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81900.9939Trimethoprim resistance transposon Tn4003 from Staphylococcus aureus encodes genes for a dihydrofolate reductase and thymidylate synthetase flanked by three copies of IS257. Trimethoprim resistance mediated by the Staphylococcus aureus multi-resistance plasmid pSK1 is encoded by a structure with characteristics of a composite transposon which we have designated Tn4003. Nucleotide sequence analysis of Tn4003 revealed it to be 4717 bp in length and to contain three copies of the insertion element IS257 (789-790 bp), the outside two of which are flanked by directly repeated 8-bp target sequences. IS257 has imperfect terminal inverted repeats of 27-28 bp and encodes for a putative transposase with two potential alpha-helix-turn-alpha-helix DNA recognition motifs. IS257 shares sequence similarities with members of the IS15 family of insertion sequences from Gram-negative bacteria and with ISS1 from Streptococcus lactis. The central region of the transposon contains the dfrA gene that specifies the S1 dihydrofolate reductase (DHFR) responsible for trimethoprim resistance. The S1 enzyme shows sequence homology with type I and V trimethoprim-resistant DHFRs from Gram-negative bacteria and with chromosomally encoded DHFRs from Gram-positive and Gram-negative bacteria. 5' to dfrA is a thymidylate synthetase gene, designated thyE.19892548057
301810.9936The large Bacillus plasmid pTB19 contains two integrated rolling-circle plasmids carrying mobilization functions. Plasmid pTB19 is a 27-kb plasmid originating from a thermophilic Bacillus species. It was shown previously that pTB19 contains an integrated copy of the rolling-circle type plasmid pTB913. Here we describe the analysis of a 4324-bp region of pTB19 conferring resistance to tetracycline. The nucleotide sequence of this region revealed all the characteristics of a second plasmid replicating via the rolling-circle mechanism. This sequence contained (i) the tetracycline resistance marker of pTB19, which is highly similar to other tetL-genes of gram-positive bacteria; (ii) a hybrid mob gene, which bears relatedness to both the mob-genes of pUB110 and pTB913; (iii) a palU type minus origin identical to those of pUB110 and pTB913; and (iv) a plus origin of replication similar to that of pTB913. A repB-type replication initiation gene sequence identical to that of pTB913 was present, which lacked the middle part (492 bp), thus preventing autonomous replication of this region. The hybrid mob gene was functional in conjugative mobilization of plasmids between strains of Bacillus subtilis.19911946749
82020.9930Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram-negative bacteria. A class II Tn3-type transposable element, designated Tn5393 and located on plasmid pEa34 from streptomycin-resistant strain CA11 of Erwinia amylovora, was identified by its ability to move from pEa34 to different sites in plasmids pGEM3Zf(+) and pUCD800. Nucleotide sequence analysis reveals that Tn5393 consists of 6,705 bp with 81-bp terminal inverted repeats and generates 5-bp duplications of the target DNA following insertion. Tn5393 contains open reading frames that encode a putative transposase (tnpA) and resolvase (tnpR) of 961 and 181 amino acids, respectively. The two open reading frames are separated by a putative recombination site (res) consisting of 194 bp. Two streptomycin resistance genes, strA and strB, were identified on the basis of their DNA sequence homology to streptomycin resistance genes in plasmid RSF1010. StrA is separated from tnpR by a 1.2-kb insertion element designated IS1133. The tnpA-res-tnpR region of Tn5393 was detected in Pseudomonas syringae pv. papulans Psp36 and in many other gram-negative bacteria harboring strA and strB. Except for some strains of Erwinia herbicola, these other gram-negative bacteria lacked insertion sequence IS1133. The prevalence of strA and strB could be accounted for by transposition of Tn5393 to conjugative plasmids that are then disseminated widely among gram-negative bacteria.19938380801
301730.9927The ancient small mobilizable plasmid pALWED1.8 harboring a new variant of the non-cassette streptomycin/spectinomycin resistance gene aadA27. The small mobilizable plasmid pALWED1.8 containing a novel variant of the streptomycin/spectinomycin resistance gene aadA27 was isolated from the permafrost strains of Acinetobacter lwoffii. The 4135bp plasmid carries mobА and mobC genes that mediate its mobilization by conjugative plasmids. The nucleotide sequences of mobА and mobC are similar to those of mobilization genes of the modern plasmid pRAY* and its variants, which contain aadB gene, and are widespread among the pathogenic strains of Acinetobacter baumannii. Almost identical pALWED1.8 variants were detected in modern environmental Аcinetobacter strains. A highly similar plasmid was revealed in a strain of Acinetobacter parvus isolated from mouse intestine. Furthermore, we discovered six previously unidentified variants of plasmids related to pALWED1.8 and pRAY* in public databases. In contrast to most known variants of aadA which are cassette genes associated with integrons, the aadA27 variant harbored by pALWED1.8 is a non-cassette, autonomously transcribed gene. Non-cassette aadA genes with 96% sequence identity to aadA27 were detected in the chromosomes of Acinetobacter gyllenbergii and several uncharacterized strains of Аcinetobacter sp. Moreover, we revealed that the autonomous aadA-like genes are present in the chromosomes of many gram-positive and gram-negative bacteria. The phylogenetic analysis of amino acid sequences of all identified AadA proteins showed the following: (i) cassette aadA genes form a separate monophyletic group and mainly reside on plasmids and (ii) chromosomal non-cassette aadA genes are extremely diverse and can be inherited both vertical and via horizontal gene transfer.201626896789
301340.9926Nucleotide sequence and organization of the multiresistance plasmid pSCFS1 from Staphylococcus sciuri. OBJECTIVES: The multiresistance plasmid pSCFS1 from Staphylococcus sciuri was sequenced completely and analysed with regard to its gene organization and the putative role of a novel ABC transporter in antimicrobial resistance. METHODS: Plasmid pSCFS1 was transformed into Staphylococcus aureus RN4220, overlapping restriction fragments were cloned into Escherichia coli plasmid vectors and sequenced. For further analysis of the ABC transporter, a approximately 3 kb EcoRV-HpaI fragment was cloned into the staphylococcal plasmid pT181MCS and the respective S. aureus RN4220 transformants were subjected to MIC determination. RESULTS: A total of 14 ORFs coding for proteins of >100 amino acids were detected within the 17 108 bp sequence of pSCFS1. Five of them showed similarity to recombination/mobilization genes while another two were similar to plasmid replication genes. In addition to the previously described genes cfr for chloramphenicol/florfenicol resistance and erm(33) for inducible resistance to macrolide-lincosamide-streptogramin B resistance, a Tn554-like spectinomycin resistance gene and Tn554-related transposase genes were identified. Moreover, a novel ABC transporter was detected and shown to mediate low-level lincosamide resistance. CONCLUSION: Plasmid pSCFS1 is composed of various parts which show similarity to sequences known to occur on plasmids or transposons of Gram-positive, but also Gram-negative bacteria. It is likely that pSCFS1 represents the result of inter-plasmid recombination events also involving the truncation of a Tn554-like transposon.200415471995
34950.9926Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor.19902172217
42960.9926An integrative vector exploiting the transposition properties of Tn1545 for insertional mutagenesis and cloning of genes from gram-positive bacteria. We have constructed and used an integrative vector, pAT112, that takes advantage of the transposition properties (integration and excision) of transposon Tn1545. This 4.9-kb plasmid is composed of: (i) the replication origin of pACYC184; (ii) the attachment site (att) of Tn1545; (iii) erythromycin-and kanamycin-resistance-encoding genes for selection in Gram- and Gram+ bacteria; and (iv) the transfer origin of IncP plasmid RK2, which allows mobilization of the vector from Escherichia coli to various Gram+ recipients. Integration of pAT112 requires the presence of the transposon-encoded integrase, Int-Tn, in the new host. This vector retains the insertion specificity of the parental element Tn1545 and utilises it to carry out insertional mutagenesis, as evaluated in Enterococcus faecalis. Since pAT112 contains the pACYC184 replicon and lacks most of the restriction sites that are commonly used for molecular cloning, a gene from a Gram+ bacterium disrupted with this vector can be recovered in E. coli by cleavage of genomic DNA, intramolecular ligation and transformation. Regeneration of the gene, by excision of pAT112, can be obtained in an E. coli strain expressing the excisionase and integrase of Tn1545. The functionality of this system was illustrated by characterization of an IS30-like structure in the chromosome of En. faecalis. Derivatives pAT113 and pAT114 contain ten unique cloning sites that allow screening of recombinants having DNA inserts by alpha-complementation in E. coli carrying the delta M15 deletion of lacZ alpha. These vectors are useful to clone and introduce foreign genes into the genomes of Gram+ bacteria.19911657722
305970.9925Genome Analysis of Kingella kingae Strain KWG1 Reveals How a β-Lactamase Gene Inserted in the Chromosome of This Species. We describe the genome of a penicillinase-producing Kingella kingae strain (KWG1), the first to be isolated in continental Europe, whose bla(TEM-1) gene was, for the first time in this species, found to be chromosomally inserted. The bla(TEM) gene is located in an integrative and conjugative element (ICE) inserted in Met-tRNA and comprising genes that encode resistance to sulfonamides, streptomycin, and tetracycline. This ICE is homologous to resistance-conferring plasmids of K. kingae and other Gram-negative bacteria.201626574009
34880.9924Conjugative DNA transfer in Streptomyces by TraB: is one protein enough? Antibiotic-producing soil bacteria of the genus Streptomyces form a huge natural reservoir of antibiotic resistance genes for the dissemination within the soil community. Streptomyces plasmids encode a unique conjugative DNA transfer system clearly distinguished from classical conjugation involving a single-stranded DNA molecule and a type IV protein secretion system. Only a single plasmid-encoded protein, TraB, is sufficient to translocate a double-stranded DNA molecule into the recipient in Streptomyces matings. TraB is a hexameric pore-forming ATPase that resembles the chromosome segregator protein FtsK and translocates DNA by recognizing specific 8-bp repeats present in the plasmid clt locus. Mobilization of chromosomal genes does not require integration of the plasmid, because TraB also recognizes clt-like sequences distributed all over the chromosome.201223082971
300990.9924Identification of a novel conjugative plasmid carrying the multiresistance gene cfr in Proteus vulgaris isolated from swine origin in China. The multiresistance gene cfr has a broad host range encompassing both Gram-positive and Gram-negative bacteria, and can be located on the chromosomes or on plasmids. In this study, a novel conjugative plasmid carrying cfr, designated as pPvSC3, was characterized in a Proteus vulgaris strain isolated from swine in China. Plasmid pPvSC3 is 284,528 bp in size and harbors 10 other antimicrobial resistance genes, making it a novel plasmid that differs from all known plasmids due to its unique backbone and repA gene. BLAST analysis of the plasmid sequence shows no significant homology to any known plasmid backbone, but shows high level homology to Providencia rettgeri strain CCBH11880 Contig_9, a strain isolated from surgical wound in Brazil, 2014. There are two resistance-determining regions in pPvSC3, a cfr-containing region and a multidrug-resistant (MDR) region. The cfr-containing region is flanked by IS26, which could be looped out via IS26-mediated recombination. The MDR region harbors 10 antimicrobial resistance genes carried by various DNA segments that originated from various sources. Plasmid pPvSC3 could be successfully transferred to Escherichia coli by conjugation. In summary, we have characterized a novel conjugative plasmid pPvSC3 carrying the multiresistance gene cfr and 10 other antimicrobial resistance genes, and consider that this novel type of plasmid deserves attention.201931499097
3008100.9924Sequence of conjugative plasmid pIP1206 mediating resistance to aminoglycosides by 16S rRNA methylation and to hydrophilic fluoroquinolones by efflux. Self-transferable IncFI plasmid pIP1206, isolated from an Escherichia coli clinical isolate, carries two new resistance determinants: qepA, which confers resistance to hydrophylic fluoroquinolones by efflux, and rmtB, which specifies a 16S rRNA methylase conferring high-level aminoglycoside resistance. Analysis of the 168,113-bp sequence (51% G+C) revealed that pIP1206 was composed of several subregions separated by copies of insertion sequences. Of 151 open reading frames, 56 (37%) were also present in pRSB107, isolated from a bacterium in a sewage treatment plant. pIP1206 contained four replication regions (RepFIA, RepFIB, and two partial RepFII regions) and a transfer region 91% identical with that of pAPEC-O1-ColBM, a plasmid isolated from an avian pathogenic E. coli. A putative oriT region was found upstream from the transfer region. The antibiotic resistance genes tet(A), catA1, bla(TEM-1), rmtB, and qepA were clustered in a 33.5-kb fragment delineated by two IS26 elements that also carried a class 1 integron, including the sulI, qacEDelta1, aad4, and dfrA17 genes and Tn10, Tn21, and Tn3-like transposons. The plasmid also possessed a raffinose operon, an arginine deiminase pathway, a putative iron acquisition gene cluster, an S-methylmethionine metabolism operon, two virulence-associated genes, and a type I DNA restriction-modification (R-M) system. Three toxin/antitoxin systems and the R-M system ensured stabilization of the plasmid in the host bacteria. These data suggest that the mosaic structure of pIP1206 could have resulted from recombination between pRSB107 and a pAPEC-O1-ColBM-like plasmid, combined with structural rearrangements associated with acquisition of additional DNA by recombination and of mobile genetic elements by transposition.200818458128
3021110.9923Sequencing and comparative analysis of IncP-1α antibiotic resistance plasmids reveal a highly conserved backbone and differences within accessory regions. Although IncP-1 plasmids are important for horizontal gene transfer among bacteria, in particular antibiotic resistance spread, so far only three plasmids from the subgroup IncP-1α have been completely sequenced. In this study we doubled this number. The three IncP-1α plasmids pB5, pB11 and pSP21 were isolated from bacteria of two different sewage treatment plants and sequenced by a combination of next-generation and capillary sequencing technologies. A comparative analysis including the previously analysed IncP-1α plasmids RK2, pTB11 and pBS228 revealed a highly conserved plasmid backbone (at least 99.9% DNA sequence identity) comprising 54 core genes. The accessory elements of the plasmid pB5 constitute a class 1 integron interrupting the parC gene and an IS6100 copy inserted into the integron. In addition, the tetracycline resistance genes tetAR and the ISTB11-like element are located between the klc operon and the trfA-ssb operon. Plasmid pB11 is loaded with a Tn5053-like mercury resistance transposon between the parCBA and parDE operons and contains tetAR that are identical to those identified in plasmid pB5 and the insertion sequence ISSP21. Plasmid pSP21 harbours an ISPa7 element in a Tn402 transposon including a class 1 integron between the partitioning genes parCBA and parDE. The IS-element ISSP21 (99.89% DNA sequence identity to ISSP21 from pB11), inserted downstream of the tetR gene and a copy of ISTB11 (identical to ISTB11 on pTB11) inserted between the genes pncA and pinR. On all three plasmids the accessory genes are almost always located between the backbone modules confirming the importance of the backbone functions for plasmid maintenance. The striking backbone conservation among the six completely sequenced IncP-1α plasmids is in contrast to the much higher diversity within the IncP-1β subgroup.201121115076
3019120.9923Identification and Characterization of New Resistance-Conferring SGI1s (Salmonella Genomic Island 1) in Proteus mirabilis. Salmonella genomic island 1 (SGI1) is a resistance-conferring chromosomal genomic island that contains an antibiotic resistance gene cluster. The international spread of SGI1-containing strains drew attention to the role of genomic islands in the dissemination of antibiotic resistance genes in Salmonella and other Gram-negative bacteria. In this study, five SGI1 variants conferring multidrug and heavy metal resistance were identified and characterized in Proteus mirabilis strains: SGI1-PmCAU, SGI1-PmABB, SGI1-PmJN16, SGI1-PmJN40, and SGI1-PmJN48. The genetic structures of SGI1-PmCAU and SGI1-PmABB were identical to previously reported SGI1s, while structural analysis showed that SGI1-PmJN16, SGI1-PmJN40, and SGI1-PmJN48 are new SGI1 variants. SGI1-PmJN16 is derived from SGI1-Z with the MDR region containing a new gene cassette array dfrA12-orfF-aadA2-qacEΔ1-sul1-chrA-orf1. SGI1-PmJN40 has an unprecedented structure that contains two right direct repeat sequences separated by a transcriptional regulator-rich DNA fragment, and is predicted to form two different extrachromosomal mobilizable DNA circles for dissemination. SGI1-PmJN48 lacks a common ORF S044, and its right junction region exhibits a unique genetic organization due to the reverse integration of a P. mirabilis chromosomal gene cluster and the insertion of part of a P. mirabilis plasmid, making it the largest known SGI1 to date (189.1 kb). Further mobility functional analysis suggested that these SGIs can be excised from the chromosome for transfer between bacteria, which promotes the horizontal transfer of antibiotic and heavy metal resistance genes. The identification and characterization of the new SGI1 variants in this work suggested the diversity of SGI1 structures and their significant roles in the evolution of bacteria.201830619228
821130.9922DNA probes for studying streptothricin resistance evolution in enteric bacteria. Probes for the detection of streptothricin resistance genes have been derived from recombinant plasmids. These include the streptothricin resistance gene probe sat 1/2 derived from Tn 1826 and specific for both the sat-1 determinant of Tn 1825 and the sat-2 determinant of Tn 1826, and the probe sat D derived from and specific for the sat-1 determinant of transposon Tn 1825. A third streptothricin resistance gene probe, sat 3, represents the streptothricin resistance determinant sat-3 of the IncQ R plasmid pIE639. Hybridization studies did not reveal any sequence homology between sat-3 and the transposon-localized sat-1 and sat-2 determinants. Moreover, non of the different sat-determinants isolated from plasmids of gram negative bacteria hybridized with the analogous resistance determinant of Streptomyces noursei, which had been cloned and named nat by Krügel et al. (Gene, 1988, 62, 209-214). The sat 1/2 probe in combination with the sat D probe proved to be suitable for the identification and the differentiation of sat-1 and sat-2 determinants in different genetic environments. Streptothricin resistance genes related to those present on transposons Tn 1825 and Tn 1826 have been detected by hybridization with the probe sat 1/2 on plasmids isolated a long time ago before the application of streptothricins. The sat-3 determinant appears to be exclusively associated with the IncQ plasmid pIE639.19902166786
3036140.9921Complete nucleotide sequences of 84.5- and 3.2-kb plasmids in the multi-antibiotic resistant Salmonella enterica serovar Typhimurium U302 strain G8430. The multi-antibiotic resistant (MR) Salmonella enterica serovar Typhimurium phage type U302 strain G8430 exhibits the penta-resistant ACSSuT-phenotype (ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline), and is also resistant to carbenicillin, erythromycin, kanamycin, and gentamicin. Two plasmids, 3.2- and 84.5-kb in size, carrying antibiotic resistance genes were isolated from this strain, and the nucleotide sequences were determined and analyzed. The 3.2-kb plasmid, pU302S, belongs to the ColE1 family and carries the aph(3')-I gene (Kan(R)). The 84.5-kb plasmid, pU302L, is an F-like plasmid and contains 14 complete IS elements and multiple resistance genes including aac3, aph(3')-I, sulII, tetA/R, strA/B, bla(TEM-1), mph, and the mer operon. Sequence analyses of pU302L revealed extensive homology to various plasmids or transposons, including F, R100, pHCM1, pO157, and pCTX-M3 plasmids and TnSF1 transposon, in regions involved in plasmid replication/maintenance functions and/or in antibiotic resistance gene clusters. Though similar to the conjugative plasmids F and R100 in the plasmid replication regions, pU302L does not contain oriT and the tra genes necessary for conjugal transfer. This mosaic pattern of sequence similarities suggests that pU302L acquired the resistance genes from a variety of enteric bacteria and underscores the importance of a further understanding of horizontal gene transfer among the enteric bacteria.200716828159
491150.9921Class II broad-spectrum mercury resistance transposons in Gram-positive bacteria from natural environments. We have studied the mechanisms of the horizontal dissemination of a broad-spectrum mercury resistance determinant among Bacillus and related species. This mer determinant was first described in Bacillus cereus RC607 from Boston Harbor, USA, and was then found in various Bacillus and related species in Japan, Russia and England. We have shown that the mer determinant can either be located at the chromosome, or on a plasmid in the Bacillus species, and is carried by class II mercury resistance transposons: Tn5084 from B. cereus RC607 and B. cereus VKM684 (ATCC10702) and Tn5085 from Exiguobacterium sp. TC38-2b. Tn5085 is identical in nucleotide sequence to TnMERI1, the only other known mer transposon from Bacillus species, but it does not contain an intron like TnMERI1. Tn5085 is functionally active in Escherichia coli. Tn5083, which we have isolated from B. megaterium MK64-1, contains an RC607-like mer determinant, that has lost some mercury resistance genes and possesses a merA gene which is a novel sequence variant that has not been previously described. Tn5083 and Tn5084 are recombinants, and are comprised of fragments from several transposons including Tn5085, and a relative of a putative transposon from B. firmus (which contains similar genes to the cadmium resistance operon of Staphylococcus aureus), as well as others. The sequence data showed evidence for recombination both between transposition genes and between mer determinants.200111446519
3043160.9920The role of insertions, deletions, and substitutions in the evolution of R6 related plasmids encoding aminoglycoside transferase ANT-(2"). In 7% of gram-negative bacteria resistance to gentamicin is mainly mediated by plasmid-encoded aminoglycoside transferase ANT-(2"). The genome organization of 15 aadB plasmids (42-110 kb) was analyzed by restriction and hybridization techniques. They appeared to be IncFII-like replicons but were distinct from R6 by virtue of small substitutions in the transfer region. Aminoglycoside resistance genes aadB and aadA were located on Tn21 related elements. Only one of them was able to transpose its resistance genes mer sul aadA and aadB ( Tn4000 ), the other elements were naturally occurring defective transposons. In some of these structures deletions were identified at the termini, at sul, aadA , mer or transposition function--insertions adjacent to aadA or mer. The mode of these rearrangements and their site-specificity were considered with respect to the evolution of the Tn21 transposon family.19846328217
3024170.9920Identification of ISVlu1-derived translocatable units containing optrA and/or fexA genes generated by homologous or illegitimate recombination in Lactococcus garvieae of porcine origin. The optrA gene encodes an ABC-F protein which confers cross-resistance to oxazolidinones and phenicols. Insertion sequence ISVlu1, a novel ISL3-family member, was recently reported to be involved in the transmission of optrA in Vagococcus lutrae. However, the role of ISVlu1 in mobilizing resistance genes has not yet fully explored. In this study, two complete and three truncated copies of ISVlu1 were found on plasmid pBN62-optrA from Lactococcus garvieae. Analysis of the genetic context showed that both optrA and the phenicols resistance gene fexA were flanked by the complete or truncated ISVlu1 copies. Moreover, three different-sized ISVlu1-based translocatable units (TUs) carrying optrA and/or fexA, were detected from pBN62-optrA. Sequence analysis revealed that the TU-optrA was generated by homologous recombination while TU-fexA and TU-optrA+fexA were the products of illegitimate recombinations. Importantly, conjugation assays confirmed that pBN62-optrA was able to successfully transfer into the recipient Enterococcus faecalis JH2-2. To our knowledge, this is the first report about an optrA-carrying plasmid in L. garvieae which could horizontally transfer into other species. More importantly, the ISVlu1-flanked genetic structures containing optrA and/or fexA were also observed in bacteria of different species, which underlines that ISVlu1 is highly active and plays a vital role in the transfer of some important resistance genes, such as optrA and fexA.202438479301
430180.9920Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6. DNA fragments generated by the EcoRI of HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColE1 or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants. Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable supporting replication of a linked ColE1 plasmid in polA- bacteria, were also identified.1978672900
9874190.9920Genomic islands related to Salmonella genomic island 1; integrative mobilisable elements in trmE mobilised in trans by A/C plasmids. Salmonella genomic island 1 (SGI1), an integrative mobilisable element (IME), was first reported 20 years ago, in the multidrug resistant Salmonella Typhimurium DT104 clone. Since this first report, many variants and relatives have been found in Salmonella enterica and Proteus mirabilis. Thanks to whole genome sequencing, more and more complete sequences of SGI1-related elements (SGI1-REs) have been reported in these last few years among Gammaproteobacteria. Here, the genetic organisation and main features common to SGI1-REs are summarised to help to classify them. Their integrases belong to the tyrosine-recombinase family and target the 3'-end of the trmE gene. They share the same genetic organisation (integrase and excisionase genes, replicase module, SgaCD-like transcriptional activator genes, traN, traG, mpsB/mpsA genes) and they harbour AcaCD binding sites promoting their excision, replication and mobilisation in presence of A/C plasmid. SGI1-REs are mosaic structures suggesting that recombination events occurred between them. Most of them harbour a multiple antibiotic resistance (MAR) region and the plasticity of their MAR region show that SGI1-REs play a key role in antibiotic resistance and might help multiple antibiotic resistant bacteria to adapt to their environment. This might explain the emergence of clones with SGI1-REs.202133582118