Project description:The ribonucleases (RNases) E and J are essential in Escherichia coli and Bacillus subtilis, respectively. Sinorhizobium meliloti contains both, the rne gene encoding RNase E and the rnj gene encoding RNase J. The transcriptomes of the S. meliloti Rm2011 wild type, and rne and rnj mutants were compared.
Project description:Sinorhizobium meliloti strain 1021, a nitrogen-fixing, root-nodulating bacterial microsymbiont of alfalfa, has a 3.5 Mbp circular chromosome and two megaplasmids including 1.3 Mbp pSymA carrying nonessential 'accessory' genes for nitrogen fixation (nif), nodulation and host specificity (nod). A related bacterium, psyllid-vectored 'Ca. Liberibacter asiaticus,' is an obligate phytopathogen with a reduced genome that was previously analyzed for genes orthologous to genes on the S. meliloti circular chromosome. In general, proteins encoded by pSymA genes are more similar in sequence alignment to those encoded by S. meliloti chromosomal orthologs than to orthologous proteins encoded by genes carried on the 'Ca. Liberibacter asiaticus' genome. Only two 'Ca. Liberibacter asiaticus' proteins were identified as having orthologous proteins encoded on pSymA but not also encoded on the chromosome of S. meliloti. These two orthologous gene pairs encode a Na(+)/K+ antiporter (shared with intracellular pathogens of the family Bartonellacea) and a Co++, Zn++ and Cd++ cation efflux protein that is shared with the phytopathogen Agrobacterium. Another shared protein, a redox-regulated K+ efflux pump may regulate cytoplasmic pH and homeostasis. The pSymA and 'Ca. Liberibacter asiaticus' orthologs of the latter protein are more highly similar in amino acid alignment compared with the alignment of the pSymA-encoded protein with its S. meliloti chromosomal homolog. About 182 pSymA encoded proteins have sequence similarity (? E-10) with 'Ca. Liberibacter asiaticus' proteins, often present as multiple orthologs of single 'Ca. Liberibacter asiaticus' proteins. These proteins are involved with amino acid uptake, cell surface structure, chaperonins, electron transport, export of bioactive molecules, cellular homeostasis, regulation of gene expression, signal transduction and synthesis of amino acids and metabolic cofactors. The presence of multiple orthologs defies mutational analysis and is consistent with the hypothesis that these proteins may be of particular importance in host/microbe interaction and their duplication likely facilitates their ongoing evolution.
Project description:We present the complete nucleotide sequence of the multipartite genome of Sinorhizobium/Ensifer meliloti GR4, a predominant rhizobial strain in an agricultural field site. The genome (total size, 7.14 Mb) consists of five replicons: one chromosome, two expected symbiotic megaplasmids (pRmeGR4c and pRmeGR4d), and two accessory plasmids (pRmeGR4a and pRmeGR4b).
Project description:BACKGROUND: Sinorhizobium meliloti is a model system for the studies of symbiotic nitrogen fixation. An extensive polymorphism at the genetic and phenotypic level is present in natural populations of this species, especially in relation with symbiotic promotion of plant growth. AK83 and BL225C are two nodule-isolated strains with diverse symbiotic phenotypes; BL225C is more efficient in promoting growth of the Medicago sativa plants than strain AK83. In order to investigate the genetic determinants of the phenotypic diversification of S. meliloti strains AK83 and BL225C, we sequenced the complete genomes for these two strains. RESULTS: With sizes of 7.14 Mbp and 6.97 Mbp, respectively, the genomes of AK83 and BL225C are larger than the laboratory strain Rm1021. The core genome of Rm1021, AK83, BL225C strains included 5124 orthologous groups, while the accessory genome was composed by 2700 orthologous groups. While Rm1021 and BL225C have only three replicons (Chromosome, pSymA and pSymB), AK83 has also two plasmids, 260 and 70 Kbp long. We found 65 interesting orthologous groups of genes that were present only in the accessory genome, consequently responsible for phenotypic diversity and putatively involved in plant-bacterium interaction. Notably, the symbiosis inefficient AK83 lacked several genes required for microaerophilic growth inside nodules, while several genes for accessory functions related to competition, plant invasion and bacteroid tropism were identified only in AK83 and BL225C strains. Presence and extent of polymorphism in regulons of transcription factors involved in symbiotic interaction were also analyzed. Our results indicate that regulons are flexible, with a large number of accessory genes, suggesting that regulons polymorphism could also be a key determinant in the variability of symbiotic performances among the analyzed strains. CONCLUSIONS: In conclusions, the extended comparative genomics approach revealed a variable subset of genes and regulons that may contribute to the symbiotic diversity.
Project description:l-Carnitine is a trimethylammonium compound mostly known for its contribution to fatty acid transport into mitochondria. In bacteria, it is synthesized from ?-butyrobetaine (GBB) and can be used as a carbon source. l-Carnitine can be formed directly by GBB hydroxylation or synthesized via a biosynthetic route analogous to fatty acid degradation. However, this multistep pathway has not been experimentally characterized. In this work, we identified by gene context analysis a cluster of l-carnitine anabolic genes next to those involved in its catabolism and proceeded to the complete in vitro characterization of l-carnitine biosynthesis and degradation in Sinorhizobium meliloti The five enzymes catalyzing the seven steps that convert GBB to glycine betaine are described. Metabolomic analysis confirmed the multistage synthesis of l-carnitine in GBB-grown cells but also revealed that GBB is synthesized by S. meliloti To our knowledge, this is the first report of aerobic GBB synthesis in bacteria. The conservation of l-carnitine metabolism genes in different bacterial taxonomic classes underscores the role of l-carnitine as a ubiquitous nutrient.IMPORTANCE The experimental characterization of novel metabolic pathways is essential for realizing the value of genome sequences and improving our knowledge of the enzymatic capabilities of the bacterial world. However, 30% to 40% of genes of a typical genome remain unannotated or associated with a putative function. We used enzyme kinetics, liquid chromatography-mass spectroscopy (LC-MS)-based metabolomics, and mutant phenotyping for the characterization of the metabolism of l-carnitine in Sinorhizobium meliloti to provide an accurate annotation of the corresponding genes. The occurrence of conserved gene clusters for carnitine metabolism in soil, plant-associated, and marine bacteria underlines the environmental abundance of carnitine and suggests this molecule might make a significant contribution to ecosystem nitrogen and carbon cycling.
Project description:During development of the symbiotic soil bacterium Sinorhizobium meliloti into nitrogen-fixing bacteroids, DNA replication and cell division cease and the cells undergo profound metabolic and morphological changes. Regulatory genes controlling the early stages of this process have not been identified. As a first step in the search for regulators of these events, we report the isolation and characterization of a ctrA gene from S. meliloti. We show that the S. meliloti CtrA belongs to the CtrA-like family of response regulators found in several alpha-proteobacteria. In Caulobacter crescentus, CtrA is essential and is a global regulator of multiple cell cycle functions. ctrA is also an essential gene in S. meliloti, and it is expressed similarly to the autoregulated C. crescentus ctrA in that both genes have complex promoter regions which bind phosphorylated CtrA.
Project description:Sinorhizobium meliloti enters into beneficial symbiotic interactions with Medicago species of legumes. Bacterial exopolysaccharides play critical signaling roles in infection thread initiation and growth during the early stages of root nodule formation. After endocytosis of S. meliloti by plant cells in the developing nodule, plant-derived nodule-specific cysteine-rich (NCR) peptides mediate terminal differentiation of the bacteria into nitrogen-fixing bacteroids. Previous transcriptional studies showed that the intensively studied cationic peptide NCR247 induces expression of the exo genes that encode the proteins required for succinoglycan biosynthesis. In addition, genetic studies have shown that some exo mutants exhibit increased sensitivity to the antimicrobial action of NCR247. Therefore, we investigated whether the symbiotically active S. meliloti exopolysaccharide succinoglycan can protect S. meliloti against the antimicrobial activity of NCR247. We discovered that high-molecular-weight forms of succinoglycan have the ability to protect S. meliloti from the antimicrobial action of the NCR247 peptide but low-molecular-weight forms of wild-type succinoglycan do not. The protective function of high-molecular-weight succinoglycan occurs via direct molecular interactions between anionic succinoglycan and the cationic NCR247 peptide, but this interaction is not chiral. Taken together, our observations suggest that S. meliloti exopolysaccharides not only may be critical during early stages of nodule invasion but also are upregulated at a late stage of symbiosis to protect bacteria against the bactericidal action of cationic NCR peptides. Our findings represent an important step forward in fully understanding the complete set of exopolysaccharide functions during legume symbiosis.IMPORTANCE Symbiotic interactions between rhizobia and legumes are economically important for global food production. The legume symbiosis also is a major part of the global nitrogen cycle and is an ideal model system to study host-microbe interactions. Signaling between legumes and rhizobia is essential to establish symbiosis, and understanding these signals is a major goal in the field. Exopolysaccharides are important in the symbiotic context because they are essential signaling molecules during early-stage symbiosis. In this study, we provide evidence suggesting that the Sinorhizobium meliloti exopolysaccharide succinoglycan also protects the bacteria against the antimicrobial action of essential late-stage symbiosis plant peptides.
Project description:Sinorhizobium meliloti is a nitrogen-fixing bacterial symbiont of alfalfa and related legumes. Symbiotic infection by S. meliloti requires an osmosensory two-component system composed of the response regulator FeuP and the sensor kinase FeuQ. The FeuPQ pathway positively regulates transcription of multiple genes including ndvA, which encodes the cyclic glucan exporter. Here we show that proper regulation of this signalling pathway is essential for cell viability. Without the small 83 amino acid protein FeuN, S. meliloti cells are unable to grow, and this phenotype is dependent on the FeuPQ pathway. Using Escherichia coli as a heterologous system, we show that expression of feuP and feuQ leads to a dramatic increase in ndvA promoter activity, but that simultaneous expression of feuN abrogates this effect. Random mutagenesis of the feuPQ bicistron revealed a defined region of the FeuQ protein in and around its two predicted transmembrane domains that are required for FeuN-dependent signalling modulation. Marker enzyme fusion experiments indicate that most of the FeuN polypeptide is localized to the periplasm. Our data support a model in which FeuN interacts directly with FeuQ to attenuate phosphorylation of FeuP, and that without this activity, hyperactive signalling through FeuPQ results in cessation of growth or death.
Project description:We report here the complete genome sequence of the salt-tolerant Sinorhizobium meliloti strain AK21, isolated from nodules of Medicago sativa L. subsp. ambigua inhabiting the northern Aral Sea Region. This genome (7.36?Mb) consists of a chromosome and four accessory plasmids, two of which are the symbiotic megaplasmids pSymA and pSymB.
Project description:In this work, DNA microarrays were used to investigate genome-wide transcriptional responses of Sinorhizobium meliloti to a sudden increase in external osmolarity elicited by addition of either NaCl or sucrose to exponentially growing cultures. A time course of the response within the first 4 h after the osmotic shock was established. We found that there was a general redundancy in the differentially expressed genes after NaCl or sucrose addition. Both kinds of stress resulted in induction of a large number of genes having unknown functions and in repression of many genes coding for proteins with known functions. There was a strong replicon bias in the pattern of the osmotic stress response; whereas 64% of the upregulated genes had a plasmid localization, 85% of the downregulated genes were chromosomal. Among the pSymB osmoresponsive genes, 83% were upregulated, suggesting the importance of this plasmid for S. meliloti osmoadaptation. Indeed, we identified a 200-kb region in pSymB needed for adaptation to saline shock which has a high density of osmoregulated genes.