Erythromycin resistance-conferring plasmid pRSB105, isolated from a sewage treatment plant, harbors a new macrolide resistance determinant, an integron-containing Tn402-like element, and a large region of unknown function.

The erythromycin resistance plasmid pRSB105 was previously isolated from an activated sludge bacterial community of a municipal wastewater treatment plant. Compilation of the complete pRSB105 nucleotide sequence revealed that the plasmid is 57,137 bp in size and has a mean G+C content of 56.66 mol%. The pRSB105 backbone is composed of two different replication and/or partitioning modules and a functional mobilization region encoding the mobilization genes mobCDE and mobBA. The first replicon (Rep1) is nearly identical to the corresponding replication module of the multiresistance plasmid pRSB101 isolated from an unknown activated sludge bacterium. Accordingly, pRSB101 and pRSB105 are sister plasmids belonging to a new plasmid family. The second replicon (Rep2) of pRSB105 was classified as a member of the IncP-6 group. While Rep1 confers replication ability only in gamma-proteobacteria, Rep2 extents the host range of the plasmid since it is also functional in the beta-proteobacterium Ralstonia eutropha. Plasmid pRSB105 harbors the macrolide resistance genes mel and mph, encoding, respectively, a predicted ABC-type efflux permease and a macrolide-2'-phosphotransferase. Erythromycin resistance is mainly attributed to mel, whereas mph contributes to erythromycin resistance to a lesser extent. The second resistance region, represented by an integron-containing Tn402-like element, includes a beta-lactam (oxa10) and a trimethoprim (dfrB2) resistance gene cassette. In addition to antibiotic resistance modules, pRSB105 encodes a functional restriction/modification system and two nonresistance regions of unknown function. The presence of different mobile genetic elements that flank resistance and nonresistance modules on pRSB105 indicates that these elements were involved in acquisition of accessory plasmid modules. Comparative genomics of pRSB105 and related plasmids elucidated that pRSB105 evolved by integration of distinct modules from different plasmid sources, including Pseudomonas aeruginosa plasmids, and thus represents a mosaic plasmid.

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 120 592 bp IncF plasmid pRSB107 isolated from a sewage-treatment plant encodes nine different antibiotic-resistance determinants, two iron-acquisition systems and other putative virulence-associated functions.

The antibiotic-multiresistance IncF plasmid pRSB107 was isolated by a transformation-based approach from activated-sludge bacteria of a wastewater-treatment plant. It confers resistance to ampicillin, penicillin G, chloramphenicol, erythromycin, kanamycin, neomycin, streptomycin, sulfonamides, tetracycline and trimethoprim and against mercuric ions. Complete sequencing of this plasmid revealed that it is 120 592 bp in size and has a G+C content of 53.1 mol%. The plasmid backbone is composed of three replicons, RepFIA, RepFIB and RepFII, which are almost identical to corresponding regions located on the F-plasmid and on R100. The three replicons encode replication initiation (rep) and replication control, multimer resolution (mrs), post-segregational killing of plasmid-free cells (psk) and active plasmid partitioning (sopABC locus). Part of the F-leading region and remnants of the F-homologous DNA-transfer (tra) module complete the pRSB107 backbone. Plasmid pRSB107 contains a complex, highly mosaic 35 991 bp antibiotic-resistance region consisting of a Tn21- and a Tn10-derivative and a chloramphenicol-resistance module. The Tn21 derivative is composed of a mercury-resistance region (mer), a Tn4352B-like kanamycin/neomycin-resistance transposon, a streptomycin/sulfonamide-resistance module, remnants of the beta-lactam-resistance transposon Tn1, a macrolide-resistance module flanked by copies of IS26 and IS6100, remnants of Tn402 integrating a class 1 integron and the Tn21-specific transposition module. A truncated version of the tetracycline-resistance transposon Tn10 and the chloramphenicol acetyltransferase gene catA complete the pRSB107 resistance region. In addition to antibiotic resistance, pRSB107 encodes the following putative virulence-associated functions: (i) an aerobactin iron-acquisition siderophore system (iuc/iut); (ii) a putative high-affinity Fe(2+) uptake system which was previously identified on a pathogenicity island of Yersinia pestis and in the genome of the phytopathogen Erwinia carotovora subsp. atroseptica SCRI1043; (iii) an sn-glycerol-3-phosphate transport system (ugp); and (iv) the virulence-associated genes vagCD having a possible function in stable plasmid inheritance. All the accessory modules are framed by insertion sequences, indicating that pRSB107 was gradually assembled by integration of different horizontally acquired DNA segments via transposition or homologous recombination.

The complete sequences of plasmids pB2 and pB3 provide evidence for a recent ancestor of the IncP-1beta group without any accessory genes.

The nucleotide sequences of the broad-host-range antibiotic resistance plasmids pB2 (61 kb) and pB3 (56 kb), which were isolated from a wastewater treatment plant, were determined and analysed. Both have a nearly identical IncP-1beta backbone, which diverged early from the sequenced IncP-1beta plasmids R751, pB10, pJP4, pADP1 and pUO1. In contrast to the latter plasmids, the pB2 and pB3 backbone does not seem to have undergone any deletions. The complete partition gene parA is located downstream of the mating pair formation (trb) module. A 14.4 kb or 19.0 kb mobile genetic element is present between traC and parA of pB3 and pB2, respectively. This region is typical for insertions in IncP-1beta plasmids, but the insertion site is unique. Both elements differ only by a duplication in pB2 of a tetA(C)-tetR-tnpA(IS26) fragment. The 5 bp target site duplication and the 26 bp inverted repeats flanking the mobile genetic elements are still intact, indicating that the insertion occurred recently. The element consists of three nested transposable elements: (i) a relict of a Tn402-like transposon with a gene for a new class D beta-lactamase (bla(NPS-2)); (ii) within that, another Tn402-like element with a class 1 integron harbouring the gene cassettes cmlA1 for a chloramphenicol efflux protein and aadA2 encoding a streptomycin/spectinomycin adenylyltransferase, and a copy of IS6100; (iii) into the integrase gene intI1 a tetracycline resistance module tetA(C)-tetR flanked by copies of IS26 is inserted. Interestingly, in contrast to all other IncP-1beta plasmids analysed so far, the oriV region between trfA and klcA is not interrupted by accessory genes, and there is no indication that previously inserted accessory genes have subsequently been deleted. The genes kluAB are also missing in that region and should thus be considered acquired genes. These findings, together with the fact that IncP-1beta plasmids acquired accessory elements at various positions in the backbone, suggest that IncP-1beta plasmids without any accessory genes exist in microbial communities. They must occasionally acquire accessory genes by transposition events, resulting in those plasmids that have been found based on selectable phenotypic traits.

The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa.

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101-pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1beta) and RK2 (IncP-1alpha), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.

The Sinorhizobium meliloti insertion sequence (IS) element ISRm14 is related to a previously unrecognized IS element located adjacent to the Escherichia coli locus of enterocyte effacement (LEE) pathogenicity island.

ISRm14 is 2695 basepairs (bp) in size and bordered by 22 bp imperfect inverted repeats (IRs). A 9-bp target sequence is duplicated upon ISRm14 transposition. The DNA strand that putatively encodes the transposase enzyme carries three open reading frames (ORFs) designated ORFs1 to 3, which specify putative proteins of 15. 9 kDa, 13.1 kDa, and 61.1 kDa, respectively. According to its structural characteristics, ISRm14 belongs to the recently proposed IS66 family of IS elements. The ORFs1 to 3 encoded putative proteins displayed significant similarities to ORFs of the previously unrecognized IS element ISEc8, which is inserted adjacent to the locus of enterocyte effacement (LEE) pathogenicity island of Escherichia coli EDL933. Analyses of the distribution of ISRm14 in a natural S. meliloti population showed its widespread occurrence in 66% of the strains tested with a copy number ranging from 1 to 6.