Yolanda Mora

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.

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

ABSTRACT 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.

Diversification of DNA Sequences in the Symbiotic Genome of Rhizobium etli

Bacteria of the genus Rhizobium and related genera establish nitrogen-fixing symbioses with the roots of leguminous plants. The genetic elements that participate in the symbiotic process are usually compartmentalized in the genome, either as independent replicons (symbiotic plasmids) or as symbiotic regions or islands in the chromosome. The complete nucleotide sequence of the symbiotic plasmid of Rhizobium etli model strain CFN42, symbiont of the common bean plant, has been reported. To better understand the basis of DNA sequence diversification of this symbiotic compartment, we analyzed the distribution of single-nucleotide polymorphisms in homologous regions from different Rhizobium etli strains. The distribution of polymorphisms is highly asymmetric in each of the different strains, alternating regions containing very few changes with regions harboring an elevated number of substitutions. The regions showing high polymorphism do not correspond with discrete genetic elements and are not the same in the different strains, indicating that they are not hypervariable regions of functional genes. Most interesting, some highly polymorphic regions share exactly the same nucleotide substitutions in more than one strain. Furthermore, in different regions of the symbiotic compartment, different sets of strains share the same substitutions. The data indicate that the majority of nucleotide substitutions are spread in the population by recombination and that the contribution of new mutations to polymorphism is relatively low. We propose that the horizontal transfer of homologous DNA segments among closely related organisms is a major source of genomic diversification.

Engineering the nifH Promoter Region and Abolishing Poly-β-Hydroxybutyrate Accumulation in Rhizobium etli Enhance Nitrogen Fixation in Symbiosis with Phaseolus vulgaris

ABSTRACT Rhizobium etli, as well as some other rhizobia, presents nitrogenase reductase (nifH) gene reiterations. Several R. etli strains studied in this laboratory showed a unique organization and contained two complete nifHDK operons (copies a and b) and a truncated nifHD operon (copy c). Expression analysis of lacZ fusion demonstrated that copies a and b in strain CFN42 are transcribed at lower levels than copy c, although this copy has no discernible role during nitrogen fixation. To increase nitrogenase production, we constructed a chimeric nifHDK operon regulated by the strong nifHc promoter sequence and expressed it in symbiosis with the common bean plant (Phaseolus vulgaris), either cloned on a stably inherited plasmid or incorporated into the symbiotic plasmid (pSym). Compared with the wild-type strain, strains with the nitrogenase overexpression construction assayed in greenhouse experiments had, increased nitrogenase activity (58% on average), increased plant weight (32% on average), increased nitrogen content in plants (15% at 32 days postinoculation), and most importantly, higher seed yield (36% on average), higher nitrogen content (25%), and higher nitrogen yield (72% on average) in seeds. Additionally, expression of the chimeric nifHDK operon in a poly-β-hydroxybutyrate-negative R. etli strain produced an additive effect in enhancing symbiosis. To our knowledge, this is the first report of increased seed yield and nutritional content in the common bean obtained by using only the genetic material already present in Rhizobium.

Nitrogen regulation of glutamine synthetase in Neurospora crassa.

A higher activity of glutamine synthetase (EC 6.3.1.2) was found in Neurospora crassa when NH4+ was limiting as nitrogen source than when glutamate was limiting. When glutamate, glutamine or NH4+ were in excess, a lower activity was found. Immunological titration and sucrose gradient sedimentation of the enzyme established that under all these conditions enzyme activity corresponded to enzyme concentration and that the octamer was the predominant oligomeric form. When N. crassa was shifted from nitrogen-limiting substrates to excess product as nitrogen source, the concentration of glutamine synthetase was adjusted with kinetics that closely followed dilution by growth. When grown on limiting amounts of glutamate, a lower oligomer was present in addition to the octameric form of the enzyme. When the culture was shifted to excess NH4+, glutamine accululated at a high rate; nevertheless, there was only a slow decrease in enzyme activity and no modification of the oligomeric pattern.

Systems biology of bacterial nitrogen fixation: High-throughput technology and its integrative description with constraint-based modeling

BackgroundBacterial 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.ResultsIn 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.ConclusionsIn 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.

Regulation and Symbiotic Role of nirK and norC Expression in Rhizobium etli

Rhizobium etli CFN42 is unable to use nitrate for respiration and lacks nitrate reductase activity as well as the nap or nar genes encoding respiratory nitrate reductase. However, genes encoding proteins closely related to denitrification enzymes, the norCBQD gene cluster and a novel nirKnirVnnrRnnrU operon are located on pCFN42f. In this study, we carried out a genetic and functional characterization of the reductases encoded by the R. etli nirK and norCB genes. By gene fusion expression analysis in free-living conditions, we determined that R. etli regulates its response to nitric oxide through NnrR via the microaerobic expression mediated by FixKf. Interestingly, expression of the norC and nirK genes displays a different level of dependence for NnrR. A null mutation in nnrR causes a drastic drop in the expression of norC, while nirK still exhibits significant expression. A thorough analysis of the nirK regulatory region revealed that this gene is under both positive and negative regulation. Functional analysis carried out in this work demonstrated that reduction of nitrite and nitric oxide in R. etli requires the reductase activities encoded by the norCBQD and nirK genes. Levels of nitrosylleghemoglobin complexes in bean plants exposed to nitrate are increased in a norC mutant but decreased in a nirK mutant. The nitrate-induced decline in nitrogenase-specific activity observed in both the wild type and the norC mutant was not detected in the nirK mutant. This data indicate that bacterial nitrite reductase is an important contributor to the formation of NO in bean nodules in response to nitrate.

Novel reiterated Fnr-type proteins control the production of the symbiotic terminal oxidase cbb3 in Rhizobium etli CFN42.

Symbiotic nitrogen-fixing bacteria express a terminal oxidase with a high oxygen affinity, the cbb3-type oxidase encoded by the fixNOQP operon. Previously, we have shown that, in Rhizobium etli CFN42, the repeatedfixNOQP operons (fixNOQPd and fixNOQPf) have a differential role in nitrogen fixation. Only the fixNOQPd operon is required for the establishment of an effective symbiosis; microaerobic induction of this operon is under the control of at least three transcriptional regulators, FixKf, FnrNd, and FnrNchr, belonging to the Crp/Fnr family. In this work, we describe two novel Crp/Fnr-type transcriptional regulators (StoRd and StoRf, symbiotic terminal oxidase regulators) that play differential roles in the control of key genes for nitrogen fixation. Mutations either in stoRd or stoRf enhance the microaerobic expression of both fixNOQP reiterations, increasing also the synthesis of the cbb3-type oxidase in nodules. Despite their structural similarity, a differential role of these genes was also revealed, since a mutation in stoRd but not in stoRf enhanced both the expression of fixKf and the nitrogen-fixing capacity of R. etli CFN42.

Nitrogen-Fixing Rhizobial Strains Isolated from Common Bean Seeds: Phylogeny, Physiology, and Genome Analysis

ABSTRACT Rhizobial bacteria are commonly found in soil but also establish symbiotic relationships with legumes, inhabiting the root nodules, where they fix nitrogen. Endophytic rhizobia have also been reported in the roots and stems of legumes and other plants. We isolated several rhizobial strains from the nodules of noninoculated bean plants and looked for their provenance in the interiors of the seeds. Nine isolates were obtained, covering most known bean symbiont species, which belong to the Rhizobium and Sinorhizobium groups. The strains showed several large plasmids, except for a Sinorhizobium americanum isolate. Two strains, one Rhizobium phaseoli and one S. americanum strain, were thoroughly characterized. Optimal symbiotic performance was observed for both of these strains. The S. americanum strain showed biotin prototrophy when subcultured, as well as high pyruvate dehydrogenase (PDH) activity, both of which are key factors in maintaining optimal growth. The R. phaseoli strain was a biotin auxotroph, did not grow when subcultured, accumulated a large amount of poly-β-hydroxybutyrate, and exhibited low PDH activity. The physiology and genomes of these strains showed features that may have resulted from their lifestyle inside the seeds: stress sensitivity, a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, a homocitrate synthase (usually present only in free-living diazotrophs), a hydrogenase uptake cluster, and the presence of prophages. We propose that colonization by rhizobia and their presence in Phaseolus seeds may be part of a persistence mechanism that helps to retain and disperse rhizobial strains.

Functional Modules, Structural Topology, and Optimal Activity in Metabolic Networks

Modular organization in biological networks has been suggested as a natural mechanism by which a cell coordinates its metabolic strategies for evolving and responding to environmental perturbations. To understand how this occurs, there is a need for developing computational schemes that contribute to integration of genomic-scale information and assist investigators in formulating biological hypotheses in a quantitative and systematic fashion. In this work, we combined metabolome data and constraint-based modeling to elucidate the relationships among structural modules, functional organization, and the optimal metabolic phenotype of Rhizobium etli, a bacterium that fixes nitrogen in symbiosis with Phaseolus vulgaris. To experimentally characterize the metabolic phenotype of this microorganism, we obtained the metabolic profile of 220 metabolites at two physiological stages: under free-living conditions, and during nitrogen fixation with P. vulgaris. By integrating these data into a constraint-based model, we built a refined computational platform with the capability to survey the metabolic activity underlying nitrogen fixation in R. etli. Topological analysis of the metabolic reconstruction led us to identify modular structures with functional activities. Consistent with modular activity in metabolism, we found that most of the metabolites experimentally detected in each module simultaneously increased their relative abundances during nitrogen fixation. In this work, we explore the relationships among topology, biological function, and optimal activity in the metabolism of R. etli through an integrative analysis based on modeling and metabolome data. Our findings suggest that the metabolic activity during nitrogen fixation is supported by interacting structural modules that correlate with three functional classifications: nucleic acids, peptides, and lipids. More fundamentally, we supply evidence that such modular organization during functional nitrogen fixation is a robust property under different environmental conditions.