Genomic studies of nitrogen-fixing rhizobial strains from Phaseolus vulgaris seeds and nodules.

BACKGROUND: Rhizobia are soil bacteria that establish symbiotic relationships with legumes and fix nitrogen in root nodules. We recently reported that several nitrogen-fixing rhizobial strains, belonging to Rhizobium phaseoli, R. trifolii, R. grahamii and Sinorhizobium americanum, were able to colonize Phaseolus vulgaris (common bean) seeds. To gain further insight into the traits that support this ability, we analyzed the genomic sequences and proteomes of R. phaseoli (CCGM1) and S. americanum (CCGM7) strains from seeds and compared them with those of the closely related strains CIAT652 and CFNEI73, respectively, isolated only from nodules. RESULTS: In a fine structural study of the S. americanum genomes, the chromosomes, megaplasmids and symbiotic plasmids were highly conserved and syntenic, with the exception of the smaller plasmid, which appeared unrelated. The symbiotic tract of CCGM7 appeared more disperse, possibly due to the action of transposases. The chromosomes of seed strains had less transposases and strain-specific genes. The seed strains CCGM1 and CCGM7 shared about half of their genomes with their closest strains (3353 and 3472 orthologs respectively), but a large fraction of the rest also had homology with other rhizobia. They contained 315 and 204 strain-specific genes, respectively, particularly abundant in the functions of transcription, motility, energy generation and cofactor biosynthesis. The proteomes of seed and nodule strains were obtained and showed a particular profile for each of the strains. About 82 % of the proteins in the comparisons appeared similar. Forty of the most abundant proteins in each strain were identified; these proteins in seed strains were involved in stress responses and coenzyme and cofactor biosynthesis and in the nodule strains mainly in central processes. Only 3 % of the abundant proteins had hypothetical functions. CONCLUSIONS: Functions that were enriched in the genomes and proteomes of seed strains possibly participate in the successful occupancy of the new niche. The genome of the strains had features possibly related to their presence in the seeds. This study helps to understand traits of rhizobia involved in seed adaptation.

High-throughput transcriptome sequencing and comparative analysis of Escherichia coli and Schizosaccharomyces pombe in respiratory and fermentative growth.

In spite of increased complexity in eukaryotes compared to prokaryotes, several basic metabolic and regulatory processes are conserved. Here we explored analogies in the eubacteria Escherichia coli and the unicellular fission yeast Schizosaccharomyces pombe transcriptomes under two carbon sources: 2% glucose; or a mix of 2% glycerol and 0.2% sodium acetate using the same growth media and growth phase. Overall, twelve RNA-seq libraries were constructed. A total of 593 and 860 genes were detected as differentially expressed for E. coli and S. pombe, respectively, with a log2 of the Fold Change ≥ 1 and False Discovery Rate ≤ 0.05. In aerobic glycolysis, most of the expressed genes were associated with cell proliferation in both organisms, including amino acid metabolism and glycolysis. In contrast in glycerol/acetate condition, genes related to flagellar assembly and membrane proteins were differentially expressed such as the general transcription factors fliA, flhD, flhC, and flagellum assembly genes were detected in E. coli, whereas in S. pombe genes for hexose transporters, integral membrane proteins, galactose metabolism, and ncRNAs related to cellular stress were overexpressed. In general, our study shows that a conserved "foraging behavior" response is observed in these eukaryotic and eubacterial organisms in gluconeogenic carbon sources.

Transcriptomic analysis of Burkholderia cenocepacia CEIB S5-2 during methyl parathion degradation.

Methyl parathion (MP) is a highly toxic organophosphorus pesticide associated with water, soil, and air pollution events. The identification and characterization of microorganisms capable of biodegrading pollutants are an important environmental task for bioremediation of pesticide impacted sites. The strain Burkholderia cenocepacia CEIB S5-2 is a bacterium capable of efficiently hydrolyzing MP and biodegrade p-nitrophenol (PNP), the main MP hydrolysis product. Due to the high PNP toxicity over microbial living forms, the reports on bacterial PNP biodegradation are scarce. According to the genomic data, the MP- and PNP-degrading ability observed in B. cenocepacia CEIB S5-2 is related to the presence of the methyl parathion-degrading gene (mpd) and the gene cluster pnpABA'E1E2FDC, which include the genes implicated in the PNP degradation. In this work, the transcriptomic analysis of the strain in the presence of MP revealed the differential expression of 257 genes, including all genes implicated in the PNP degradation, as well as a set of genes related to the sensing of environmental changes, the response to stress, and the degradation of aromatic compounds, such as translational regulators, membrane transporters, efflux pumps, and oxidative stress response genes. These findings suggest that these genes play an important role in the defense against toxic effects derived from the MP and PNP exposure. Therefore, B. cenocepacia CEIB S5-2 has a great potential for application in pesticide bioremediation approaches due to its biodegradation capabilities and the differential expression of genes for resistance to MP and PNP.

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.

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.

Transcriptional analysis reveals the metabolic state of Burkholderia zhejiangensis CEIB S4-3 during methyl parathion degradation.

Burkholderia zhejiangensis CEIB S4-3 has the ability to degrade methyl parathion (MP) and its main hydrolysis byproduct p-nitrophenol (PNP). According to genomic data, several genes related with metabolism of MP and PNP were identified in this strain. However, the metabolic state of the strain during the MP degradation has not been evaluated. In the present study, we analyzed gene expression changes during MP hydrolysis and PNP degradation through a transcriptomic approach. The transcriptional analysis revealed differential changes in the expression of genes involved in important cellular processes, such as energy production and conversion, transcription, amino acid transport and metabolism, translation, ribosomal structure and biogenesis, among others. Transcriptomic data also exhibited the overexpression of both PNP-catabolic gene clusters (pnpABA'E1E2FDC and pnpE1E2FDC) present in the strain. We found and validated by quantitative reverse transcription polymerase chain reaction the expression of the methyl parathion degrading gene, as well as the genes responsible for PNP degradation contained in two clusters. This proves the MP degradation pathway by the strain tested in this work. The exposure to PNP activates, in the first instance, the expression of the transcriptional regulators multiple antibiotic resistance regulator and Isocitrate Lyase Regulator (IclR), which are important in the regulation of genes from aromatic compound catabolism, as well as the expression of genes that encode transporters, permeases, efflux pumps, and porins related to the resistance to multidrugs and other xenobiotics. In the presence of the pesticide, 997 differentially expressed genes grouped in 104 metabolic pathways were observed. This report is the first to describe the transcriptomic analysis of a strain of B. zhejiangensis during the biodegradation of PNP.

Eco-evolutionary feedbacks drive species interactions.

In the biosphere, many species live in close proximity and can thus interact in many different ways. Such interactions are dynamic and fall along a continuum between antagonism and cooperation. Because interspecies interactions are the key to understanding biological communities, it is important to know how species interactions arise and evolve. Here, we show that the feedback between ecological and evolutionary processes has a fundamental role in the emergence and dynamics of species interaction. Using a two-species artificial community, we demonstrate that ecological processes and rapid evolution interact to influence the dynamics of the symbiosis between a eukaryote (Saccharomyces cerevisiae) and a bacterium (Rhizobium etli). The simplicity of our experimental design enables an explicit statement of causality. The niche-constructing activities of the fungus were the key ecological process: it allowed the establishment of a commensal relationship that switched to ammensalism and provided the selective conditions necessary for the adaptive evolution of the bacteria. In this latter state, the bacterial population radiates into more than five genotypes that vary with respect to nutrient transport, metabolic strategies and global regulation. Evolutionary diversification of the bacterial populations has strong effects on the community; the nature of interaction subsequently switches from ammensalism to antagonism where bacteria promote yeast extinction. Our results demonstrate the importance of the evolution-to-ecology pathway in the persistence of interactions and the stability of communities. Thus, eco-evolutionary dynamics have the potential to transform the structure and functioning of ecosystems. Our results suggest that these dynamics should be considered to improve our understanding of beneficial and detrimental host-microbe interactions.

Identification of putative ortholog gene blocks involved in gestant and lactating mammary gland development: a rodent cross-species microarray transcriptomics approach.

The mammary gland (MG) undergoes functional and metabolic changes during the transition from pregnancy to lactation, possibly by regulation of conserved genes. The objective was to elucidate orthologous genes, chromosome clusters and putative conserved transcriptional modules during MG development. We analyzed expression of 22,000 transcripts using murine microarrays and RNA samples of MG from virgin, pregnant, and lactating rats by cross-species hybridization. We identified 521 transcripts differentially expressed; upregulated in early (78%) and midpregnancy (89%) and early lactation (64%), but downregulated in mid-lactation (61%). Putative orthologous genes were identified. We mapped the altered genes to orthologous chromosomal locations in human and mouse. Eighteen sets of conserved genes associated with key cellular functions were revealed and conserved transcription factor binding site search entailed possible coregulation among all eight block sets of genes. This study demonstrates that the use of heterologous array hybridization for screening of orthologous gene expression from rat revealed sets of conserved genes arranged in chromosomal order implicated in signaling pathways and functional ontology. Results demonstrate the utilization power of comparative genomics and prove the feasibility of using rodent microarrays to identification of putative coexpressed orthologous genes involved in the control of human mammary gland development.

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

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.

Engineering the nifH promoter region and abolishing poly-beta-hydroxybutyrate accumulation in Rhizobium etli enhance nitrogen fixation in symbiosis with Phaseolus vulgaris.

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-beta-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.