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34600.9213Horizontal transfer of CS1 pilin genes of enterotoxigenic Escherichia coli. CS1 is one of a limited number of serologically distinct pili found in enterotoxigenic Escherichia coli (ETEC) strains associated with disease in people. The genes for the CS1 pilus are on a large plasmid, pCoo. We show that pCoo is not self-transmissible, although our sequence determination for part of pCoo shows regions almost identical to those in the conjugative drug resistance plasmid R64. When we introduced R64 into a strain containing pCoo, we found that pCoo was transferred to a recipient strain in mating. Most of the transconjugant pCoo plasmids result from recombination with R64, leading to acquisition of functional copies of all of the R64 transfer genes. Temporary coresidence of the drug resistance plasmid R64 with pCoo leads to a permanent change in pCoo so that it is now self-transmissible. We conclude that when R64-like plasmids are transmitted to an ETEC strain containing pCoo, their recombination may allow for spread of the pCoo plasmid to other enteric bacteria.200415126486
299710.9130Genomic Characterization of Multidrug-Resistant Escherichia coli BH100 Sub-strains. The rapid emergence of multidrug-resistant (MDR) bacteria is a global health problem. Mobile genetic elements like conjugative plasmids, transposons, and integrons are the major players in spreading resistance genes in uropathogenic Escherichia coli (UPEC) pathotype. The E. coli BH100 strain was isolated from the urinary tract of a Brazilian woman in 1974. This strain presents two plasmids carrying MDR cassettes, pBH100, and pAp, with conjugative and mobilization properties, respectively. However, its transposable elements have not been characterized. In this study, we attempted to unravel the factors involved in the mobilization of virulence and drug-resistance genes by assessing genomic rearrangements in four BH100 sub-strains (BH100 MG2014, BH100 MG2017, BH100L MG2017, and BH100N MG2017). Therefore, the complete genomes of the BH100 sub-strains were achieved through Next Generation Sequencing and submitted to comparative genomic analyses. Our data shows recombination events between the two plasmids in the sub-strain BH100 MG2017 and between pBH100 and the chromosome in BH100L MG2017. In both cases, IS3 and IS21 elements were detected upstream of Tn21 family transposons associated with MDR genes at the recombined region. These results integrated with Genomic island analysis suggest pBH100 might be involved in the spreading of drug resistance through the formation of resistance islands. Regarding pathogenicity, our results reveal that BH100 strain is closely related to UPEC strains and contains many IS3 and IS21-transposase-enriched genomic islands associated with virulence. This study concludes that those IS elements are vital for the evolution and adaptation of BH100 strain.202033584554
82220.9118Exoglucanase-encoding genes from three Wickerhamomyces anomalus killer strains isolated from olive brine. Wickerhamomyces anomalus killer strains are important for fighting pathogenic yeasts and for controlling harmful yeasts and bacteria in the food industry. Targeted disruption of key genes in β-glucan synthesis of a sensitive Saccharomyces cerevisiae strain conferred resistance to the toxins of W. anomalus strains BS91, BCA15 and BCU24 isolated from olive brine. Competitive inhibition of the killing activities by laminarin and pustulan refer to β-1,3- and β-1,6-glucans as the main primary toxin targets. The extracellular exoglucanase-encoding genes WaEXG1 and WaEXG2 from the three strains were sequenced and were found to display noticeable similarities to those from known potent W. anomalus killer strains.201323148020
53230.9118Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain.199910514571
82540.9112Attaching 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
82650.9112Sequence 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
53560.9112Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria. Improved broad-host-range plasmid vectors were constructed based on existing plasmids RSF1010 and RK404. The new plasmids pDSK509, pDSK519, and pRK415, have several additional cloning sites and improved antibiotic-resistance genes which facilitate subcloning and mobilization into various Gram-negative bacteria. Several new polylinker sites were added to the Escherichia coli plasmids pUC118 and pUC119, resulting in the new plasmids, pUC128 and pUC129. These plasmids facilitate the transfer of cloned DNA fragments to the broad-host-range vectors. Finally, the broad-host-range cosmid cloning vector pLAFR3 was improved by the addition of a double cos casette to generate the new plasmid, pLAFR5. This latter cosmid simplifies vector preparation and has permitted the rapid cloning of genomic DNA fragments generated with Sau3A. The resulting clones may be introduced into other Gram-negative bacteria by conjugation.19882853689
300870.9108Sequence 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
139580.9108Emerging Multidrug-Resistant Hybrid Pathotype Shiga Toxin-Producing Escherichia coli O80 and Related Strains of Clonal Complex 165, Europe. Enterohemorrhagic Escherichia coli serogroup O80, involved in hemolytic uremic syndrome associated with extraintestinal infections, has emerged in France. We obtained circularized sequences of the O80 strain RDEx444, responsible for hemolytic uremic syndrome with bacteremia, and noncircularized sequences of 35 O80 E. coli isolated from humans and animals in Europe with or without Shiga toxin genes. RDEx444 harbored a mosaic plasmid, pR444_A, combining extraintestinal virulence determinants and a multidrug resistance-encoding island. All strains belonged to clonal complex 165, which is distantly related to other major enterohemorrhagic E. coli lineages. All stx-positive strains contained eae-ξ, ehxA, and genes characteristic of pR444_A. Among stx-negative strains, 1 produced extended-spectrum β-lactamase, 1 harbored the colistin-resistance gene mcr1, and 2 possessed genes characteristic of enteropathogenic and pyelonephritis E. coli. Because O80-clonal complex 165 strains can integrate intestinal and extraintestinal virulence factors in combination with diverse drug-resistance genes, they constitute dangerous and versatile multidrug-resistant pathogens.201830457551
305990.9107Genome 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
1396100.9106Genomic Characterization of hlyF-positive Shiga Toxin-Producing Escherichia coli, Italy and the Netherlands, 2000-2019. Shiga toxin-producing Escherichia coli (STEC) O80:H2 has emerged in Europe as a cause of hemolytic uremic syndrome associated with bacteremia. STEC O80:H2 harbors the mosaic plasmid pR444_A, which combines several virulence genes, including hlyF and antimicrobial resistance genes. pR444_A is found in some extraintestinal pathogenic E. coli (ExPEC) strains. We identified and characterized 53 STEC strains with ExPEC-associated virulence genes isolated in Italy and the Netherlands during 2000-2019. The isolates belong to 2 major populations: 1 belongs to sequence type 301 and harbors diverse stx(2) subtypes, the intimin variant eae-ξ, and pO157-like and pR444_A plasmids; 1 consists of strains belonging to various sequence types, some of which lack the pO157 plasmid, the locus of enterocyte effacement, and the antimicrobial resistance-encoding region. Our results showed that STEC strains harboring ExPEC-associated virulence genes can include multiple serotypes and that the pR444_A plasmid can be acquired and mobilized by STEC strains.202133622476
3021110.9104Sequencing 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
3060120.9102Integron mobilization unit as a source of mobility of antibiotic resistance genes. Antibiotic resistance genes are spread mostly through plasmids, integrons (as a form of gene cassettes), and transposons in gram-negative bacteria. We describe here a novel genetic structure, named the integron mobilization unit (IMU), that has characteristics similar to those of miniature inverted transposable elements (MITEs). Two IMUs (288 bp each) were identified from a carbapenem-resistant Enterobacter cloacae isolate that formed a composite structure encompassing a defective class 1 integron containing the carbapenem resistance gene bla(GES-5). This beta-lactamase gene was located on a 7-kb IncQ-type plasmid named pCHE-A, which was sequenced completely. The plasmid pCHE-A was not self conjugative but was mobilizable, and it was successfully transferred from E. cloacae to Pseudomonas aeruginosa. The in silico analysis of the extremities of the IMU elements identified similarities with those of insertion sequence ISSod9 from Shewanella oneidensis MR-1. The mobilization of the IMU composite structure was accomplished by using the transposase activity of ISSod9 that was provided in trans. This is the first identification of MITE-type structures as a source of gene mobilization, implicating here a clinically relevant antibiotic resistance gene.200919332679
6131130.9100Draft Genome Sequence of Eggerthia catenaformis Strain MAR1 Isolated from Saliva of Healthy Humans. Here, we report the draft genome sequence of Eggerthia catenaformis MAR1 isolated during a screen for d-cycloserine-resistant bacteria from the saliva of healthy humans. Analysis of the genome reveals that the strain has the potential to be a human pathogen and carries genes related to virulence and antibiotic resistance.201728705984
3036140.9100Complete 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
3038150.9099Biotinylated probes for epidemiological studies of drug resistance in Salmonella krefeld. A gene probe for ampicillin resistance and one for sulphonamide resistance were prepared to study the origin and the relation of multiple drug resistances in Salmonella krefeld. The resistance genes were cloned into the pACYC184 vector of Escherichia coli from a common plasmid of S. krefeld that encoded for resistance to ampicillin, chloramphenicol, kanamycin, streptomycin, sulphonamide and tetracycline resistance. Restriction map analysis and deletion analysis of a recombinant plasmid (pACSS1) showed that the gene determining ampicillin resistance was located on a 1.34 and 1.12 kb PstI fragment, and that the gene for sulphonamide resistance was located on a 0.85 kb PstI fragment. These fragments were used as probes. Their specificity was tested by colony hybridization with various bacterial species, including sensitive and resistance S. krefeld isolates. Further study indicated that the ampicillin resistance gene probe reacted with the gene for TEM-1 beta-lactamase and that the gene probe for sulphonamide resistance reacted with the gene for type II dihydropteroate synthase. The two probes were sufficiently specific to allow study of the epidemiology of resistance in S. krefeld and other enteric bacteria.19902190970
3018160.9099The 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
347170.9097A novel plasmid gene involved in bacteriophage PRD1 infection and conjugative host-range. PRD1 infects bacteria carrying IncN plasmids by binding to their conjugative pili. Mutations in a plasmid locus kikA close to the pilus region result in PRD1 resistance and reduced conjugation proficiency to Klebsiella but not to Escherichia coli. One of the two genes of kikA is sufficient to restore both normal phenotypes. PRD1 binds to cells carrying the mutant plasmid but fails to inject its genome.19968812786
359180.9097Construction of shuttle cloning vectors for Bacteroides fragilis and use in assaying foreign tetracycline resistance gene expression. Shuttle vectors capable of replication in both Escherichia coli and Bacteroides fragilis have been developed. Conjugal transfer of these plasmids from E. coli to B. fragilis is facilitated by inclusion of the origin of transfer of the IncP plasmid RK2. The vectors pDK1 and pDK2 provide unique sites for cloning selectable markers in Bacteroides. pOA10 is a cosmid vector containing the replication region of pCP1 necessary for maintenance in Bacteroides. pDK3, pDK4.1, and pDK4.2 contain the Bacteroides clindamycin resistance gene allowing selection and maintenance in B. fragilis of plasmids containing inserted DNA fragments. pDK3 was used to test the expression in B. fragilis of five foreign tetracycline resistance (TcR) genes. The tetA, -B, and -C markers from facultative gram-negative bacteria, as well as a TcR determinant from Clostridium perfringens, did not express TcR in B. fragilis. The tetM gene, originally described in streptococci, encoded a small but reproducible increase of TcR in Bacteroides. These studies demonstrate the utility of shuttle vectors for introducing cloned genes into Bacteroides and underscore the differences in gene expression in these anaerobes.19883071818
348190.9096Conjugative 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