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82600.8514Sequence identity with type VIII and association with IS176 of type IIIc dihydrofolate reductase from Shigella sonnei. An uncommon dihydrofolate reductase (DHFR), type IIIc, was coded for by Shigella sonnei that harbors plasmid pBH700 and that was isolated in North Carolina. The trimethoprim resistance gene carried on pBH700 was subcloned and sequenced. The nucleotide sequence of the gene encoding type IIIc DHFR was identical to the gene encoding type VIII DHFR. The type IIIc amino acid sequence was approximately 50% similar to those of DHFRs commonly found in enteric bacteria. Furthermore, this gene was flanked by IS176 (IS26), an insertion sequence usually associated with those of aminoglycoside resistance genes. The gene for type IIIc DHFR was located by hybridization within a 1,993-bp PstI fragment in each of eight conjugative plasmids from geographically diverse strains of S. sonnei. Each plasmid also conferred resistance to ampicillin, streptomycin, and sulfamethoxazole and belonged to incompatibility group M. Plasmids carrying this new trimethoprim resistance gene, which is uniquely associated with IS176, have disseminated throughout the United States.19957695291
81910.8407Trimethoprim 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
81220.8369Characterization of plQ5 plasmid originating fromKlebsiella pneumoniae. plQ5 plasmid consists of a group of genes specifying resistance to ampicillin, chloramphenicol, carbencillin, kanamycin and trimethoprim-sulphamethoxazole. It is isolated inKlebslella pneumoniae ZD532, is about 26.8 Kb and is freely transmissible to various bacterial species of Gram-negative bacteria. Physical characterization revealed that plQ5 plasmid has a single site forHindill,BamHI,EcoRI and two sites forBglII restriction enzyme.199024429982
306130.8336Tetracycline-resistance encoding plasmids from Paenibacillus larvae, the causal agent of American foulbrood disease, isolated from commercial honeys. Paenibacillus larvae, the causal agent of American foulbrood disease in honeybees, acquires tetracycline-resistance via native plasmids carrying known tetracycline-resistance determinants. From three P. larvae tetracycline-resistant strains isolated from honeys, 5-kb-circular plasmids with almost identical sequences, designated pPL373 in strain PL373, pPL374 in strain PL374, and pPL395 in strain PL395, were isolated. These plasmids were highly similar (99%) to small tetracycline-encoding plasmids (pMA67, pBHS24, pBSDMV46A, pDMV2, pSU1, pAST4, and pLS55) that replicate by the rolling circle mechanism. Nucleotide sequences comparisons showed that pPL373, pPL374, and pPL395 mainly differed from the previously reported P. larvae plasmid pMA67 in the oriT region and mob genes. These differences suggest alternative mobilization and/or conjugation capacities. Plasmids pPL373, pPL374, and pPL395 were individually transferred by electroporation and stably maintained in tetracycline-susceptible P. larvae NRRL B-14154, in which they autonomously replicated. The presence of nearly identical plasmids in five different genera of gram-positive bacteria, i.e., Bhargavaea, Bacillus, Lactobacillus, Paenibacillus, and Sporosarcina, inhabiting diverse ecological niches provides further evidence of the genetic transfer of tetracycline resistance among environmental bacteria from soils, food, and marine habitats and from pathogenic bacteria such as P. larvae.201425296446
81540.8321The sequence of the mer operon of pMER327/419 and transposon ends of pMER327/419, 330 and 05. Three different, independently isolated mercury-resistance-conferring plasmids, pMER327/419, pMER330 and pMER05, from cultures originating from the river Mersey (UK), contain identical regulatory merR genes and transposon ends. The mer determinant from pMER327/419 contains an additional potential ORF (ORF F) located between merP and merA when compared with the archetypal Tn501. Although these plasmids confer narrow-spectrum resistance (resistance to Hg2+, but not organomercurials) their merR genes encode a potential organomercurial-sensing protein. Transposition of the mer of pMER05 into plasmid RP4 was demonstrated and, as with Tn502 and Tn5053, insertion occurred at a specific region. The sequence of pMER05 is identical at the 'left' and 'right' termini and across merR to Tn5053, which was independently isolated from the chromosome of a Xanthomonas sp. bacteria from the Khaidarkan mercury mine in Kirgizia, former Soviet Union [Kholodii et al., J. Mol. Biol. 230 (1993a) 1103-1107]. The transpositional unit of pMER05 is, like that of Tn5053, bounded by DNA homologous to the imperfect 25-bp inverted repeats (IR) of the In2 integron, which brackets antibiotic-resistance cassettes in Tn21 subgroup transposons. At one end of the transposable element, and internal to the In2-like IR, is a 38-bp IR which closely resembles the IR that bounds Tn21.19948063107
82050.8321Nucleotide 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
53460.8313Plasmid shuttle vector with two insertionally inactivable markers for coryneform bacteria. A new shuttle vector pCEM500 replicating in Escherichia coli and in Brevibacterium flavum was constructed. It carries two antibiotic resistance determinants (Kmr/Gmr from plasmid pSa of Gram-negative bacteria and Smr/Spr from plasmid pCG4 of Corynebacterium glutamicum) which are efficiently expressed in both hosts and can be inactivated by insertion of DNA fragments into the unique restriction endonuclease sites located within them. This vector was found to be stably maintained in B. flavum and can be used for transfer of the cloned genes into this amino-acid-producing coryneform bacterium.19902148164
53070.8306Location of the genes for anthranilate synthase in Streptomyces venezuelae ISP5230: genetic mapping after integration of the cloned genes. The anthranilate synthase (trpEG) genes in Streptomyces venezuelae ISP5230 were located by allowing a segregationally unstable plasmid carrying cloned S. venezuelae trpEG DNA and a thiostrepton resistance (tsr) marker to integrate into the chromosome. The integrated tsr was mapped by conjugation and transduction to a location close to tyr-2, between arg-6 and trpA13. A genomic DNA fragment containing trpC from S. venezuelae ISP5230 was cloned by complementation of a trpC mutation in Streptomyces lividans. Evidence from restriction enzyme analysis of the cloned DNA fragments, from Southern hybridization using the cloned trp DNA as probes, and from cotransduction frequencies, placed trpEG at a distance of 12-45 kb from the trpCBA cluster. The overall arrangement of tryptophan biosynthesis genes in the S. venezuelae chromosome differs from that in other bacteria examined so far.19938515229
50380.8298Interaction of the chromosomal Tn 551 with two thermosensitive derivatives, pS1 and p delta D, of the plasmid pI9789 in Staphylococcus aureus. The plasmid pI9789::Tn552 carries genes conferring resistance to penicillins and to cadmium, mercury and arsenate ions. The presence of Tn551 at one location in the chromosome of Staphylococcus aureus enhances the frequency of suppression of thermosensitivity of replication of the plasmids pS1 and p delta D which are derivatives of pI9789::Tn552. Bacteriophage propagated on the bacteria in which thermosensitivity of replication had been suppressed was used to transduce cadmium resistance to S. aureus PS80N. The cadmium-resistant transductants obtained carried plasmid pS1 or p delta D with a copy of Tn551 inserted into a specific site on pS1 but into several different sites on p delta D. The possible mechanisms of the suppression are discussed.19957758929
82590.8297Attaching effacement of the rabbit enterocyte brush border is encoded on a single 96.5-kilobase-pair plasmid in an enteropathogenic Escherichia coli O111 strain. An enteropathogenic Escherichia coli (EPE) O111 serotype a,b,H- strain carried the following four plasmids: pLV501 (96.5 kilobase pairs [kbp]) specifying resistance to chloramphenicol, tetracycline, and kanamycin; pLV502 (8 kbp) specifying ampicillin resistance; pLV503 (1.9 kbp) specifying streptomycin resistance; and pLV504 (80 kbp) with no resistance markers. This EPEC attached to HEp-2 cells to produce localized clumps of bacteria (localized adhesion) and attached intimately to the enterocyte surface, leading to loss of the brush border (attaching effacement). Plasmid pLV501 was also found to specify the ability to produce localized adhesion on HEp-2 cells and attaching effacement in a rabbit ileal explant model system. Restriction maps showed considerable dissimilarities between pLV501 and pMAR-2, an EPEC plasmid carrying the EPEC adherence factor (EAF) genes. Furthermore, pLV501 did not hybridize with the EAF probe, whereas pLV504 did. There was sequence homology between pLV501 and large plasmids in all seven other well-characterized EPEC, only five of which hybridized with the EAF probe. These findings indicate that pLV501 carries at least one of the genes responsible for production of the brush border damage characteristic of EPEC.19902182541
3018100.8295The 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
816110.8286High-Level Nickel Resistance in Alcaligenes xylosoxydans 31A and Alcaligenes eutrophus KTO2. Two new nickel-resistant strains of Alcaligenes species were selected from a large number (about 400) of strains isolated from ecosystems polluted by heavy metals and were studied on the physiological and molecular level. Alcaligenes xylosoxydans 31A is a heterotrophic bacterium, and Alcaligenes eutrophus KTO2 is an autotrophic aerobic hydrogen-oxidizing bacterium. Both strains carry-among other plasmids-a megaplasmid determining resistance to 20 to 50 mM NiCl(2) and 20 mM CoCl(2) (when growing in defined Tris-buffered media). Megaplasmids pTOM8, pTOM9 from strain 31A, and pGOE2 from strain KTO2 confer nickel resistance to the same degree to transconjugants of all strains of A. eutrophus tested but were not transferred to Escherichia coli. However, DNA fragments carrying the nickel resistance genes, cloned into broad-hostrange vector pVDZ'2, confer resistance to A. eutrophus derivatives as well as E. coli. The DNA fragments of both bacteria, TBA8, TBA9, and GBA (14.5-kb BamHI fragments), appear to be identical. They share equal size, restriction maps, and strong DNA homology but are largely different from fragment HKI of nickel-cobalt resistance plasmid pMOL28 of A. eutrophus CH34.199116348590
817120.8283Mercury resistance transposons in Bacilli strains from different geographical regions. A total of 65 spore-forming mercury-resistant bacteria were isolated from natural environments worldwide in order to understand the acquisition of additional genes by and dissemination of mercury resistance transposons across related Bacilli genera by horizontal gene movement. PCR amplification using a single primer complementary to the inverted repeat sequence of TnMERI1-like transposons showed that 12 of 65 isolates had a transposon-like structure. There were four types of amplified fragments: Tn5084, Tn5085, Tn(d)MER3 (a newly identified deleted transposon-like fragment) and Tn6294 (a newly identified transposon). Tn(d)MER3 is a 3.5-kb sequence that carries a merRETPA operon with no merB or transposase genes. It is related to the mer operon of Bacillus licheniformis strain FA6-12 from Russia. DNA homology analysis shows that Tn6294 is an 8.5-kb sequence that is possibly derived from Tn(d)MER3 by integration of a TnMERI1-type transposase and resolvase genes and in addition the merR2 and merB1 genes. Bacteria harboring Tn6294 exhibited broad-spectrum mercury resistance to organomercurial compounds, although Tn6294 had only merB1 and did not have the merB2 and merB3 sequences for organomercurial lyases found in Tn5084 of B. cereus strain RC607. Strains with Tn6294 encode mercuric reductase (MerA) of less than 600 amino acids in length with a single N-terminal mercury-binding domain, whereas MerA encoded by strains MB1 and RC607 has two tandem domains. Thus, Tn(d)MER3 and Tn6294 are shorter prototypes for TnMERI1-like transposons. Identification of Tn6294 in Bacillus sp. from Taiwan and in Paenibacillus sp. from Antarctica indicates the wide horizontal dissemination of TnMERI1-like transposons across bacterial species and geographical barriers.201626802071
403130.8277Nucleotide sequence and expression of the mercurial-resistance operon from Staphylococcus aureus plasmid pI258. The mercurial-resistance determinant from Staphylococcus aureus plasmid pI258 is located on a 6.4-kilobase-pair Bgl II fragment. The determinant was cloned into both Bacillus subtilis and Escherichia coli. Mercury resistance was found only in B. subtilis. The 6404-base-pair DNA sequence of the Bgl II fragment was determined. The mer DNA sequence includes seven open reading frames, two of which have been identified by homology with the merA (mercuric reductase) and merB (organomercurial lyase) genes from the mercurial-resistance determinants of Gram-negative bacteria. Whereas 40% of the amino acid residues overall were identical between the pI258 merA polypeptide product and mercuric reductases from Gram-negative bacteria, the percentage identity in the active-site positions and those thought to be involved in NADPH and FAD contacts was above 90%. The 216 amino acid organomercurial lyase sequence was 39% identical with that from a Serratia plasmid, with higher conservation in the middle of the sequences and lower homologies at the amino and carboxyl termini. The remaining five open reading frames in the pI258 mer sequence have no significant homologies with the genes from previously sequenced Gram-negative mer operons.19873037534
3049140.8272Characterisation of plasmids purified from Acetobacter pasteurianus 2374. Four cryptic plasmids pAP1, pAP2, pAP3, and pAP4 with their replication regions AP were isolated from Gram-negative bacteria Acetobacter pasteurianus 2374 and characterised by sequence analyses. All plasmids were carrying the kanamycin resistance gene. Three of four plasmids pAP2, pAP3, and pAP4 encode an enzyme that confers ampicillin resistance to host cells. Moreover, the tetracycline resistance gene was identified only in pAP2 plasmid. All plasmids are capable to coexist with each other in Acetobacter cells. On the other hand, the coexistence of more than one plasmid is excluded in Escherichia coli. The nucleotide sequence of replication regions showed significant homology. The nucleotide and protein sequence analyses of resistance genes of all plasmids were compared with transposons Tn3, Tn10, and Tn903 which revealed significant differences in the primary structure, however no functional changes of gene were obtained.200314511653
818150.8267Characterization of a staphylococcal plasmid related to pUB110 and carrying two novel genes, vatC and vgbB, encoding resistance to streptogramins A and B and similar antibiotics. We isolated and sequenced a plasmid, named pIP1714 (4,978 bp), which specifies resistance to streptogramins A and B and the mixture of these compounds. pIP1714 was isolated from a Staphylococcus cohnii subsp. cohnii strain found in the environment of a hospital where pristinamycin was extensively used. Resistance to both compounds and related antibiotics is encoded by two novel, probably cotranscribed genes, (i) vatC, encoding a 212-amino-acid (aa) acetyltransferase that inactivates streptogramin A and that exhibits 58.2 to 69.8% aa identity with the Vat, VatB, and SatA proteins, and (ii) vgbB, encoding a 295-aa lactonase that inactivates streptogramin B and that shows 67% aa identity with the Vgb lactonase. pIP1714 includes a 2,985-bp fragment also found in two rolling-circle replication and mobilizable plasmids, pUB110 and pBC16, from gram-positive bacteria. In all three plasmids, the common fragment was delimited by two direct repeats of four nucleotides (GGGC) and included (i) putative genes closely related to repB, which encodes a replication protein, and to pre(mob), which encodes a protein required for conjugative mobilization and site-specific recombination, and (ii) sequences very similar to the double- and single-strand origins (dso, ssoU) and the recombination site, RSA. The antibiotic resistance genes repB and pre(mob) carried by each of these plasmids were found in the same transcriptional orientation.19989661023
407160.8261Molecular cloning and characterization of two lincomycin-resistance genes, lmrA and lmrB, from Streptomyces lincolnensis 78-11. Two different lincomycin-resistance determinants (lmrA and lmrB) from Streptomyces lincolnensis 78-11 were cloned in Streptomyces lividans 66 TK23. The gene lmrA was localized on a 2.16 kb fragment, the determined nucleotide sequence of which encoded a single open reading frame 1446 bp long. Analysis of the deduced amino acid sequence suggested the presence of 12 membrane-spanning domains and showed significant similarities to the methylenomycin-resistance protein (Mmr) from Streptomyces coelicolor, the QacA protein from Staphylococcus aureus, and several tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria, as well as to some sugar-transport proteins from Escherichia coli. The lmrB gene was actively expressed from a 2.7 kb fragment. An open reading frame of 837 bp could be localized which encoded a protein that was significantly similar to 23S rRNA adenine(2058)-N-methyltransferases conferring macrolide-lincosamide-streptogramin resistance. LmrB also had putative rRNA methyltransferase activity since lincomycin resistance of ribosomes was induced in lmrB-containing strains. Surprisingly, both enzymes, LmrA and LmrB, had a substrate specificity restricted to lincomycin and did not cause resistance to other lincosamides such as celesticetin and clindamycin, or to macrolides.19921328813
5873170.8257pDB2011, a 7.6 kb multidrug resistance plasmid from Listeria innocua replicating in Gram-positive and Gram-negative hosts. pDB2011, a multidrug resistance plasmid isolated from the foodborne Listeria innocua strain TTS-2011 was sequenced and characterized. Sequence analysis revealed that pDB2011 had a length of 7641 bp and contained seven coding DNA sequences of which two were annotated as replication proteins, one as a recombination/mobilization protein and one as a transposase. Furthermore, pDB2011 harbored the trimethoprim, spectinomycin and macrolide-lincosamide-streptogramin B resistance genes dfrD, spc and erm(A), respectively. However, pDB2011 was only associated with trimethoprim and spectinomycin resistance phenotypes and not with phenotypic resistance to erythromycin. A region of the plasmid encoding the resistance genes spc and erm(A) plus the transposase was highly similar to Staphylococcus aureus transposon Tn554. The dfrD gene was 100% identical to dfrD found in a number of Listeria monocytogenes isolates. Additionally, assessment of the potential host range of pDB2011 revealed that the plasmid was able to replicate in Lactococcus lactis subsp. cremoris MG1363 as well as in Escherichia coli MC1061 and DH5α. This study reports the first multidrug resistance plasmid in L. innocua. A large potential for dissemination of pDB2011 is indicated by its host range of both Gram-positive and Gram-negative bacteria.201323774482
5209180.8256Complete Nucleotide Sequence of pGA45, a 140,698-bp IncFIIY Plasmid Encoding bla IMI-3-Mediated Carbapenem Resistance, from River Sediment. Plasmid pGA45 was isolated from the sediments of Haihe River using Escherichia coli CV601 (gfp-tagged) as recipients and indigenous bacteria from sediment as donors. This plasmid confers reduced susceptibility to imipenem which belongs to carbapenem group. Plasmid pGA45 was fully sequenced on an Illumina HiSeq 2000 sequencing system. The complete sequence of plasmid pGA45 was 140,698 bp in length with an average G + C content of 52.03%. Sequence analysis shows that pGA45 belongs to IncFIIY group and harbors a backbone region which shares high homology and gene synteny to several other IncF plasmids including pNDM1_EC14653, pYDC644, pNDM-Ec1GN574, pRJF866, pKOX_NDM1, and pP10164-NDM. In addition to the backbone region, plasmid pGA45 harbors two notable features including one bla IMI-3-containing region and one type VI secretion system region. The bla IMI-3-containing region is responsible for bacteria carbapenem resistance and the type VI secretion system region is probably involved in bacteria virulence, respectively. Plasmid pGA45 represents the first complete nucleotide sequence of the bla IMI-harboring plasmid from environment sample and the sequencing of this plasmid provided insight into the architecture used for the dissemination of bla IMI carbapenemase genes.201626941718
821190.8254DNA 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