Sequence Analysis of the 144-Kilobase Accessory Plasmid pSmeSM11a, Isolated from a Dominant Sinorhizobium meliloti Strain Identified during a Long-Term Field Release Experiment

The genome of Sinorhizobium meliloti type strain Rm1021 consists of three replicons: the chromosome and two megaplasmids, pSymA and pSymB. Additionally, many indigenous S. meliloti strains possess one or more smaller plasmids, which represent the accessory genome of this species. Here we describe the complete nucleotide sequence of an accessory plasmid, designated pSmeSM11a, that was isolated from a dominant indigenous S. meliloti subpopulation in the context of a long-term field release experiment with genetically modified S. meliloti strains. Sequence analysis of plasmid pSmeSM11a revealed that it is 144,170 bp long and has a mean G+C content of 59.5 mol%. Annotation of the sequence resulted in a total of 160 coding sequences. Functional predictions could be made for 43% of the genes, whereas 57% of the genes encode hypothetical or unknown gene products. Two plasmid replication modules, one belonging to the repABC replicon family and the other belonging to the plasmid type A replicator region family, were identified. Plasmid pSmeSM11a contains a mobilization (mob) module composed of the type IV secretion system-related genes traG and traA and a putative mobC gene. A large continuous region that is about 42 kb long is very similar to a corresponding region located on S. meliloti Rm1021 megaplasmid pSymA. Single-base-pair deletions in the homologous regions are responsible for frameshifts that result in nonparalogous coding sequences. Plasmid pSmeSM11a carries additional copies of the nodulation genes nodP and nodQ that are responsible for Nod factor sulfation. Furthermore, a tauD gene encoding a putative taurine dioxygenase was identified on pSmeSM11a. An acdS gene located on pSmeSM11a is the first example of such a gene in S. meliloti. The deduced acdS gene product is able to deaminate 1-aminocyclopropane-1-carboxylate and is proposed to be involved in reducing the phytohormone ethylene, thus influencing nodulation events. The presence of numerous insertion sequences suggests that these elements mediated acquisition of accessory plasmid modules.     

Novel DNA Sequences from Natural Strains of the Nitrogen-Fixing Symbiotic Bacterium Sinorhizobium meliloti

Variation in genome size and content is common among bacterial strains. Identifying these naturally occurring differences can accelerate our understanding of bacterial attributes, such as ecological specialization and genome evolution. In this study, we used representational difference analysis to identify potentially novel sequences not present in the sequenced laboratory strain Rm1021 of the nitrogen-fixing bacterium Sinorhizobium meliloti. Using strain Rm1021 as the driver and the type strain of S. meliloti ATCC 9930, which has a genome size ~370 kilobases bigger than that of strain Rm1021, as the tester, we identified several groups of sequences in the ATCC 9930 genome not present in strain Rm1021. Among the 85 novel DNA fragments examined, 55 showed no obvious homologs anywhere in the public databases. Of the remaining 30 sequences, 24 contained homologs to the Rm1021 genome as well as unique segments not found in Rm1021, 3 contained sequences homologous to those published for another S. meliloti strain but absent in Rm1021, 2 contained sequences homologous to other symbiotic nitrogen-fixing bacteria (Rhizobium etli and Bradyrhizobium japonicum), and 1 contained a sequence homologous to a gene in a non-nitrogen-fixing species, Pseudomonas sp. NK87. Using PCR, we assayed the distribution of 12 of the above 85 novel sequences in a collection of 59 natural S. meliloti strains. The distribution varied widely among the 12 novel DNA fragments, from 1.7% to 72.9%. No apparent correlation was found between the distribution of these novel DNA sequences and their genotypes obtained using multilocus enzyme electrophoresis. Our results suggest potentially high rates of gene gain and loss in S. meliloti genomes.     

Entropy-driven motility of Sinorhizobium meliloti on a semi-solid surface

Sinorhizobium meliloti growing on soft agar can exhibit an unusual surface spreading behaviour that differs from other bacterial surface motilities. Bacteria in the colony secrete an exopolysaccharide-rich mucoid fluid that expands outward on the surface, carrying within it a suspension of actively dividing cells. The moving slime disperses the cells in complex and dynamic patterns indicative of simultaneous bacterial growth, swimming and aggregation. We find that while flagellar swimming is required to maintain the cells in suspension, the spreading and the associated pattern formation are primarily driven by the secreted exopolysaccharide EPS II, which creates two entropy-increasing effects: an osmotic flow of water from the agar to the mucoid fluid and a crowding or depletion attraction between the cells. Activation of these physical/chemical phenomena may be a useful function for the high molecular weight EPS II, a galactoglucan whose biosynthesis is tightly regulated by the ExpR/SinI/SinR quorum-sensing system: unlike bacterial colonies that spread via bacterium-generated, physical propulsive forces, S. meliloti under quorum conditions may use EPS II to activate purely entropic forces within its environment, so that it can disperse by passively ‘surfing’ on those forces.     

Sensory transduction to the flagellar motor of Sinorhizobium meliloti

Molecular mechanisms that govern chemotaxis and motility in the nitrogen-fixing soil bacterium, Sinorhizobium meliloti, are distinguished from the well-studied taxis systems of enterobacteria by new features. (i) In addition to six transmembrane chemotaxis receptors, S. meliloti has two cytoplasmic receptor proteins, McpY (methyl-accepting chemotaxis protein) and IcpA (internal chemotaxis protein). (ii) The tactic response is mediated by two response regulators, CheY1 and CheY2, but no phosphatase, CheZ. Phosphorylated CheY2 (CheY2-P) is the main regulator of motor function, whereas CheY1 assumes the role of a 'sink' for phosphate that is shuttled from CheY2-P back to CheA. This phospho-transfer from surplus CheY2-P to CheA to CheY1 replaces CheZ phosphatase. (iii) S. meliloti flagella have a complex structure with three helical ribbons that render the filaments rigid and unable to undergo polymorphic transitions from right- to left-handedness. Flagella rotate only clockwise and their motors can increase and decrease rotary speed. Hence, directional changes of a swimming cell occur during slow-down, when several flagella rotate at different speed. Two novel motility proteins, the periplasmic MotC and the cytoplasmic MotD, are essential for motility and rotary speed variation. A model consistent with these data postulates a MotC-mediated gating of the energizing MotA-MotB proton channels leading to variations in flagellar rotary speed.     

A Proteomic Approach Towards the Analysis of Salt Tolerance in Rhizobium etli and Sinorhizobium meliloti Strains

Soluble proteins from the salt-tolerant Rhizobium etli strain EBRI 26 were separated by two-dimensional (2D) gel electrophoresis and visualised by Commassie staining. Six proteins are highly expressed after induction by 4% NaCl compared to the non-salt-stressed cells. These proteins have pI between 5 and 5.5 and masses of approximately 22, 25, 40, 65, 70, and 95 kDa. These proteins were analysed by Matrix-assisted laser adsorption ionization time of flight (MALDI-TOF) after digestion with trypsin. Despite having very good peptide mass fingerprint data, these proteins could not be identified, because the genome sequence of R. etli is not yet published. In a second approach, soluble proteins from salt-induced or non-salt-induced cultures from R. etli strain EBRI 26 were separately labelled with different fluorescent cyano-dyes prior to 2D difference in gel electrophoresis. Results revealed that 49 proteins are differentially expressed after the addition of sodium chloride. Fourteen proteins are overexpressed and 35 were downregulated. The genome of Sinorhizobium meliloti, a closely related species to R. etli, has been published. Similar experiments using Sinorhizobium meliloti strain 2011 identified four overexpressed and six downregulated proteins. Among the overexpressed protein is a carboxynospermidin decarboxylase, which plays an important role in the biosynthesis of spermidin (polyamine). The enzyme catalase is among the downregulated proteins. These proteins may play a role in salt tolerance.     

Construction and Environmental Release of a Sinorhizobium meliloti Strain Genetically Modified To Be More Competitive for Alfalfa Nodulation

Highly efficient nitrogen-fixing strains selected in the laboratory often fail to increase legume production in agricultural soils containing indigenous rhizobial populations because they cannot compete against these populations for nodule formation. We have previously demonstrated, with a Sinorhizobium meliloti PutA⁻ mutant strain, that proline dehydrogenase activity is required for colonization and therefore for the nodulation efficiency and competitiveness of S. meliloti on alfalfa roots (J. I. Jiménez-Zurdo, P. van Dillewijn, M. J. Soto, M. R. de Felipe, J. Olivares, and N. Toro, Mol. Plant-Microbe Interact. 8:492–498, 1995). In this work, we investigated whether the putA gene could be used as a means of increasing the competitiveness of S. meliloti strains. We produced a construct in which a constitutive promoter was placed 190 nucleotides upstream from the start codon of the putA gene. This resulted in an increase in the basal expression of this gene, with this increase being even greater in the presence of the substrate proline. We found that the presence of multicopy plasmids containing this putA gene construct increased the competitiveness of S. meliloti in microcosm experiments in nonsterile soil planted with alfalfa plants subjected to drought stress only during the first month. We investigated whether this construct also increased the competitiveness of S. meliloti strains under agricultural conditions by using it as the inoculum in a contained field experiment at León, Spain. We found that the frequency of nodule occupancy was higher with inoculum containing the modified putA gene for samples that were analyzed after 34 days but not for samples that were analyzed later.     

Development of a lab-made microarray for analyzing the genetic diversity of nitrogen fixing symbionts Sinorhizobium meliloti and Sinorhizobium medicae

Some bacterial species, like nitrogen-fixing Sinorhizobium that interact with Medicago plants, are prone to frequent horizontal gene transfers. Investigation of their genetic structure requires to study polymorphism patterns at many loci. Although DNA microarrays represent a method of choice for high throughput analysis of polymorphisms, this technology yet remains an expensive and heavy approach, thus depriving most of research groups from this powerful tool. In an attempt to overcome this limitation, we have developed a simple genotyping procedure by DNA microarrays, and have evaluated its ability to characterize a Sinorhizobium population. Thirty 18- to 24-mer oligonucleotide probes were designed to target the most frequent mutations in three polymorphic loci of Sinorhizobium meliloti and S. medicae. Probe hybridization efficiency was compared on two spotting surfaces: nylon membranes and epoxy-coated glass slides. Epoxy-coated glass slides revealed more sensitive than nylon membranes and allowed discrimination of single mismatches. Using this procedure, an uncharacterized population consisting of 33 S. meliloti/S. medicae isolates was successfully genotyped.     

转座子挽救法对苜蓿中华根瘤菌与耐盐有关基因的定位

用含Tn5转座子的质粒pRL1063a诱变苜蓿中华根瘤菌(Sinorhizobium meliloti)042BM,得到盐敏感突变株042BML-2。采用转座子挽救法对Tn5插入位点两边的序列进行克隆与测序,获得了1179bp的转座子插入位点侧翼DNA序列。在GenBank中进行序列同源性和基因定位分析,结果表明：转座子插入在一个功能未知的基因内部,此基因长6270bp。研究证明：该基因与042BM的耐盐性有关,并定名为rtsC。氨基酸疏水性分析表明,在RtsC蛋白的N端有两个跨膜区,该蛋白与细菌趋化性相关蛋白的功能域有同源性。并对RtsC蛋白在苜蓿中华根瘤菌042BM耐盐性中的作用进行了讨论。     

Effect of a Sinorhizobium meliloti Strain with a Modified putA Gene on the Rhizosphere Microbial Community of Alfalfa

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.     

Effect of a Sinorhizobium meliloti strain with a modified putA gene on the rhizosphere microbial community of alfalfa

The success of a rhizobial inoculant in the soil depends to a large extent on its capacity to compete against indigenous strains. M403, a Sinorhizobium meliloti strain with enhanced competitiveness for nodule occupancy, was recently constructed by introducing a plasmid containing an extra copy of a modified putA (proline dehydrogenase) gene. This strain and M401, a control strain carrying the same plasmid without the modified gene, were used as soil inoculants for alfalfa in a contained field release experiment at León, Spain. In this study, we determined the effects of these two strains on the indigenous microbial community. 16S rRNA genes were obtained from the rhizosphere of alfalfa inoculated with strain M403 or strain M401 or from noninoculated plants by amplification of DNA from soil with bacterial group-specific primers. These genes were analyzed and compared by restriction fragment length polymorphism and temperature gradient gel electrophoresis. The results allowed us to differentiate between alterations in the microbial community apparently caused by inoculation and by the rhizosphere effect and seasonal fluctuations induced by the alfalfa plants and by the environment. Only moderate inoculation-dependent effects could be detected, while the alfalfa plants appeared to have a much stronger influence on the microbial community.     

Development of a cultivation-independent approach for the study of genetic diversity of Sinorhizobium meliloti populations

The development of a species-specific marker for the analysis of the genetic polymorphism of the nitrogen-fixing symbiotic bacterium Sinorhizobium meliloti directly from environmental DNA is reported. The marker is based on terminal-restriction fragment length polymorphism (T-RFLP) methodology targeting specifically the 16S-23S Ribosomal Intergenic Spacer of S. meliloti. Species-specificity and polymorphism of the marker were tested on DNA extracted from soil samples and from a collection of 130 S. meliloti bacterial isolates. These primers and the T-RFLP approach proved useful for the detection and analysis of polymorphism of S. meliloti populations.     

Regulatory and DNA Repair Genes Contribute to the Desiccation Resistance of Sinorhizobium meliloti Rm1021

Sinorhizobium meliloti can form a nitrogen-fixing symbiotic relationship with alfalfa after bacteria in the soil infect emerging root hairs of the growing plant. To be successful at this, the bacteria must be able to survive in the soil between periods of active plant growth, including when conditions are dry. The ability of S. meliloti to withstand desiccation has been known for years, but genes that contribute to this phenotype have not been identified. Transposon mutagenesis was used in combination with novel screening techniques to identify four desiccation-sensitive mutants of S. meliloti Rm1021. DNA sequencing of the transposon insertion sites identified three genes with regulatory functions (relA, rpoE2, and hpr) and a DNA repair gene (uvrC). Various phenotypes of the mutants were determined, including their behavior on several indicator media and in symbiosis. All of the mutants formed an effective symbiosis with alfalfa. To test the hypothesis that UvrC-related excision repair was important in desiccation resistance, uvrA, uvrB, and uvrC deletion mutants were also constructed. These strains were sensitive to DNA damage induced by UV light and 4-NQO and were also desiccation sensitive. These data indicate that uvr gene-mediated DNA repair and the regulation of stress-induced pathways are important for desiccation resistance.     

Insights into the history of a bacterial group II intron remnant from the genomes of the nitrogen-fixing symbionts Sinorhizobium meliloti and Sinorhizobium medicae

Group II introns are self-splicing catalytic RNAs that act as mobile retroelements. In bacteria, they are thought to be tolerated to some extent because they self-splice and home preferentially to sites outside of functional genes, generally within intergenic regions or in other mobile genetic elements, by mechanisms including the divergence of DNA target specificity to prevent target site saturation. RmInt1 is a mobile group II intron that is widespread in natural populations of Sinorhizobium meliloti and was first described in the GR4 strain. Like other bacterial group II introns, RmInt1 tends to evolve toward an inactive form by fragmentation, with loss of the 3′ terminus. We identified genomic evidence of a fragmented intron closely related to RmInt1 buried in the genome of the extant S. meliloti/S. medicae species. By studying this intron, we obtained evidence for the occurrence of intron insertion before the divergence of ancient rhizobial species. This fragmented group II intron has thus existed for a long time and has provided sequence variation, on which selection can act, contributing to diverse genetic rearrangements, and to generate pan-genome divergence after strain differentiation. The data presented here suggest that fragmented group II introns within intergenic regions closed to functionally important neighboring genes may have been microevolutionary forces driving adaptive evolution of these rhizobial species.