Analysis of a Bacteroides Conjugative Transposon Using a Novel “Targeted Capture” Model System

Smith, C. Jeffrey; Parker, A C; Bacic, Melissa K.

doi:10.1006/plas.2001.1528

Cited by 7 publications

(7 citation statements)

References 23 publications

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“…The relevant bacterial strains and plasmids used in this study are described in Table 1. For many of the studies described in this report, the pFD699 model system was used (1,35). Plasmid pFD699 is a chimera of an E. coli plasmid and CTn341, and it is a fully functional conjugative transposon in Bacteroides strains but a stable plasmid in E. coli.…”

Section: Methodsmentioning

confidence: 99%

Genetic and Functional Analyses of the mob Operon on Conjugative Transposon CTn 341 from Bacteroides spp

2010

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Bacteroides are Gram-negative anaerobes indigenous to the intestinal tract of humans, and they are important opportunistic pathogens. Mobile genetic elements, such as conjugative transposons (CTns), have contributed to an increase in antibiotic resistance in these organisms. CTns are self-transmissible elements that belong to the superfamily of integrative and conjugative elements (ICEs). CTn341 is 52 kb; it encodes tetracycline resistance and its transfer is induced by tetracycline. The mobilization region of CTn341 was shown to be comprised of a three-gene operon, mobABC, and the transfer origin, oriT. The three genes code for a nicking accessory protein, a relaxase, and a VirD4-like coupling protein, respectively. The Mob proteins were predicted to mediate the formation of the relaxosome complex, nick DNA at the oriT, and shuttle the DNA/protein complex to the mating-pore apparatus. The results of mutational studies indicated that the three genes are required for maximal transfer of CTn341. Mob gene transcription was induced by tetracycline, and this regulation was mediated through the two-component regulatory system, RteAB. The oriT region of CTn341 was located within 100 bp of mobA, and a putative Bacteroides consensus nicking site was observed within this region. Mutation of the putative nick site resulted in a loss of transfer. This study demonstrated a role of the mobilization region for transfer of Bacteroides CTns and that tetracycline induction occurs for the mob gene operon, as for the tra gene operon(s), as shown previously.

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Section: Methodsmentioning

confidence: 99%

Genetic and Functional Analyses of the mob Operon on Conjugative Transposon CTn 341 from Bacteroides spp

2010

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“…Previously, CTn341 was captured in E. coli on a low-copy-number plasmid by using a simple strategy in which an E. coli plasmid bearing a portion of the CTn341 tetQ gene was inserted by singlecrossover, homologous recombination into CTn341 (41). The resulting construct, pFD699, is a stable plasmid in E. coli, and by using RK231 it can be mobilized from E. coli into Bacteroides hosts, where it does not replicate as a plasmid but is a fully functional conjugative transposon.…”

Section: Resultsmentioning

confidence: 99%

“…Chromosomal DNA from Bacteroides species was isolated from total cell lysates by a previously described method (39). Southern blot analysis of chromosomal DNA was carried out as described previously by using nylon membranes probed with biotinylated probes (41). Following overnight hybridization at 65°C, the membranes were washed under stringent conditions and then exposed to X-ray film for autoradiography.…”

Section: Methodsmentioning

confidence: 99%

“…Additional sequencing reactions were needed to resolve the sequence around the N terminus of the CTn341 tetQ gene, which was duplicated during the construction of pFD699 (41). In order to obtain an unaltered sequence from this region, a portion of the tetQ gene and upstream region was PCR amplified from genomic DNA of the original CTn341 host strain, Bacteroides vulgatus CLA341.…”

Section: Methodsmentioning

confidence: 99%

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Genetic and Structural Analysis of the Bacteroides Conjugative Transposon CTn341

et al. 2005

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The genetic structure and functional organization of a Bacteroides conjugative transposon (CTn), CTn341, were determined. CTn341 was originally isolated from a tetracycline-resistant clinical isolate of Bacteroides vulgatus. The element was 51,993 bp long, which included a 5-bp coupling sequence that linked the transposon ends in the circular form. There were 46 genes, and the corresponding gene products fell into three major functional groups: DNA metabolism, regulation and antibiotic resistance, and conjugation. The G؉C content and codon usage observed in the functional groups suggested that the groups belong to different genetic lineages, indicating that CTn341 is a composite, modular element. Mutational analysis of genes representing the different functional groups provided evidence for the gene assignments and showed that the basic conjugation and excision genes are conserved among Bacteroides spp. A group IIA1 intron, designated B.f.I1, was found to be inserted into the bmhA methylase gene. Reverse transcriptase PCR analysis of CTn341 RNA showed that B.fr.I1 was functional and was spliced out of the bmhA gene. Six related CTn-like elements were found in the genome sequences of Bacteroides fragilis NCTC9343 and Bacteroides thetaiotaomicron VPI5482. The putative elements were similar to CTn341 primarily in the tra and mob regions and in the exc gene, and several appeared to contain intron elements. Our data provide the first reported sequence for a complete Bacteroides CTn, and they should be of considerable benefit to further functional and genetic analyses of antibiotic resistance elements and genome evolution in Bacteroides.

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“…A transposon capture strategy for Prevotella was designed utilizing plasmid pFD665, a Bacteroides - E. coli suicide vector containing an E. coli pSC101 plasmid origin of replication and RK2 origin of transfer, and an ermF selectable marker for expression in members of the Bacteroidetes [29]. pFD665 replicates as a low-copy number plasmid in E. coli , and must integrate via homologous recombination into the chromosome to be maintained in a Bacteroidetes recipient.…”

Section: Methodsmentioning

confidence: 99%

Genetic analysis of mobile tetQ elements in oral Prevotella species

et al. 2010

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Prevotella species are members of the bacterial oral flora and are opportunistic pathogens in polymicrobial infections of soft tissues. Antibiotic resistance to tetracyclines is common in these bacteria, and the gene encoding this resistance has been previously identified as tetQ. The tetQ gene is also found on conjugative transposons in the intestinal Bacteroides species; whether these related bacteria have transmitted tetQ to Prevotella is unknown. In this study, we describe our genetic analysis of mobile tetQ elements in oral Prevotella species. Our results indicate that the mobile elements encoding tetQ in oral species are distinct from those found in the Bacteroides. The intestinal bacteria may act as a reservoir for the tetQ gene, but Prevotella has incorporated this gene into an IS21-family transposon. This transposon is present in Prevotella species from more than one geographical location, implying that the mechanism of tetQ spread between oral Prevotella species is highly conserved.

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Analysis of a Bacteroides Conjugative Transposon Using a Novel “Targeted Capture” Model System

Cited by 7 publications

References 23 publications

Genetic and Functional Analyses of the mob Operon on Conjugative Transposon CTn 341 from Bacteroides spp

Genetic and Functional Analyses of the mob Operon on Conjugative Transposon CTn 341 from Bacteroides spp

Genetic and Structural Analysis of the Bacteroides Conjugative Transposon CTn341

Genetic analysis of mobile tetQ elements in oral Prevotella species

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