizobium etli/fisiologia , Fator sigma/fisiologia , Proteínas de Bactérias/genética , Sequência de Bases , Fabaceae/microbiologia , Regulação Bacteriana da Expressão Gênica , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Estresse Oxidativo/fisiologia , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rhizobium etli/genética , Rhizobium etli/crescimento & desenvolvimento , Rhizobium etli/metabolismo , Homologia de Sequência do Ácido Nucleico , Fator sigma/genéticaRESUMO
Because Rhizobium etli CE3 is normally dependent on an external source of biotin and lacks orthodox biotin biosynthesis genes, we undertook an analysis of biotin uptake in this organism. By complementation of a Sinorhizobium meliloti bioM mutant we isolated an R. etli chromosomal region encoding homologs of the S. meliloti bioMNB genes, whose products have been implicated in intracellular biotin retention in that organism. Disruption of the R. etli bioM resulted in a mutant which took up biotin at a lower rate and accumulated significantly less biotin than the wild type. As in S. meliloti, the R. etli bioMN gene-products resemble the ATPase and permease components, respectively, of an ABC-type transporter. The bioB gene product is in fact similar to members of the BioY family, which has been postulated to function in biotin transport, and we refer to this gene as bioY. An R. etli bioY mutant exhibited lower biotin uptake than the wild-type, providing the first experimental evidence for a role of BioY in biotin transport. We show that the bioMNY operon is transcriptionally repressed by biotin. An analysis of the competitiveness of the wild-type strain versus the bioM mutant showed that the mutant had a diminished capacity to form nodules on bean plants.
Assuntos
Biotina/metabolismo , Óperon , Rhizobium etli/genética , Rhizobium etli/metabolismo , Adenosina Trifosfatases/genética , Clonagem Molecular , DNA Bacteriano , Repressão Enzimática , Regulação Bacteriana da Expressão Gênica , Teste de Complementação Genética , Proteínas de Membrana Transportadoras/genética , Dados de Sequência Molecular , Fixação de Nitrogênio , Transporte Proteico , Análise de Sequência de DNA , Sinorhizobium meliloti/genética , Simportadores/genéticaRESUMO
Plasmid p42a from Rhizobium etli CFN42 is self-transmissible and indispensable for conjugative transfer of the symbiotic plasmid (pSym). Most pSym transconjugants also inherit p42a. pSym transconjugants that lack p42a always contain recombinant pSyms, which we designated RpSyms*. RpSyms* do not contain some pSym segments and instead have p42a sequences, including the replication and transfer regions. These novel recombinant plasmids are compatible with wild-type pSym, incompatible with p42a, and self-transmissible. The symbiotic features of derivatives simultaneously containing a wild-type pSym and an RpSym* were analyzed. Structural analysis of 10 RpSyms* showed that 7 shared one of the two pSym-p42a junctions. Sequencing of this common junction revealed a 53-bp region that was 90% identical in pSym and p42a, including a 5-bp central region flanked by 9- to 11-bp inverted repeats reminiscent of bacterial and phage attachment sites. A gene encoding an integrase-like protein (intA) was localized downstream of the attachment site on p42a. Mutation or the absence of intA abolished pSym transfer from a recA mutant donor. Complementation with the wild-type intA gene restored transfer of pSym. We propose that pSym-p42a cointegration is required for pSym transfer; cointegration may be achieved either through homologous recombination among large reiterated sequences or through IntA-mediated site-specific recombination between the attachment sites. Cointegrates formed through the site-specific system but resolved through RecA-dependent recombination or vice versa generate RpSyms*. A site-specific recombination system for plasmid cointegration is a novel feature of these large plasmids and implies that there is unique regulation which affects the distribution of pSym in nature due to the role of the cointegrate in conjugative transfer.