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 3,908,022bp in size and codes for 3785 predicted protein coding sequences. The size of megaplasmid pSmeSM11c is 1,633,319bp and it contains 1760 predicted protein coding sequences whereas megaplasmid pSmeSM11d is 1,632,395bp 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.

Comparative genomic hybridisation and ultrafast pyrosequencing revealed remarkable differences between the Sinorhizobium meliloti genomes of the model strain Rm1021 and the field isolate SM11.

Genomic variation between the Sinorhizobium meliloti model strain Rm1021 and the field isolate SM11 was assessed by using the genome-wide S. meliloti Rm1021 Sm6k-oligonucleotide microarray in a comparative genomic hybridisation experiment. Several gene clusters present in the Rm1021 genome are missing in the SM11 genome. In detail, three missing gene clusters were identified for the chromosome, five for megaplasmid pSymA and two for megaplasmid pSymB. To confirm these hybridisation results, the draft genome sequence of the S. meliloti field isolate SM11 was established by 454-pyrosequencing. Three sequencing runs on the ultrafast Genome Sequencer 20 System yielded 112.5 million bases. These could be assembled into 905 larger contigs resulting in a nearly 15-fold coverage of the 7.1Mb SM11 genome. The missing gene regions identified by comparative genomic hybridisation could be confirmed by the results of the 454-sequencing project. An in-depth analysis of these gene regions resulted in the following findings: (i) a complete type I restriction/modification system encoded by a composite transposon is absent in the chromosome of strain SM11. (ii) Most of the Rm1021 denitrification genes and the complete siderophore biosynthesis operon were found to be missing on SM11 megaplasmid pSymA. (iii) S. meliloti SM11 megaplasmid pSymB lacks a complete cell surface carbohydrate synthesis gene cluster. (iv) Several genes that are absent in the SM11 genome could be assigned to insertion sequences and transposons.

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.

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.

Sequence analysis of the 181-kb accessory plasmid pSmeSM11b, isolated from a dominant Sinorhizobium meliloti strain identified during a long-term field release experiment.

The 181 251 bp accessory plasmid pSmeSM11b of Sinorhizobium meliloti strain SM11, belonging to a dominant indigenous S. meliloti subpopulation identified during a long-term field release experiment, was sequenced. This plasmid has 166 coding sequences (CDSs), 42% of which encode proteins with homology to proteins of known function. Plasmid pSmeSM11b is a member of the repABC replicon family and contains a large gene region coding for a conjugation system similar to that of other self-transmissible plasmids in Rhizobium and Agrobacterium. Another pSmeSM11b gene region, possibly involved in sugar metabolism and polysaccharide catabolism, resembled a region of S. meliloti 1021 megaplasmid pSymB and in the genome of Sinorhizobium medicae WSM419. Another module of plasmid pSmeSM11b encodes proteins similar to those of the nitrogen-fixing actinomycete Frankia CcI3, and which are likely to be involved in the synthesis of a secondary metabolite. Several ORFs of pSmeSM11b were predicted to play a role in nonribosomal peptide synthesis. Plasmid pSmeSM11b has many mobile genetic elements, which contribute to the mosaic composition of the plasmid.

Mobilizable IncQ-related plasmid carrying a new quinolone resistance gene, qnrS2, isolated from the bacterial community of a wastewater treatment plant.

Plasmid-encoded quinolone resistance was previously reported for different bacteria isolated from patients not only in the United States and Asia but also in Europe. Here we describe the isolation, by applying a new selection strategy, of the quinolone resistance plasmid pGNB2 from an activated sludge bacterial community of a wastewater treatment plant in Germany. The hypersensitive Escherichia coli strain KAM3 carrying a mutation in the multidrug efflux system genes acrAB was transformed with total plasmid DNA preparations isolated from activated sludge bacteria and subsequently selected on medium containing the fluoroquinolone norfloxacin. This approach resulted in the isolation of plasmid pGNB2 conferring decreased susceptibility to nalidixic acid and to different fluoroquinolones. Analysis of the pGNB2 nucleotide sequence revealed that it is 8,469 bp in size and has a G+C content of 58.2%. The plasmid backbone is composed of a replication initiation module (repA-repC) belonging to the IncQ-family and a two-component mobilization module that confers horizontal mobility to the plasmid. In addition, plasmid pGNB2 carries an accessory module consisting of a transposon Tn1721 remnant and the quinolone resistance gene, qnrS2, that is 92% identical to the qnrS gene located on plasmid pAH0376 from Shigella flexneri 2b. QnrS2 belongs to the pentapeptide repeat protein family and is predicted to protect DNA-gyrase activity against quinolones. This is not only the first report on a completely sequenced plasmid mediating quinolone resistance isolated from an environmental sample but also on the first qnrS-like gene detected in Europe.