Expression of Escherichia coli K-12 arginine genes in Pseudomonas fluorescens.
(17/3280)
Escherichia coli argE and argH gene products were detected in Pseudomonas fluorescens argH122 carrying the E. coli F110 plasmid. (+info)
Replication mechanism and sequence analysis of the replicon of pAW63, a conjugative plasmid from Bacillus thuringiensis.
(18/3280)
A 5.8-kb fragment of the large conjugative plasmid pAW63 from Bacillus thuringiensis subsp. kurstaki HD73 containing all the information for autonomous replication was cloned and sequenced. By deletion analysis, the pAW63 replicon was reduced to a 4.1-kb fragment harboring four open reading frames (ORFs). Rep63A (513 amino acids [aa]), encoded by the largest ORF, displayed strong similarity (40% identity) to the replication proteins from plasmids pAMbeta1, pIP501, and pSM19035, indicating that the pAW63 replicon belongs to the pAMbeta1 family of gram-positive theta-replicating plasmids. This was confirmed by the facts that no single-stranded DNA replication intermediates could be detected and that replication was found to be dependent on host-gene-encoded DNA polymerase I. An 85-bp region downstream of Rep63A was also shown to have strong similarity to the origins of replication of pAMbeta1 and pIP501, and it is suggested that this region contains the bona fide pAW63 ori. The protein encoded by the second large ORF, Rep63B (308 aa), was shown to display similarity to RepB (34% identity over 281 aa) and PrgP (32% identity over 310 aa), involved in copy control of the Enterococcus faecalis plasmids pAD1 and pCF10, respectively. No significant similarity to known proteins or DNA sequences could be detected for the two smallest ORFs. However, the location, size, hydrophilicity, and orientation of ORF6 (107 codons) were analogous to those features of the putative genes repC and prgO, which encode stability functions on plasmids pAD1 and pCF10, respectively. The cloned replicon of plasmid pAW63 was stably maintained in Bacillus subtilis and B. thuringiensis and displayed incompatibility with the native pAW63. Hybridization experiments using the cloned replicon as a probe showed that pAW63 has similarity to large plasmids from other B. thuringiensis subsp. kurstaki strains and to a strain of B. thuringiensis subsp. alesti. (+info)
Survey of extended-spectrum beta-lactamases in clinical isolates of Escherichia coli and Klebsiella pneumoniae: prevalence of TEM-52 in Korea.
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Two hundred ninety isolates of Escherichia coli were investigated for the production of extended-spectrum beta-lactamases (ESBLs). Fourteen (4.8%) of the 290 strains were found to produce ESBLs. Each of the 14 strains produced one or two ESBLs, as follows: 10 strains produced TEM-52, 1 strain produced SHV-2a, 1 strain produced SHV-12, 1 strain produced a CMY-1-like enzyme, and 1 strain expressed SHV-2a and a CMY-1-like enzyme. Another two strains for which the MICs of ceftazidime and cefoxitin were high, were probable AmpC enzyme hyperproducers. Because of the high prevalence of TEM-52 in E. coli isolates, we further investigated the TEM-type ESBLs produced by Klebsiella pneumoniae in order to observe the distribution of TEM-52 enzymes among Enterobacteriaceae in Korea. All TEM enzymes produced by 12 strains of K. pneumoniae were identified as TEM-52. To evaluate the genetic relatedness among the organisms, ribotyping of TEM-52-producing E. coli and K. pneumoniae was performed. The ribotyping profiles of the organisms showed similar but clearly different patterns. In conclusion, TEM-52 is the most prevalent TEM-type ESBL in Korea. (+info)
Conjugal transfer but not quorum-dependent tra gene induction of pTiC58 requires a solid surface.
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Donors of Agrobacterium tumefaciens harboring a transfer-constitutive derivative of the nopaline-type Ti plasmid pTiC58 transferred this element at frequencies 3 to 4 orders of magnitude higher in matings conducted on solid surfaces than in those conducted in liquid medium. However, as measured with a lacZ reporter fusion, the tra genes of the wild-type Ti plasmid were inducible by opines to indistinguishable levels on solid and in liquid medium. Donors induced in liquid transferred the Ti plasmid at high frequency when mated with recipients on solid medium. We conclude that while formation of stable mating pairs and subsequent transfer of the Ti plasmid is dependent on a solid stratum, the regulatory system can activate tra gene expression to equivalent levels in liquid and on solid surfaces. (+info)
Erythromycin-resistant Neisseria gonorrhoeae and oral commensal Neisseria spp. carry known rRNA methylase genes.
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Two Neisseria gonorrhoeae isolates from Seattle and two isolates from Uruguay were resistant to erythromycin (MIC, 4 to 16 microg/ml) and had reduced susceptibility to azithromycin (MIC, 1 to 4 microg/ml) due to the presence of the self-mobile rRNA methylase gene(s) ermF or ermB and ermF. The two Seattle isolates and one isolate from Uruguay were multiresistant, carrying either the 25.2-MDa tetM-containing plasmid (Seattle) or a beta-lactamase plasmid (Uruguay). Sixteen commensal Neisseria isolates (10 Neisseria perflava-N. sicca, 2 N. flava, and 4 N. mucosa) for which erythromycin MICs were 4 to 16 microg/ml were shown to carry one or more known rRNA methylase genes, including ermB, ermC, and/or ermF. Many of these isolates also were multiresistant and carried the tetM gene. This is the first time that a complete transposon or a complete conjugative transposon carrying an antibiotic resistance gene has been described for the genus Neisseria. (+info)
Conjugative mobilization of the rolling-circle plasmid pIP823 from Listeria monocytogenes BM4293 among gram-positive and gram-negative bacteria.
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We determined the sequence and genetic organization of plasmid pIP823, which contains the dfrD gene; dfrD confers high-level trimethoprim resistance to Listeria monocytogenes BM4293 by synthesis of dihydrofolate reductase type S2. pIP823 possessed all the features of the pUB110/pC194 plasmid family, whose members replicate by the rolling-circle mechanism. The rep gene encoded a protein identical to RepU, the protein required for initiation of the replication of plasmids pTB913 from a thermophilic Bacillus sp. and pUB110 from Staphylococcus aureus. The mob gene encoded a protein with a high degree of amino acid identity with the Mob proteins involved in conjugative mobilization and interplasmidic recombination of pTB913 and pUB110. The host range of pIP823 was broad and included L. monocytogenes, Enterococcus faecalis, S. aureus, Bacillus subtilis, and Escherichia coli. In all these species, pIP823 replicated by generating single-stranded DNA and was stable. Conjugative mobilization of pIP823 was obtained by self-transferable plasmids between L. monocytogenes and E. faecalis, between L. monocytogenes and E. coli, and between strains of E. coli, and by the streptococcal conjugative transposon Tn1545 from L. monocytogenes to E. faecalis, and from L. monocytogenes and E. faecalis to E. coli. These data indicate that the gene flux observed in nature from gram-positive to gram-negative bacteria can occur by conjugative mobilization. Our results suggest that dissemination of trimethoprim resistance in Listeria spp. and acquisition of other antibiotic resistance determinants in this species can be anticipated. (+info)
Structural and functional roles of the surface-exposed loops of the beta-barrel membrane protein OmpA from Escherichia coli.
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The N-terminal domain of the OmpA protein from Escherichia coli, consisting of 170 amino acid residues, is embedded in the outer membrane, in the form of an antiparallel beta-barrel whose eight transmembrane beta-strands are connected by three short periplasmic turns and four relatively large surface-exposed hydrophilic loops. This protein domain serves as a paradigm for the study of membrane assembly of integral beta-structured membrane proteins. In order to dissect the structural and functional roles of the surface-exposed loops, they were shortened separately and in all possible combinations. All 16 loop deletion mutants assembled into the outer membrane with high efficiency and adopted the wild-type membrane topology. This systematic approach proves the absence of topogenic signals (e.g., in the form of loop sizes or charge distributions) in these loops. The shortening of surface-exposed loops did not reduce the thermal stability of the protein. However, none of the mutant proteins, with the exception of the variant with the fourth loop shortened, served as a receptor for the OmpA-specific bacteriophage K3. Furthermore, all loops were necessary for the OmpA protein to function in the stabilization of mating aggregates during F conjugation. An OmpA deletion variant with all four loops shortened, consisting of only 135 amino acid residues, constitutes the smallest beta-structured integral membrane protein known to date. These results represent a further step toward the development of artificial outer membrane proteins. (+info)
Plasmid heterogeneity and identification of a Tn5281-like element in clinical isolates of high-level gentamicin-resistant Enterococcus faecium isolated in the UK.
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Ten clinical isolates of high-level gentamicin-resistant (HLGR) Enterococcus faecium, collected from six hospitals throughout the UK, were studied to determine whether HLGR was attributed to widespread dissemination of a single plasmid or whether different plasmid types were implicated in the dissemination of this phenotype. HLGR was attributed to the presence of the AAC6'-APH2" bifunctional aminoglycoside modifying enzyme. The aac6'-aph2" gene was present on a 70 kb plasmid in all ten isolates. Conjugation studies indicated that the HLGR marker could transfer with varying frequency, with or without the associated 70 kb plasmid. Detailed molecular genetic analysis suggested that four of the isolates harboured a transposon similar to Tn5281, originally identified in Enterococcus faecalis strain HH22 isolated in the USA. The UK transposon, however, lacked the two symmetrically located HaeIII sites found in Tn5281. The six remaining isolates appeared to have a Tn5281-truncated structure in which the aac6'-aph2" gene is flanked by an IS256 element at the 5' end. Further studies with nine restriction endonucleases showed that the aac6'-aph2" gene was associated with two different plasmid types in E. faecium. Pulsed-field gel electrophoresis (PFGE) analysis identified three different patterns. The four E. faecium isolates harbouring the Tn5281-like structure were indistinguishable from each other, while the remaining six isolates exhibited two distinct PFGE patterns. This is the first study to indicate that there is heterogeneity among the plasmids that confer the HLGR phenotype in E. faecium isolates in the UK and to report on the presence of a transposon, similar to Tn5281, in E. faecium harbouring the aac6'-aph2" gene. (+info)