中华根瘤菌自体诱导物合成酶基因的筛选及其在大肠杆菌中的表达

通过携带有mariner转座子的质粒pJZ290随机插入诱变中华根瘤菌(Sinorhizobium meliloti)建立突变子文库,并从中筛选到自体诱导物(autoinducer,AI)部分缺失突变株YW1。Arbitrary PCR扩增、DNA测序得到YW1基因组DNA中mariner转座子两端侧翼序列,经DNA序列拼接在GenBank上进行同源性分析后获得一个621bp的完整的开放阅读框(ORF),该ORF编码的酶具有206个氨基酸,与草木樨中华根瘤菌(Sinorhizobium medicae)WSM419的LuxI类自体诱导物合成酶(autoinducer synthase)TraI的同源性高达99%。因此,也将该基因命名为traⅠ。将该基因克隆到广宿主范围表达载体pYC12并在大肠杆菌Escherichia coli DH5α中成功表达,C18反相薄层层析(TLC)在阳性重组子培养上清中检测到四种自体诱导物分子,其中的两种正是AI缺失突变株YW1所缺失的AI,这些结果表明该traⅠ基因在苜蓿中华根瘤菌负责合成两种自体诱导物分子,为进一步研究其群体感应系统奠定了理论基础。     

鸡眼草根瘤菌的１６SrDNA全序列分析

Based on the previous studies on numerical taxonomy, SDS-PAGE of whole-cell protein and DNA hybridization, the rhizobial strains isolated from Kummerowia sp. in semi-arid area of North-west constituted a new subgroup, the 16S rDNA sequence of representative strain SH714 were tested. The unrooted phylogenetic tree was produced. In this tree, the strain SH714 with Sinorhizobium xinjiangensis, S. fredii, S. meliloti, S. medicae, S. saheli and S. teranga constituted a branch of Sinorhizobium. Within this branch, the similarity valuse of 16S rDNA sequence between strain SH714 and S. xinjiangesis, S. fredii, S. meliloti, S. medicae, S. saheli and S. teranga were 97.4%, 97.5%, 96.8%, 96.7%, 97.2% and 95.6% respectively, the values were more than 95%, this indicated that these known species should belong to the same genus. The values of DNA homology between type strains of these species were less than 70%. Thus, the strain SH714 represented a new rhizobial species, and there were some diversity between SH714 and known rhizobial species in phenotypic feature and composition of protein.     

The lipid lysyl-phosphatidylglycerol is present in membranes of Rhizobium tropici CIAT899 and confers increased resistance to polymyxin B under acidic growth conditions

Lysyl-phosphatidylglycerol (LPG) is a well-known membrane lipid in several gram-positive bacteria but is almost unheard of in gram-negative bacteria. In Staphylococcus aureus, the gene product of mprF is responsible for LPG formation. Low pH-inducible genes, termed IpiA, have been identified in the gram-negative alpha-proteobacteria Rhizobium tropici and Sinorhizobium medicae in screens for acid-sensitive mutants and they encode homologs of MprF. An analysis of the sequenced bacterial genomes reveals that genes coding for homologs of MprF from S. aureus are present in several classes of organisms throughout the bacterial kingdom. In this study, we show that the expression of lpiA from R. tropici in the heterologous hosts Escherichia coli and Sinorhizobium meliloti causes formation of LPG. A wild-type strain of R. tropici forms LPG (about 1% of the total lipids) when the cells are grown in minimal medium at pH 4.5 but not when grown in minimal medium at neutral pH or in complex tryptone yeast (TY) medium at either pH. LPG biosynthesis does not occur when lpiA is deleted and is restored upon complementation of lpiA-deficient mutants with a functional copy of the lpiA gene. When grown in the low-pH medium, lpiA-deficient rhizobial mutants are over four times more susceptible to the cationic peptide polymyxin B than the wild type.     

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.     

Genetic relationship of Sinorhizobium meliloti and Sinorhizobium medicae strains isolated from Caucasian region

Sinorhizobium meliloti and Sinorhizobium medicae are two closely related species of the genus Sinorhizobium showing a similar host range, nodulating leguminous species of the genera Medicago, Melilotus and Trigonella, but their phylogenic relationship has not been elucidated yet. In this paper we report the application of three different molecular markers, (i) RFLP of nodD genes, (ii) 16S-23S rDNA intergenic gene spacer fingerprinting and (iii) amplification fragment length polymorphism to S. meliloti and S. medicae strains isolated from the Caucasian area, which is the region of origin of the host plant Medicago. The analysis of data could suggest the origin of S. medicae strains from an ancestral S. meliloti population.     

Stable low molecular weight RNA profiling showed variations within Sinorhizobium meliloti and Sinorhizobium medicae nodulating different legumes from the alfalfa cross-inoculation group

Four different low molecular weight (LMW) RNA profiles, designated I-IV, among 179 isolates from Medicago, Melilotus and Trigonella species growing in a field site in Northern Spain were identified. From sequence analysis of the 16S rRNA, atpD and recA genes as well as DNA-DNA hybridization analysis with representatives of each LMW RNA profile it was evident that isolates with LMW RNA profiles I and II belonged to Sinorhizobium meliloti and those displaying profiles III and IV to Sinorhizobium medicae. Therefore, two distinct LMW RNA electrophoretic mobility profiles were found within each of these two species. Collectively, LMW RNA profiles I and II (identified as S. meliloti) were predominant in Melilotus alba, Melilotus officinalis and Medicago sativa. Profiles III and IV (identified as S. medicae) were predominant in Melilotus parviflora, Medicago sphaerocarpa, Medicago lupulina and Trigonella foenum-graecum. All the four LMW RNA profiles were identified among isolates from Trigonella monspelliaca nodules. These results revealed a different specificity by the hosts of the alfalfa cross-inoculation group towards the two bacterial species found in this study.     

The model legume Medicago truncatula A17 is poorly matched for N2 fixation with the sequenced microsymbiont Sinorhizobium meliloti 1021

Medicago truncatula (barrel medic) A17 is currently being sequenced as a model legume, complementing the sequenced root nodule bacterial strain Sinorhizobium meliloti 1021 (Sm1021). In this study, the effectiveness of the Sm1021-M. truncatula symbiosis at fixing N(2) was evaluated. N(2) fixation effectiveness was examined with eight Medicago species and three accessions of M. truncatula with Sm1021 and two other Sinorhizobium strains. Plant shoot dry weights, plant nitrogen content and nodule distribution, morphology and number were analysed. Compared with nitrogen-fed controls, Sm1021 was ineffective or partially effective on all hosts tested (excluding M. sativa), as measured by reduced dry weights and shoot N content. Against an effective strain, Sm1021 on M. truncatula accessions produced more nodules, which were small, pale, more widely distributed on the root system and with fewer infected cells. The Sm1021-M. truncatula symbiosis is poorly matched for N(2) fixation and the strain could possess broader N(2) fixation deficiencies. A possible origin for this reduction in effectiveness is discussed. An alternative sequenced strain, effective at N(2) fixation on M. truncatula A17, is Sinorhizobium medicae WSM419.     

Genetic characterization of fast-growing rhizobia able to nodulate Prosopis alba in North Spain

Prosopis is a Mimosaceae legume tree indigenous to South America and not naturalized in Europe. In this work 18 rhizobial strains nodulating Prosopis alba roots were isolated from a soil in North Spain that belong to eight different randomly amplified polymorphic DNA groups phylogenetically related to Sinorhizobium medicae, Sinorhizobium meliloti and Rhizobium giardinii according to their intergenic spacer and 16S rRNA gene sequences. The nodC genes of isolates close to S. medicae and S. meliloti were identical to those of S. medicae USDA 1,037(T) and S. meliloti LMG 6,133(T) and accordingly all these strains were able to nodulate both alfalfa and Prosopis. These nodC genes were phylogenetically divergent from those of the isolates close to R. giardinii that were identical to that of R. giardinii H152(T) and therefore all these strains formed nodules in common beans and Prosopis. The nodC genes of the strains isolated in Spain were phylogenetically divergent from that carried by Mesorhizobium chacoense Pr-5(T) and Sinorhizobium arboris LMG 1,4919(T) nodulating Prosopis in America and Africa, respectively. Therefore, Prosopis is a promiscuous host which can establish symbiosis with strains carrying very divergent nodC genes and this promiscuity may be an important advantage for this legume tree to be used in reforestation.     

Differential regulation of a family of apyrase genes from Medicago truncatula

Four putative apyrase genes were identified from the model legume Medicago truncatula. Two of the genes identified from M. truncatula (Mtapy1 and Mtapy4) are expressed in roots and are inducible within 3 h after inoculation with Sinorhizobium meliloti. The level of mRNA expression of the other two putative apyrases, Mtapy2 and Mtapy3, was unaffected by rhizobial inoculation. Screening of a bacterial artificial chromosome library of M. truncatula genomic DNA showed that Mtapy1, Mtapy3, and Mtapy4 are present on a single bacterial artificial chromosome clone. This apyrase cluster was mapped to linkage group seven. A syntenic region on soybean linkage group J was found to contain at least two apyrase genes. Screening of nodulation deficient mutants of M. truncatula revealed that two such mutants do not express apyrases to any detectable level. The data suggest a role for apyrases early in the nodulation response before the involvement of root cortical cell division leading to the nodule structure.     

Horizontal gene transfer and homologous recombination drive the evolution of the nitrogen-fixing symbionts of Medicago species

Using nitrogen-fixing Sinorhizobium species that interact with Medicago plants as a model system, we aimed at clarifying how sex has shaped the diversity of bacteria associated with the genus Medicago on the interspecific and intraspecific scales. To gain insights into the diversification of these symbionts, we inferred a topology that includes the different specificity groups which interact with Medicago species, based on sequences of the nodulation gene cluster. Furthermore, 126 bacterial isolates were obtained from two soil samples, using Medicago truncatula and Medicago laciniata as host plants, to study the differentiation between populations of Sinorhizobium medicae, Sinorhizobium meliloti bv. meliloti, and S. meliloti bv. medicaginis. The former two can be associated with M. truncatula (among other species of Medicago), whereas the last organism is the specific symbiont of M. laciniata. These bacteria were characterized using a multilocus sequence analysis of four loci, located on the chromosome and on the two megaplasmids of S. meliloti. The phylogenetic results reveal that several interspecific horizontal gene transfers occurred during the diversification of Medicago symbionts. Within S. meliloti, the analyses show that nod genes specific to different host plants have spread to different genetic backgrounds through homologous recombination, preventing further divergence of the different ecotypes. Thus, specialization to different host plant species does not prevent the occurrence of gene flow among host-specific biovars of S. meliloti, whereas reproductive isolation between S. meliloti bv. meliloti and S. medicae is maintained even though these bacteria can cooccur in sympatry on the same individual host plants.     

Nitrogen-fixing sinorhizobia with Medicago laciniata constitute a novel biovar (bv. medicaginis) of S. meliloti

Sixty-eight new rhizobial isolates were obtained from root-nodules of Medicago laciniata and from Mediterranean soils in Tunisia and France. All of them were identified as Sinorhizobium meliloti on the basis of PCR-RFLP analyses of 16S rDNA and the intergenic spacer sequence between 16S and 23S rDNAs. DNA/DNA hybridization, phenotypic characterization and 16S rRNA gene sequencing led to the conclusion that they belong the same taxon. All new isolates shared the ability to nodulate and fix nitrogen with M. laciniata except 11 of them not capable of fixing nitrogen with this plant and originating from French soils containing no efficiently adapted symbionts with M. laciniata. The nitrogen-fixing rhizobia on M. laciniata differed markedly from the other S. meliloti or Sinorhizobium medicae isolates and references in their symbiotic traits such as nifDK RFLP diversity, nodA sequences and nitrogen effectiveness with tree other different annual Medicago species (M. truncatula, M. polymorpha and M. sauvagei). Two infrasubspecific (biovar) divisions are therefore proposed within S. meliloti: bv. medicaginis for Sinorhizobium efficient on M. laciniata and bv. meliloti for the classically known S. meliloti group represented by the strains ATCC9930(T) and RCR 2011 efficient on M. sativa.     

Population genetic structure of Sinorhizobium meliloti and S. medicae isolated from nodules of Medicago spp. in Mexico

We studied the genetic structure of 176 bacterial isolates from nodules of Medicago sativa, M. lupulina and M. polymorpha in fifteen sites distributed in three localities in Mexico. The strains were characterized by multilocus enzyme electrophoresis, plasmid profiles, PCR restriction fragment length polymorphism of 16S rRNA genes and of the intergenic spacer between 16S and 23S rRNA genes, and partial sequences of glnII, recA and nodB. Most of the strains were classified as Sinorhizobium meliloti, and a high genetic diversity was recorded. Six strains were classified as Sinorhizobium medicae, with no genetic variation. Phylogenetic and population genetic analyses revealed evidence of frequent recombination and migration within species.     

Diversity of Sinorhizobium strains nodulating Medicago sativa from different Iranian regions

Alfalfa is believed to have originated in north-western Iran and has a long history of coexistence with its bacterial symbiont Sinorhizobium in soils of Iran. However, little is known about the diversity of Sinorhizobium strains nodulating Iranian alfalfa genotypes. In this study, Sinorhizobium populations were sampled from eight different Iranian sites using three cultivars of Medicago sativa as trap host plants. A total of 982 rhizobial strains were isolated and species were identified showing a large prevalence of Sinorhizobium meliloti over Sinorhizobium medicae. Analysis of salt tolerance demonstrated a great phenotypic diversity. The genetic diversity of the Sinorhizobium isolates was analysed using BOX-PCR and enterobacterial repetitive intergenic consensus (ERIC)-PCR. Patterns ofBOX-PCR fingerprinting were statistically analysed with AMOVA to evaluate the role of plant variety and site of origin in the genetic variance observed. Results indicated that most of the total molecular variance was attributable to divergence among strains isolated from different sites and cultivars (intrapopulation, strain-by-strain variance). Moreover, the analysis showed the presence of two geographic populations (west and northwest), indicating that the effect of the site of origin could be more relevant in shaping population genetic diversity than the effect of cultivar or individual plant.     

Multilocus sequence typing as an approach for population analysis of Medicago-nodulating rhizobia

Multilocus sequence typing (MLST), a sequence-based method to characterize bacterial genomes, was used to examine the genetic structure in a large collection of Medicago-nodulating rhizobial strains. This is the first study where MLST has been applied in conjunction with eBURST analysis to determine the population genetic structure of nonpathogenic bacteria recovered from the soil environment. Sequence variation was determined in 10 chromosomal loci of 231 strains that predominantly originated from southwest Asia. Genetic diversity for each locus ranged from 0.351 to 0.819, and the strains examined were allocated to 91 different allelic profiles or sequence types (STs). The genus Medicago is nodulated by at least two groups of rhizobia with divergent chromosomes that have been classified as Sinorhizobium meliloti and Sinorhizobium medicae. Evidence was obtained that the degree of genetic exchange among the chromosomes across these groups is limited. The symbiosis with Medicago polymorpha of nine strains placed in one of these groups, previously identified as S. medicae, ranged from ineffective to fully effective, indicating that there was no strong relationship between symbiotic phenotype and chromosomal genotype.     

<Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>-induced chitinase gene expression in<Emphasis Type="Italic"> Medicago truncatula</Emphasis> ecotype R108-1: a comparison between symbiosis-specific class V and defence-related class IV chitinases

The Medicago truncatula (Gaertn.) ecotypes Jemalong A17 and R108-1 differ in Sinorhizobium meliloti-induced chitinase gene expression. The pathogen-inducible class IV chitinase gene, Mtchit 4, was strongly induced during nodule formation of the ecotype Jemalong A17 with the S. meliloti wild-type strain 1021. In the ecotype R108-1, the S. meliloti wild types Sm1021 and Sm41 did not induce Mtchit 4 expression. On the other hand, expression of the putative class V chitinase gene, Mtchit 5, was found in roots of M. truncatula cv. R108-1 nodulated with either of the rhizobial strains. Mtchit 5 expression was specific for interactions with rhizobia. It was not induced in response to fungal pathogen attack, and not induced in roots colonized with arbuscular mycorrhizal (AM) fungi. Elevated Mtchit 5 gene expression was first detectable in roots forming nodule primordia. In contrast to Mtchit 4, expression of Mtchit 5 was stimulated by purified Nod factors. Conversely, Mtchit 4 expression was strongly elevated in nodules formed with the K-antigen-deficient mutant PP699. Expression levels of Mtchit 5 were similarly increased in nodules formed with PP699 and its parental wild-type strain Sm41. Phylogenetic analysis of the deduced amino acid sequences of Mtchit 5 (calculated molecular weight = 41,810 Da, isoelectric point pH 7.7) and Mtchit 4 (calculated molecular weight 30,527 Da, isoelectric point pH 4.9) revealed that the putative Mtchit 5 chitinase forms a separate clade within class V chitinases of plants, whereas the Mtchit 4 chitinase clusters with pathogen-induced class IV chitinases from other plants. These findings demonstrate that: (i) Rhizobium-induced chitinase gene expression in M. truncatula occurs in a plant ecotype-specific manner, (ii) Mtchit 5 is a putative chitinase gene that is specifically induced by rhizobia, and (iii) rhizobia-specific and defence-related chitinase genes are differentially influenced by rhizobial Nod factors and K antigens.