Molecular analysis of closely related copper- and streptomycin-resistance plasmids in Pseudomonas syringae pv. syringae. - Related Documents




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43501.0000Molecular analysis of closely related copper- and streptomycin-resistance plasmids in Pseudomonas syringae pv. syringae. The genetic relationship of a group of copper (Cur) and streptomycin (Smr) resistance plasmids and their Pseudomonas syringae pv. syringae hosts was examined. Each of these plasmids contained sequences homologous to the oriV and par sequences from pOSU900, a cryptic P. syringae pv. syringae plasmid. Analysis of restriction digest patterns of plasmid DNA indicated that the plasmids could be clustered into four groups; two of the groups contained multiple members which differed by only a few fragments. An analysis of the host P. syringae genotypes using the arbitrarily primed PCR technique and genomic DNA indicated that the host strains could be placed in groups similar to those resulting from analysis of plasmid DNA. Southern hybridization analyses of plasmid DNA indicated that each Smr plasmid contained sequences homologous to probes specific for the strA-strB Smr genes and the transposase and resolvase genes from Tn5393. All plasmids hybridized to two additional probes derived from P. syringae plasmid DNA, but none of the plasmids contained IS51 or IS801 sequences. Furthermore, Tn5393 was mobilized, presumably by transposition, between the incompatible plasmids pPSR5 and pPSR4 in P. syringae pv. syringae FF5. The variation in molecular structure of the closely related plasmids in this study is similar to that observed with antibiotic-resistance plasmids from clinical bacteria.19968700971
43410.9997Homologous Streptomycin Resistance Gene Present among Diverse Gram-Negative Bacteria in New York State Apple Orchards. The streptomycin resistance gene of Pseudomonas syringae pv. papulans Psp36 was cloned into Escherichia coli and used to develop a 500-bp DNA probe that is specific for streptomycin resistance in P. syringae pv. papulans. The probe is a portion of a 1-kb region shared by three different DNA clones of the resistance gene. In Southern hybridizations, the probe hybridized only with DNA isolated from streptomycin-resistant strains of P. syringae pv. papulans and not with the DNA of streptomycin-sensitive strains. Transposon insertions within the region of DNA shared by the three clones resulted in loss of resistance to streptomycin. Colony hybridization of bacteria isolated from apple leaves and orchard soil indicated that 39% of 398 streptomycin-resistant bacteria contained DNA that hybridized to the probe. These included all strains of P. syringae pv. papulans and some other fluorescent pseudomonads and nonfluorescent gram-negative bacteria, but none of the gram-positive bacteria. The same-size restriction fragments hybridized to the probe in P. syringae pv. papulans. Restriction fragment length polymorphism of this region was occasionally observed in strains of other taxonomic groups of bacteria. In bacteria other than P. syringae pv. papulans, the streptomycin resistance probe hybridized to different-sized plasmids and no relationship between plasmid size and taxonomic group or between plasmid size and orchard type, soil association, or leaf association could be detected.199116348415
44820.9995Gene-for-gene interactions of five cloned avirulence genes from Xanthomonas campestris pv. malvacearum with specific resistance genes in cotton. A total DNA clone bank of a strain of Xanthomonas campestris pv. malvacearum (Xcm) was constructed in the cosmid vector pSa747 and transfected into Escherichia coli. The Xcm strain carries at least nine identifiable avirulence (A) genes. Clones in E. coli were mated individually into a recombination-proficient Xcm isolate carrying no known A genes. Screening was for incompatibility on congenic cotton host lines that differ by single specific resistance (R) genes. Ten different cosmid clones conferring race-specific avirulence were recovered. In most cases, the same A gene clone was recovered independently several times. Using the congenic host lines and the merodiploid transconjugant pathogen strains, five of the A genes were shown to specifically interact, gene-for-gene, with individual R genes in the congenic cotton lines. Some A/R gene interactions appeared qualitatively different from others, suggesting that the physiological mechanism(s) of gene-for-gene specified incompatibility may be unique to the interactive gene pair. All A genes appeared to be chromosomally determined, three were found linked on a single 32-kilobase clone, and the rest were spaced more than 31 kilobases apart. Colinearity of the cosmid inserts with the Xcm recipient (carrying no known A genes) chromosome was demonstrated in two of the three tested. This and other evidence suggests that at least some A genes in bacteria may have the equivalent of virulence (a) alleles. The genetics of race specificity in this phytopathogenic bacterium appeared in all respects to be identical to that found in phytopathogenic fungi.198616593751
43030.9994Cloning 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
449940.9994Organization of two sulfonamide resistance genes on plasmids of gram-negative bacteria. The organization of two widely distributed sulfonamide resistance genes has been studied. The type I gene was linked to other resistance genes, like streptomycin resistance in R100 and trimethoprim resistance in R388 and other recently isolated plasmids from Sri Lanka. In R388, the sulfonamide resistance gene was transcribed from a promoter of its own, but in all other studied plasmids the linked genes were transcribed from a common promoter. This was especially established with a clone derived from plasmid R6-5, in which transposon mutagenesis showed that expression of sulfonamide resistance was completely dependent on the linked streptomycin resistance gene. The type II sulfonamide resistance gene was independently transcribed and found on two kinds of small resistance plasmids and also on large plasmids isolated from clinical material.19873032095
43350.9994Expression of the strA-strB streptomycin resistance genes in Pseudomonas syringae and Xanthomonas campestris and characterization of IS6100 in X. campestris. Expression of the strA-strB streptomycin resistance (SMr) genes was examined in Pseudomonas syringae pv. syringae and Xanthomonas campestris pv. vesicatoria. The strA-strB genes in P. syringae and X. campestris were encoded on elements closely related to Tn5393 from Erwinia amylovora and designated Tn5393a and Tn5393b, respectively. The putative recombination site (res) and resolvase-repressor (tnpR) genes of Tn5393 from E. amylovora, P syringae, and X. campestris were identical; however, IS6100 mapped within tnpR in X. campestris, and IS1133 was previously located downstream of tnpR in E. amylovora (C.-S Chiou and A. L. Jones, J. Bacteriol. 175:732-740, 1993). Transcriptional fusions (strA-strB::uidA) indicated that a strong promoter sequence was located within res in Tn5393a. Expression from this promoter sequence was reduced when the tnpR gene was present in cis position relative to the promoter. In X. campestris pv. vesicatoria, analysis of promoter activity with transcriptional fusions indicated that IS6100 increased the expression of strA-strB. Analysis of codon usage patterns and percent G+C in the third codon position indicated that IS6100 could have originated in a gram-negative bacterium. The data obtained in the present study help explain differences observed in the levels of SMr expressed by three genera which share common genes for resistance. Furthermore, the widespread dissemination of Tn5393 and derivatives in phytopathogenic prokaryotes confirms the importance of these bacteria as reservoirs of antibiotic resistance in the environment.19957487022
304360.9994The 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
49370.9994Mercury resistance transposons of gram-negative environmental bacteria and their classification. A total of 29 mercury resistance transposons were isolated from mercury-resistant environmental strains of proteobacteria collected in different parts of Eurasia and the USA and tested for hybridization with probes specific for transposase genes of known mercury resistance transposons. 9 were related to Tn21 in this test, 12 were related to Tn5053, 4 to Tn5041 and 1 to Tn5044; three transposons were negative in this test. Restriction mapping and DNA sequencing revealed that 12 transposons were identical or nearly identical to their corresponding relatives while the rest showed varying divergence from their closest relatives. Most of these previously unknown transposons apparently arose as a result of homologous or site-specific recombination. One of these, Tn5046, was completely sequenced, and shown to be a chimera with the mer operon and the transposition module derived from the transposons related to Tn5041 and to Tn5044, respectively. Transposon Tn5070, showing no hybridization with the specific probes used in this study, was also completely sequenced. The transposition module of Tn5070 was most closely related to that of Tn3 while the mer operon was most closely related to that of plasmid pMERPH. The merR of Tn5070 is transcribed in the same direction as the mer structural genes, which is typical for mer operons of gram-positive bacteria. Our data suggest that environmental bacteria may harbor many not yet recognized mercury resistance transposons and warrant their further inventory.200111763242
304680.9994Presence of STRA-STRB linked streptomycin-resistance genes in clinical isolate of Escherichia coil 2418. The streptomycin resistance of Escherichia coli 2418 strain has been shown to be associated with a 1.2-kb DNA fragment found in the naturally occurring plasmid R2418S. Here, nucleotide sequence analysis of the 1.2-kb DNA fragment revealed the presence of the strB gene which is located immediately downstream of the strA gene. Both sequences are identical to those of strA and strB genes in plasmid RSF1010. Thus, the observed resistance in the clinical isolate is due to the presence of strA-strB genes encoding streptomycin-modifying enzymes. The sequence downstream of strB gene showed a perfect homology with that of RSF1010. In addition, it contained the right inverted repeat of the transposon Tn5393 that has been suggested to be a relic of this transposon found in DNA plasmids isolated from human- and animal-associated bacteria.201021598829
44190.9994Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities. A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains. Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.19853994373
3587100.9994Distribution of the streptomycin-resistance transposon Tn5393 among phylloplane and soil bacteria from managed agricultural habitats. The distribution of the strA-strB streptomycin-resistance (Smr) genes associated with Tn5393 was examined in bacteria isolated from the phylloplane and soil of ornamental pear and tomato. Two ornamental pear nurseries received previous foliar applications of streptomycin, whereas the tomato fields had no prior exposure to streptomycin bactericides. Although the recovery of culturable Smr bacteria was generally higher from soil, the highest occurrence of Smr was observed in phylloplane bacteria of an ornamental pear nursery that received 15 annual applications of streptomycin during the previous 2 years. Twenty-two and 12% of 143 Gram-negative phylloplane and 163 Gram-negative soil isolates, respectively, contained sequences that hybridized to probes specific for the strA-strB Smr genes and for the transposase and resolvase genes of Tn5393. These sequences were located on large plasmids (> 60 kb) in 74% of the isolates. The 77 Smr Gram-positive bacteria isolated in the present study showed no homology to the Tn5393-derived probes. Although the repeated use of a single antibiotic in clinical situations is known to favor the development of strains with resistance to other antibiotics, we found no evidence that intensive streptomycin usage in agricultural habitats favors the development of resistance to tetracycline, an antibiotic also registered for disease control on plants. The detection of Tn5393 in bacteria with no prior exposure to streptomycin suggests that this transposon is indigenous to both phylloplane and soil microbial communities.19957585356
3044110.9993RSF1010 and a conjugative plasmid contain sulII, one of two known genes for plasmid-borne sulfonamide resistance dihydropteroate synthase. The nucleotide sequence of the type II sulfonamide resistance dihydropteroate synthase (sulII) gene was determined. The molecular weight determined by maxicells was 30,000, and the predicted molecular weight for the polypeptide was 28,469. Comparison with the sulI gene encoded by Tn21 showed 57% DNA similarity. The sulII-encoded polypeptide has 138 of 271 amino acids in common with the polypeptide encoded by sulI. The sulII gene is located on various IncQ (broad-host-range) plasmids and other small nonconjugative resistance plasmids. Detailed restriction maps were constructed to compare the different plasmids in which sulII is found. The large conjugative plasmid pGS05 and the IncQ plasmid RSF1010 contained identical nucleotide sequences for the sulII gene. This type of sulfonamide resistance is very frequently found among gram-negative bacteria because of its efficient spread to various plasmids.19883075438
455120.9993An inducible tellurite-resistance operon in Proteus mirabilis. Tellurite resistance (Te(r)) is widespread in nature and it is shown here that the natural resistance of Proteus mirabilis to tellurite is due to a chromosomally located orthologue of plasmid-borne ter genes found in enteric bacteria. The P. mirabilis ter locus (terZABCDE) was identified in a screen of Tn5lacZ-generated mutants of which one contained an insertion in terC. The P. mirabilis terC mutant displayed increased susceptibility to tellurite (Te(s)) and complementation with terC carried on a multicopy plasmid restored high-level Te(r). Primer extension analysis revealed a single transcriptional start site upstream of terZ, but only with RNA harvested from bacteria grown in the presence of tellurite. Northern blotting and reverse transcriptase-PCR (RT-PCR) analyses confirmed that the ter operon was inducible by tellurite and to a lesser extent by oxidative stress inducers such as hydrogen peroxide and methyl viologen (paraquat). Direct and inverted repeat sequences were identified in the ter promoter region as well as motifs upstream of the -35 hexamer that resembled OxyR-binding sequences. Finally, the 390 bp intergenic promoter region located between orf3 and terZ showed no DNA sequence identity with any other published ter sequences, whereas terZABCDE genes exhibited 73-85 % DNA sequence identity. The ter operon was present in all clinical isolates of P. mirabilis and Proteus vulgaris tested and is inferred for Morganella and Providencia spp. based on screening for high level Te(r) and preliminary PCR analysis. Thus, a chromosomally located inducible tellurite resistance operon appears to be a common feature of the genus Proteus.200312724390
379130.9993Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. A broad host range cloning vehicle that can be mobilized at high frequency into Gram-negative bacteria has been constructed from the naturally occurring antibiotic resistance plasmid RK2. The vehicle is 20 kilobase pairs in size, encodes tetracycline resistance, and contains two single restriction enzyme sites suitable for cloning. Mobilization is effected by a helper plasmid consisting of the RK2 transfer genes linked to a ColE1 replicon. By use of this plasmid vehicle, a gene bank of the DNA from a wild-type strain of Rhizobium meliloti has been constructed and established in Escherichia coli. One of the hybrid plasmids in the bank contains a DNA insert of approximately 26 kilobase pairs which has homology to the nitrogenase structural gene region of Klebsiella pneumoniae.19807012838
3045140.9993Plasmid-borne sulfonamide resistance determinants studied by restriction enzyme analysis. The relationship between sulfonamide resistance genes carried on different plasmids was investigated by restriction enzyme analysis and DNA-DNA hybridization. The results showed that sulfonamide resistance mediated by different plasmids is determined by the production of at least two different types of drug-resistant dihydropteroate synthase. Plasmids pGS01, pGS02, and R22259, found in bacteria isolated from patients in Swedish hospitals, contained identical sulfonamide resistance genes, which were also identical to those of plasmids R1, R100, R6, and R388. These latter plasmids, which have been well studied in different laboratories, were originally from clinical isolates from different parts of the world. Two other clinically isolated plasmids, pGS04 and pGS05, were shown to contain sulfonamide resistance determinants of a completely different type.19836298179
281150.9993Detection of potential transgenic plant DNA recipients among soil bacteria. The likelihood of gene transfer from transgenic plants to bacteria is dependent on gene number and the presence of homologous sequences. The large number of transgene copies in transplastomic (transgenes contained in the chloroplast genome) plant cells as well as the prokaryotic origin of the transgene, may thus significantly increase the likelihood of gene transfer to bacteria that colonize plant tissues. In order to assess the probability of such transfer, the length of homologous DNA sequences required between the transgene and the genome of the bacterial host was assessed. In addition, the probability that bacteria, which co-infect diseased plants, are transformable and have sequences similar to the flanking regions of the transgene was evaluated. Using Acinetobacter baylyi strain BD143 and transplastomic tobacco plants harboring the aadA gene (streptomycin and spectinomycin resistance), we found that sequences identical to the flanking regions containing as few as 55 nucleotides were sufficient for recombination to occur. Consequently, a collection of bacterial isolates able to colonize tobacco plant tissue infected by Ralstonia solanacearum strain K60 was obtained, screened for DNA sequence similarity with the chloroplastic genes accD and rbcL flanking the transgene, and tested for their ability to uptake extracellular DNA (broad host-range pBBR1MCS plasmids) by natural or electro-transformation. Results showed that among the 288 bacterial isolates tested, 8% presented DNA sequence similarity with one or both chloroplastic regions flanking the transgene. Two isolates, identified as Pseudomonas sp. and Acinetobacter sp., were able to integrate exogenous plasmid DNA by electro-transformation and natural transformation, respectively. Our data suggest that transplastomic plant DNA recipients might be present in soil bacterial communities.200717961481
1771160.9993Occurrence of integron-associated resistance gene cassettes located on antibiotic resistance plasmids isolated from a wastewater treatment plant. The role of a municipal wastewater treatment plant as a reservoir for bacteria carrying antibiotic resistance plasmids was analysed. Altogether, ninety-seven different multiresistance plasmids were isolated and screened by PCR for the presence of class 1 integron-specific sequences. Twelve of these plasmids were identified to carry integrons. In addition, integron-specific sequences were found on plasmid-DNA preparations from bacteria residing in activated sludge and in the final effluents of the wastewater treatment plant. Sequencing and annotation of the integrons identified nineteen different gene cassette arrays, containing twenty-one different resistance gene cassettes. These cassettes carry genes encoding eight different aminoglycoside-modifying enzymes, seven dihydrofolate reductases, three beta-lactamases, two chloramphenicol resistance proteins and two small exporter proteins. Moreover, new gene cassettes and cassettes with unknown function were identified. Eleven gene cassette combinations are described for the first time. Six integron-associated gene cassette arrays are located on self-transmissible, putative broad-host-range plasmids belonging to the IncP group. Hybridisation analyses, using the integron-specific gene cassette arrays as templates and labelled plasmid-DNA preparations from bacteria of the final effluents as hybridisation probes, revealed that bacteria containing integron-specific sequences on plasmids are released into the environment.200319719593
416170.9992Characterization of In0 of Pseudomonas aeruginosa plasmid pVS1, an ancestor of integrons of multiresistance plasmids and transposons of gram-negative bacteria. Many multiresistance plasmids and transposons of gram-negative bacteria carry related DNA elements that appear to have evolved from a common ancestor by site-specific integration of discrete cassettes containing antibiotic resistance genes or sequences of unknown function. The site of integration is flanked by conserved segments coding for an integraselike protein and for sulfonamide resistance, respectively. These segments, together with the antibiotic resistance genes between them, have been termed integrons (H. W. Stokes and R. M. Hall, Mol. Microbiol. 3:1669-1683, 1989). We report here the characterization of an integron, In0, from Pseudomonas aeruginosa plasmid pVS1, which has an unoccupied integration site and hence may be an ancestor of more complex integrons. Codon usage of the integrase (int) and sulfonamide resistance (sul1) genes carried by this integron suggests a common origin. This contrasts with the codon usage of other antibiotic resistance genes that were presumably integrated later as cassettes during the evolution and spread of these DNA elements. We propose evolutionary schemes for (i) the genesis of the integrons by the site-specific integration of antibiotic resistance genes and (ii) the evolution of the integrons of multiresistance plasmids and transposons, in relation to the evolution of transposons related to Tn21.19921310501
4528180.9992Study on the excision and integration mediated by class 1 integron in Streptococcus pneumoniae. As a novel antibiotic resistance mobile element, integron was recognized as a primary source of antibiotic genes among Gram-positive organisms for its excision and integration of exogenous genes. In this study, Streptococcus pneumoniae was subjected to investigate the excision and integration of class 1 integron with eight different plasmids. As the results indicated, excision in both att site and gene cassettes were successfully observed, which was further confirmed by integration assays and PCR amplification. The observation of class 1 integron mediated excision and integration of various exogenous antibiotics resistance genes may raise the attention of integrons as novel antibiotic resistance determinant in Gram-positive bacteria, especially in Streptococcus.201728923604
4465190.9992Genetic analyses of sulfonamide resistance and its dissemination in gram-negative bacteria illustrate new aspects of R plasmid evolution. In contrast to what has been observed for many other antibiotic resistance mechanisms, there are only two known genes encoding plasmid-borne sulfonamide resistance. Both genes, sulI and sulII, encode a drug-resistant dihydropteroate synthase enzyme. In members of the family Enterobacteriaceae isolated from several worldwide sources, plasmid-mediated resistance to sulfonamides could be identified by colony hybridization as being encoded by sulI, sulII, or both. The sulI gene was in all cases found to be located in the newly defined, mobile genetic element, recently named an integron, which has been shown to contain a site-specific recombination system for the integration of various antibiotic resistance genes. The sulII gene was almost exclusively found as part of a variable resistance region on small, nonconjugative plasmids. Colony hybridization to an intragenic probe, restriction enzyme digestion, and nucleotide sequence analysis of small plasmids indicated that the sulII gene and contiguous sequences represent an independently occurring region disseminated in the bacterial population. The sulII resistance region was bordered by direct repeats, which in some plasmids were totally or partially deleted. The prevalence of sulI and sulII could thus be accounted for by their stable integration in transposons and in plasmids that are widely disseminated among gram-negative bacteria.19911952855