Formate-dependent autotrophic growth in Sinorhizobium meliloti

It was found that S. meliloti strain SmA818, which is cured of pSymA, could not grow on defined medium containing only formate and bicarbonate as carbon sources. Growth experiments showed that Rm1021 was capable of formate/bicarbonate-dependent growth, suggesting that it was capable of autotrophic-type growth. The annotated genome of S. meliloti Rm1021 contains three formate dehydrogenase genes. A systematic disruption of each of the three formate dehydrogenase genes, as well as the genes encoding determinants of the Calvin-Benson-Bassham, cycle was carried out to determine which of these determinants played a role in growth on this defined medium. The results showed that S. meliloti is capable of formate-dependent autotrophic growth. Formate-dependent autotrophic growth is dependent on the presence of the chromosomally located fdsABCDG operon, as well as the cbb operon carried by pSymB. Growth was also dependent on the presence of either of the two triose-phosphate isomerase genes (tpiA or tpiB) that are found in the genome. In addition, it was found that fdoGHI carried by pSymA encodes a formate dehydrogenase that allows Rm1021 to carry out formate-dependent respiration. Taken together, the data allow us to present a model of how S. meliloti can grow on defined medium containing only formate and bicarbonate as carbon sources.     

Identification of Sinorhizobium meliloti LsrB regulon

Redox homeostasis determines cell fate for both prokaryotes and eukaryotes.In bacteria,redox state depends on aerobic respira-tion and antioxidant protection.Su...     

Control of Gluconate Utilization in Sinorhizobium meliloti

The Sinorhizobium meliloti megaplasmid pSymA has previously been implicated in gluconate utilization. We report a locus on pSymA encoding a putative tripartite ATP-independent periplasmic (TRAP) transporter that is required for gluconate utilization. The expression of this locus is negatively regulated by a GntR family regulator encoded adjacent to the transporter operon.     

Replication regions of Sinorhizobium meliloti plasmids

The replication (rep) regions of small plasmids from three Sinorhizobium meliloti strains were cloned by marker rescue. Two unique replication regions were identified, one of which was common to two different strains. Plasmid pBB83 carried a 7.2 kbp rep region from a 42 kbp plasmid, and pBB84 carried a 4.5 kbp rep region from a 36 kbp plasmid. The cloned rep regions were of different compatibility types, and were capable of displacing their parent plasmids from S. meliloti. Neither could function in a PolA- strain of Escherichia coli. The cloned replication regions were less stable in S. meliloti than their parent plasmids. The rep genes for each plasmid were localized to less than 2.5 kbp segments. Sequencing data revealed that the pBB83 Rep protein is uncommon, with partial identity to a protein encoded by a plasmid from S. meliloti GR4 [Mercado-Blanco, J., Olivares, J., 1994. The large nonsymbiotic plasmid pRmeGR4a of Rhizobium meliloti GR4 encodes a protein involved in replication that has homology with the RepC protein of Agrobacterium plasmids. Plasmid 32, 75-79]. However, the cloned DNA fragment also contains a truncated segment of the common repABC genes, suggesting that the parent plasmid contained two sets of replication genes. Other genes and an IS-element within the insert are most closely related to sequences derived from the Rhizobiaceae family, suggesting that the plasmid has a limited host range. In contrast, the pBB84 rep region contained genes similar to those associated with several broad host-range plasmids, and its Rep protein is related to that of a Pseudomonas aeruginosa broad host-range plasmid, pVS1 [Heeb, S., Itoh, Y., Nishijyo, T., Schnider, U., Keel, C., Wade, J., Walsh, U., O'Gara, F., Haas, D., 2000. Small, stable shuttle vectors based on the minimal pVS1 replicon for use in gram-negative, plant-associated bacteria. Mol. Plant-Microbe Interact. 13, 232-237]. The pBB84 rep region also includes a probable origin of replication, consisting of DNA boxes flanking a series of direct repeats and an AT-rich sequence.     

Identification of two quorum-sensing systems in Sinorhizobium meliloti

Sinorhizobium meliloti is a free-living soil bacterium which is capable of establishing a symbiotic relationship with the alfalfa plant (Medicago sativa). This symbiosis involves a network of bacterium-host signaling, as well as the potential for bacterium-bacterium communication, such as quorum sensing. In this study, we characterized the production of N-acyl homoserine lactones (AHLs) by two commonly used S. meliloti strains, AK631 and Rm1021. We found that AK631 produces at least nine different AHLs, while Rm1021 produces only a subset of these molecules. To address the difference in AHL patterns between the strains, we developed a novel screening method to identify the genes affecting AHL synthesis. With this screening method, chromosomal groEL (groELc) was shown to be required for synthesis of the AHLs that are unique to AK631 but not for synthesis of the AHLs that are made by both AK631 and Rm1021. We then used the screening procedure to identify a mutation in a gene homologous to traM of Agrobacterium tumefaciens, which was able to suppress the phenotype of the groELc mutation. A traR homolog was identified immediately upstream of traM, and we propose that its gene product requires a functional groELc for activity and is also responsible for inducing the synthesis of the AHLs that are unique to AK631. We show that the traR/traM locus is part of a quorum-sensing system unique to AK631 and propose that this locus is involved in regulating conjugal plasmid transfer. We also present evidence for the existence of a second quorum-sensing system, sinR/sinI, which is present in both AK631 and Rm1021.     

Sucrose Is a Nonaccumulated Osmoprotectant in Sinorhizobium meliloti

Intracellular accumulation of sucrose in response to lowered water activity seems to occur only in photosynthetic organisms. Here we demonstrate, for the first time, the potent ability of this common sugar, supplied exogenously, to reduce growth inhibition of Sinorhizobium meliloti cells in media of inhibitory osmolarity. Independently of the nature of the growth substrates and the osmotic agent, sucrose appears particularly efficient in promoting the recovery of cytoplasmic volume after plasmolysis. Surprisingly, sucrose is not accumulated by the bacteria at an osmotically efficient level. Instead, it strongly stimulates the accumulation of the main endogenous osmolytes glutamate and N-acetylglutaminylglutamine amide (NAGGN). Examining cell volume changes during the hyperosmotic treatment, we found a close correlation between the enhancement of the osmotically active solute pool and the increase in cell volume. Sucrose shares several features with ectoine, another nonaccumulated osmoprotectant for S. meliloti. Overall, osmoregulation in S. meliloti appears to be strongly divergent from that in most bacteria.     

Environmental regulation of exopolysaccharide production in Sinorhizobium meliloti

Exopolysaccharide production by Sinorhizobium meliloti is required for invasion of root nodules on alfalfa and successful establishment of a nitrogen-fixing symbiosis between the two partners. S. meliloti wild-type strain Rm1021 requires production of either succinoglycan, a polymer of repeating octasaccharide subunits, or EPS II, an exopolysaccharide of repeating dimer subunits. The reason for the production of two functional exopolysaccharides is not clear. Earlier reports suggested that low-phosphate conditions stimulate the production of EPS II in Rm1021. We found that phosphate concentrations determine which exopolysaccharide is produced by S. meliloti. The low-phosphate conditions normally found in the soil (1 to 10 microM) stimulate EPS II production, while the high-phosphate conditions inside the nodule (20 to 100 mM) block EPS II synthesis and induce the production of succinoglycan. Interestingly, the EPS II produced by S. meliloti in low-phosphate conditions does not allow the invasion of alfalfa nodules. We propose that this invasion phenotype is due to the lack of the active molecular weight fraction of EPS II required for nodule invasion. An analysis of the function of PhoB in this differential exopolysaccharide production is presented.     

Laccase activity in melanin-producing strains of Sinorhizobium meliloti

Laccase-like activity was detected in melanin-producing strains of Sinorhizobium meliloti mainly in cells at the stationary growth phase when copper was added to the medium. The laccase showed both syringaldazine and ABTS (2,2'-azino-bis-ethylbenzthiazoline-6-sulfonic acid) oxidase activities and was activated by the addition of 1.7 mM sodium dodecyl sulfate. Activity was totally inhibited by the addition of 1.0 mM EDTA, suggesting that the enzyme is a metal-dependent one. The enzyme was found to be cytosolic having an optimum pH of 5.0, an estimated molecular mass of 95 kDa and a K(m) of 4 microM for syringaldazine. Both laccase and tyrosinase activities were detected in melanin-producing S. meliloti strains. Plant growth-promoting (PGP) effect in rice by a laccase-producing S. meliloti strain when co-inoculated with Azospirillum brasilense Cd was observed. PGP effect by co-inoculation significantly increased plant yield compared to A. brasilense by itself. To the best of our knowledge this is the first report on laccase production in rhizobia and cooperation between Azospirillum and Sinorhizobium in rice.     

Quorum sensing controls exopolysaccharide production in Sinorhizobium meliloti

Sinorhizobium meliloti is a soil bacterium capable of invading and establishing a symbiotic relationship with alfalfa plants. This invasion process requires the synthesis, by S. meliloti, of at least one of the two symbiotically important exopolysaccharides, succinoglycan and EPS II. We have previously shown that the sinRI locus of S. meliloti encodes a quorum-sensing system that plays a role in the symbiotic process. Here we show that the sinRI locus exerts one level of control through regulation of EPS II synthesis. Disruption of the autoinducer synthase gene, sinI, abolished EPS II production as well as the expression of several genes in the exp operon that are responsible for EPS II synthesis. This phenotype was complemented by the addition of acyl homoserine lactone (AHL) extracts from the wild-type strain but not from a sinI mutant, indicating that the sinRI-specified AHLs are required for exp gene expression. This was further confirmed by the observation that synthetic palmitoleyl homoserine lactone (C(16:1)-HL), one of the previously identified sinRI-specified AHLs, specifically restored exp gene expression. Most importantly, the absence of symbiotically active EPS II in a sinI mutant was confirmed in plant nodulation assays, emphasizing the role of quorum sensing in symbiosis.     

New recombination methods for Sinorhizobium meliloti genetics

The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative alpha-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.     

New Recombination Methods for Sinorhizobium meliloti Genetics

The availability of bacterial genome sequences has created a need for improved methods for sequence-based functional analysis to facilitate moving from annotated DNA sequence to genetic materials for analyzing the roles that postulated genes play in bacterial phenotypes. A powerful cloning method that uses lambda integrase recombination to clone and manipulate DNA sequences has been adapted for use with the gram-negative α-proteobacterium Sinorhizobium meliloti in two ways that increase the utility of the system. Adding plasmid oriT sequences to a set of vehicles allows the plasmids to be transferred to S. meliloti by conjugation and also allows cloned genes to be recombined from one plasmid to another in vivo by a pentaparental mating protocol, saving considerable time and expense. In addition, vehicles that contain yeast Flp recombinase target recombination sequences allow the construction of deletion mutations where the end points of the deletions are located at the ends of the cloned genes. Several deletions were constructed in a cluster of 60 genes on the symbiotic plasmid (pSymA) of S. meliloti, predicted to code for a denitrification pathway. The mutations do not affect the ability of the bacteria to form nitrogen-fixing nodules on Medicago sativa (alfalfa) roots.     

苜蓿中华根瘤菌氮代谢调控相关非编码RNA的研究

为了探究非编码RNA（non-coding RNA,ncRNA）在氮代谢调控过程的作用,以苜蓿中华根瘤菌（Sinorhizobium meliloti）为出发菌株,鉴定并获得了与ntrC基因表达相关的ncRNA,利用lncPRO软件分析苜蓿中华根瘤菌中ncRNA与调控蛋白NtrC相互作用的可能性,最终确定了评分较高的4个基因,即SM2011_c06191、SM2011_c06248、SM2011_c07102和SM2011_c07132,并对其中得分最高的SM2011_c06248基因进行研究。利用Northern blot验证ncRNA的表达,发现富氮处理后其表达水平呈先升高后降低的趋势。实验构建了SM2011_c06248基因敲除菌株,通过qRT-PCR发现SM2011_c06248在自然生长条件下表达无明显规律,富氮处理后,野生型菌株ncRNA表达水平呈先降低后升高再降低的趋势,与自然生长相比,ncRNA在20 min后表达水平明显上调;野生型菌株ntrC、nar基因表达水平呈先降低后升高再降低的趋势;与野生型相比,SM2011_c06248基因敲除菌株SM∷248中ntrC、nar基因表达水平明显下调。实验中构建SM2011_c06248、NtrC双突变菌株SM∷248-NtrC和SM2011_c06248过表达菌株SM:dld-248,qRT-PCR分析表明,与野生型相比,富氮处理后,SM∷248-NtrC菌株nar基因表达量无明显变化,SM:dld-248菌株ntrC、nar基因表达水平显著上调。ncRNA SM2011_c06248可响应环境中氮元素信号变化,对ntrC、nar基因的表达有正调控作用,且ntrC对nar有上位基因效应。     

Regulation of succinoglycan and galactoglucan biosynthesis in Sinorhizobium meliloti

Sinorhizobium meliloti (Rhizobium meliloti) 2011 has the ability to produce the two acidic exopolysaccharides succinoglycan (EPS I) and galactoglucan (EPS II). EPS I is a branched heteropolysaccharide composed of octasaccharide repeating units, whereas EPS II is a linear heteropolysaccharide consisting of disaccharide subunits. The exo-exs and exp gene clusters are involved in the biosynthesis of EPSI and EPSII, respectively. EPSI and EPSII biosynthesis genes are differentially expressed resulting in a complex regulation of EPS production in S. meliloti. The phosphate concentration was identified as an important factor affecting the expression of exp genes.