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.     

Prevalence of pSmeSM11a-like plasmids in indigenous Sinorhizobium meliloti strains isolated in the course of a field release experiment with genetically modified S. meliloti strains

Plasmid pSmeSM11a, residing in the indigenous Sinorhizobium meliloti strain SM11 originating from a field in Strassmoos (Bavaria, Germany), was analysed previously at the genomic level. Thirty-seven indigenous S. meliloti strains, originating from two different locations in Germany, were screened for genes identified previously on pSmeSM11a. Seven of these strains harbour accessory plasmids that are very similar to pSmeSM11a. The identified pSmeSM11a-like plasmids are c. 130-150 kb in size and possess nearly identical restriction profiles. Up to 30 genes identified previously on pSmeSM11a could be detected on these plasmids by hybridisation experiments, e.g., the nodulation genes nodP and nodQ, the ethylene level modulation gene acdS and the taurine metabolism gene tauD. A few pSmeSM11a genes were also detected on other plasmids. The reference plasmid pSmeSM11a contains a region that is similar to a segment of S. meliloti strain Rm1021 pSymA. Regions with similarity to pSymA were also detected on the aforementioned seven pSmeSM11a-like plasmids. The specifications of these regions are nearly identical to the one on pSmeSM11a and differ from Rm1021 pSymA as determined by nucleotide sequence analysis. Two further plasmids similar to pSmeSM11a completely lack the pSymA-region. Those strains carrying accessory plasmids that contain the acdS gene encoding 1-aminocyclopropane-1-carboxylate deaminase are able to grow on 1-aminocyclopropane-1-carboxylate as the sole source of nitrogen, demonstrating functionality of the acdS gene product. About 36% of the analysed plasmids, including three pSmeSM11a-like plasmids, could be transferred to another S. meliloti recipient strain, allowing for their dissemination in S. meliloti populations.     

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.     

The complete genome sequence of the dominant Sinorhizobium meliloti field isolate SM11 extends the S. meliloti pan-genome

Isolates of the symbiotic nitrogen-fixing species Sinorhizobium meliloti usually contain a chromosome and two large megaplasmids encoding functions that are absolutely required for the specific interaction of the microsymbiont with corresponding host plants leading to an effective symbiosis. The complete genome sequence, including the megaplasmids pSmeSM11c (related to pSymA) and pSmeSM11d (related to pSymB), was established for the dominant, indigenous S. meliloti strain SM11 that had been isolated during a long-term field release experiment with genetically modified S. meliloti strains. The chromosome, the largest replicon of S. meliloti SM11, is 3,908,022 bp in size and codes for 3785 predicted protein coding sequences. The size of megaplasmid pSmeSM11c is 1,633,319 bp and it contains 1760 predicted protein coding sequences whereas megaplasmid pSmeSM11d is 1,632,395 bp in size and comprises 1548 predicted coding sequences. The gene content of the SM11 chromosome is quite similar to that of the reference strain S. meliloti Rm1021. Comparison of pSmeSM11c to pSymA of the reference strain revealed that many gene regions of these replicons are variable, supporting the assessment that pSymA is a major hot-spot for intra-specific differentiation. Plasmids pSymA and pSmeSM11c both encode unique genes. Large gene regions of pSmeSM11c are closely related to corresponding parts of Sinorhizobium medicae WSM419 plasmids. Moreover, pSmeSM11c encodes further novel gene regions, e.g. additional plasmid survival genes (partition, mobilisation and conjugative transfer genes), acdS encoding 1-aminocyclopropane-1-carboxylate deaminase involved in modulation of the phytohormone ethylene level and genes having predicted functions in degradative capabilities, stress response, amino acid metabolism and associated pathways. In contrast to Rm1021 pSymA and pSmeSM11c, megaplasmid pSymB of strain Rm1021 and pSmeSM11d are highly conserved showing extensive synteny with only few rearrangements. Most remarkably, pSmeSM11b contains a new gene cluster predicted to be involved in polysaccharide biosynthesis. Compilation of the S. meliloti SM11 genome sequence contributes to an extension of the S. meliloti pan-genome.     

The rkp-3 gene region of Sinorhizobium meliloti Rm41 contains strain-specific genes that determine K antigen structure.

The rkp-3 region is indispensable for capsular polysaccharide (K antigen) synthesis in Sinorhizobium meliloti Rm41. Strain Rm41 produces a K antigen of strain-specific structure, designated as the KR5 antigen. The data in this report show that the rkp-3 gene region comprises 10 open reading frames involved in bacterial polysaccharide synthesis and export. The predicted amino acid sequences for the rkpL-Q gene products are homologous to enzymes involved in the production of specific sugar moieties, while the putative products of the rkpRST genes show a high degree of similarity to proteins required for transporting polysaccharides to the cell surface. Southern analysis experiments using gene-specific probes suggest that genes involved in the synthesis of the precursor sugars are unique in strain Rm41, whereas sequences coding for export proteins are widely distributed among Sinorhizobium species. Mutations in the rkpL-Q genes result in a modified K antigen pattern and impaired symbiotic capabilities. On this basis, we suggest that these genes are required for the production of the KR5 antigen that is necessary for S. meliloti Rm41 exoB (AK631)-alfalfa (Medicago sativa) symbiosis.     

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.     

'Ca. Liberibacter asiaticus' proteins orthologous with pSymA-encoded proteins of Sinorhizobium meliloti: hypothetical roles in plant host interaction

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.     

冻融法转化苜蓿中华根瘤菌

对于苜蓿中华根瘤菌转化方法的研究仅限于三亲杂交法和电激转化,本实验首次将冻融法用于转化苜蓿中华根瘤菌。将携带CUS片段并且含有壮观霉素抗性的质粒用冻融法转移至野生型苜蓿中华根瘤菌菌株,得到的抗性克隆用GUS引物进行PCR扩增鉴定。经过计算,冻融法的转化率为2．2×10^5／μg。与传统方法比较,冻融法操作简单,转化过程快速,转化率较高,所需时间较短,转化成本低,是适合用于苜蓿中华根瘤菌的转化方法。     

IncP-1-beta plasmid pGNB1 isolated from a bacterial community from a wastewater treatment plant mediates decolorization of triphenylmethane dyes

Plasmid pGNB1 was isolated from bacteria residing in the activated sludge compartment of a wastewater treatment plant by using a transformation-based approach. This 60-kb plasmid confers resistance to the triphenylmethane dye crystal violet and enables its host bacterium to decolorize crystal violet. Partial sequencing of pGNB1 revealed that its backbone is very similar to that of previously sequenced IncP-1beta plasmids. The two accessory regions of the plasmid, one located downstream of the replication initiation gene trfA and the other located between the conjugative transfer modules Tra and Trb, were completely sequenced. Accessory region L1 contains a transposon related to Tn5501 and a gene encoding a Cupin 2 conserved barrel protein with an unknown function. The triphenylmethane reductase gene tmr and a truncated dihydrolipoamide dehydrogenase gene that is flanked by IS1071 and another putative insertion element were identified in accessory region L2. Subcloning of the pGNB1 tmr gene demonstrated that this gene is responsible for the observed crystal violet resistance phenotype and mediates decolorization of the triphenylmethane dyes crystal violet, malachite green, and basic fuchsin. Plasmid pGNB1 and the associated phenotype are transferable to the alpha-proteobacterium Sinorhizobium meliloti and the gamma-proteobacterium Escherichia coli. This is the first report of a promiscuous IncP-1beta plasmid isolated from the bacterial community from a wastewater treatment plant that harbors a triphenylmethane reductase gene. The pGNB1-encoded enzyme activity is discussed with respect to bioremediation of sewage polluted with triphenylmethane dyes.     

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.     

Sinorhizobium meliloti plasmid pRm1132f replicates by a rolling-circle mechanism

pRm1132f isolated from Sinorhizobium meliloti is a group III rolling-circle-replicating (RCR) plasmid. At least seven of eight open reading frames in the nucleotide sequence represented coding regions. The minimal replicon contained a rep gene and single- and double-stranded origins of replication. Detection of single-stranded plasmid DNA confirmed that pRm1132f replicated via an RCR mechanism.     

Increase in alfalfa nodulation, nitrogen fixation, and plant growth by specific DNA amplification in Sinorhizobium meliloti.

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

Proteome analysis of differentially displayed proteins as a tool for the investigation of symbiosis

Two-dimensional gel electrophoresis was used to identify differentially displayed proteins expressed during the symbiotic interaction between the bacterium Sinorhizobium meliloti strain 1021 and the legume Melilotus alba (white sweetclover). Our aim was to characterize novel symbiosis proteins and to determine how the two symbiotic partners alter their respective metabolisms as part of the interaction, by identifying gene products that are differentially present between the symbiotic and non-symbiotic states. Proteome maps from control M. alba roots, wild-type nodules, cultured S. meliloti, and S. meliloti bacteroids were generated and compared. Over 250 proteins were induced or up-regulated in the nodule, compared with the root, and over 350 proteins were down-regulated in the bacteroid form of the rhizobia, compared with cultured cells. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide mass fingerprint analysis, in conjunction with data base searching, were used to assign putative identity to nearly 100 nodule, bacterial, and bacteroid proteins. These included the previously identified nodule proteins leghemoglobin and NifH as well as proteins involved in carbon and nitrogen metabolism in S. meliloti. Bacteroid cells showed down-regulation of several proteins involved in nitrogen acquisition, including glutamine synthetase, urease, a urea-amide binding protein, and a PII isoform, indicating that the bacteroids were nitrogen proficient. The down-regulation of several enzymes involved in polyhydroxybutyrate synthesis and a cell division protein was also observed. This work shows that proteome analysis will be a useful strategy to link sequence information and functional genomics.     

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。     

Functional analysis of Sinorhizobium meliloti genes involved in biotin synthesis and transport

External biotin greatly stimulates bacterial growth and alfalfa root colonization by Sinorhizobium meliloti strain 1021. Several genes involved in responses to plant-derived biotin have been identified in this bacterium, but no genes required for biotin transport are known, and not all loci required for biotin synthesis have been assigned. Searches of the S. meliloti genome database in combination with complementation tests of Escherichia coli biotin auxotrophs indicate that biotin synthesis probably is limited in S. meliloti 1021 by the poor functioning or complete absence of several key genes. Although several open reading frames with significant similarities to genes required for synthesis of biotin in gram-positive and gram-negative bacteria were found, only bioB, bioF, and bioH were demonstrably functional in complementation tests with known E. coli mutants. No sequence or complementation evidence was found for bioA, bioC, bioD, or bioZ. In contrast to other microorganisms, the S. meliloti bioB and bioF genes are not localized in a biotin synthesis operon, but bioB is cotranscribed with two genes coding for ABC transporter-like proteins, designated here bioM and bioN. Mutations in bioM and bioN eliminated growth on alfalfa roots and reduced bacterial capacity to maintain normal intracellular levels of biotin. Taken together, these data suggest that S. meliloti normally grows on exogenous biotin using bioM and bioN to conserve biotin assimilated from external sources.     

Identification and cloning of the bacterial nodulation specificity gene in the Sinorhizobium meliloti--Medicago laciniata symbiosis

Medicago laciniata (cut-leaf medic) is an annual medic that is highly nodulation specific, nodulating only with a restricted range of Sinorhizobium meliloti. e.g., strain 102L4, but not with most strains that nodulate Medicago sativa (alfalfa), e.g., strains RCR2011 and Rm41. Our aim was to identify and clone the S. meliloti 102L4 gene implicated in the specific nodulation of M. laciniata and to characterize the adjacent nodulation (nod) region. An 11-kb EcoRI DNA fragment from S. meliloti 102L4 was shown to complement strain RCR2011 for nodulation of M. laciniata. Nucleotide sequencing revealed that this fragment contained nodABCIJ genes whose overall arrangement was similar to those found in strains RCR2011 and Rm41, which do not nodulate M. laciniata. Data for Tn5 mutagenesis of the nodABCIJ region of strain 102L4 suggested that the nodC gene was involved in the specific nodulation of M. laciniata. Tn5 insertions in the nodIJ genes gave mutants with nodulation delay phenotypes on both M. laciniata and M. sativa. Only subclones of the 11-kb DNA fragment containing a functional nodC gene from strain 102L4 were able to complement strain RCR2011 for nodulation of M. laciniata. The practical implications of these findings are discussed in the context of the development of a specific M. sativa - S. meliloti combination that excludes competition for nodulation by bacterial competitors resident in soil.     

The Sinorhizobium meliloti fur gene regulates, with dependence on Mn(II), transcription of the sitABCD operon, encoding a metal-type transporter

Sinorhizobium meliloti is an alpha-proteobacterium able to induce nitrogen-fixing nodules on roots of specific legumes. In order to propagate in the soil and for successful symbiotic interaction the bacterium needs to sequester metals like iron and manganese from its environment. The metal uptake has to be in turn tightly regulated to avoid toxic effects. In this report we describe the characterization of a chromosomal region of S. meliloti encoding the sitABCD operon and the putative regulatory fur gene. It is generally assumed that the sitABCD operon encodes a metal-type transporter and that the fur gene is involved in iron ion uptake regulation. A constructed S. meliloti sitA deletion mutant was found to be growth dependent on Mn(II) and to a lesser degree on Fe(II). The sitA promoter was strongly repressed by Mn(II), with dependence on Fur, and moderately by Fe(II). Applying a genome-wide S. meliloti microarray it was shown that in the fur deletion mutant 23 genes were up-regulated and 10 genes were down-regulated when compared to the wild-type strain. Among the up-regulated genes only the sitABCD operon could be associated with metal uptake. On the other hand, the complete rhbABCDEF operon, which is involved in siderophore synthesis, was identified among the down-regulated genes. Thus, in S. meliloti Fur is not a global repressor of iron uptake. Under symbiotic conditions the sitA promoter was strongly expressed and the S. meliloti sitA mutant exhibited an attenuated nitrogen fixation activity resulting in a decreased fresh weight of the host plant Medicago sativa.