Dataset Information

Regulation of excision genes of the Bacteroides conjugative transposon CTnDOT.

ABSTRACT: The first step in the transfer of the Bacteroides conjugative transposon CTnDOT is excision of the integrated element from the chromosome to form a circular transfer intermediate. Excision occurs only after the bacteria are exposed to tetracycline. Previously, four excision genes were identified. One was the integrase gene intDOT, which appeared to be expressed constitutively. Three other genes essential for excision (orf2c, orf2d, and exc) were found located in a cluster 13 kbp downstream of intDOT. By using uidA fusions and real-time reverse transcriptase PCR, we demonstrate here that the excision genes orf2c, orf2d, and exc are part of an operon that also contains open reading frame orf3, previously shown not to be essential for excision. We also show that operon expression is regulated at the transcriptional level in response to tetracycline. The transcript start site for the operon has been localized. Three CTnDOT regulatory genes are thought to be involved in tetracycline regulation of excision, rteA, rteB, and rteC. By placing rteC under the control of a heterologous promoter, we found that RteC alone was sufficient for induction of the orf2c operon. If, however, the rteC gene was under the control of its own promoter, it was not able to induce orf2c operon expression unless rteA and rteB were present. Thus, RteA and RteB participate in excision by stimulating transcription of rteC. Using electrophoretic mobility shift analysis, we found that a purified His(6)-tagged form of RteC bound DNA upstream of the -33 region of the promoter. Changing the sequence in the region between bp -50 and -70 reduced the expression of the orf2c operon in vivo. Taken together, our results support the hypothesis that RteC acts as a DNA-binding protein that binds upstream of the orf2c promoter and is responsible for tetracycline-regulated transcriptional regulation of the orf2c operon.

SUBMITTER: Moon K

PROVIDER: S-EPMC1196058 | biostudies-literature | 2005 Aug

REPOSITORIES: biostudies-literature

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Regulation of excision genes of the Bacteroides conjugative transposon CTnDOT.

Moon Kyung K Shoemaker Nadja B NB Gardner Jeffrey F JF Salyers Abigail A AA

Journal of bacteriology 20050801 16

The first step in the transfer of the Bacteroides conjugative transposon CTnDOT is excision of the integrated element from the chromosome to form a circular transfer intermediate. Excision occurs only after the bacteria are exposed to tetracycline. Previously, four excision genes were identified. One was the integrase gene intDOT, which appeared to be expressed constitutively. Three other genes essential for excision (orf2c, orf2d, and exc) were found located in a cluster 13 kbp downstream of in ...[more]

PMID: 16077120

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Project description:Results of a recent study of antibiotic resistance genes in human colonic Bacteroides strains suggested that gene transfer events between members of this genus are fairly common. The identification of Bacteroides isolates that carried an erythromycin resistance gene, ermG, whose DNA sequence was 99% identical to that of an ermG gene found previously only in gram-positive bacteria raised the further possibility that conjugal elements were moving into Bacteroides species from other genera. Six of seven ermG-containing Bacteroides strains tested were able to transfer ermG by conjugation. One of these strains was chosen for further investigation. Results of pulsed-field gel electrophoresis experiments showed that the conjugal element carrying ermG in this strain is an integrated element about 75 kb in size. Thus, the element appears to be a conjugative transposon (CTn) and was designated CTnGERM1. CTnGERM1 proved to be unrelated to the predominant type of CTn found in Bacteroides isolates-CTns of the CTnERL/CTnDOT family-which sometimes carry another type of erm gene, ermF. A 19-kbp segment of DNA from CTnGERM1 was cloned and sequenced. A 10-kbp portion of this segment hybridized not only to DNA from all the ermG-containing strains but also to DNA from strains that did not carry ermG. Thus, CTnGERM1 seems to be part of a family of CTns, some of which have acquired ermG. The percentage of G+C content of the ermG region was significantly lower than that of the chromosome of Bacteroides species-an indication that CTnGERM1 may have entered Bacteroides strains from some other bacterial genus. A survey of strains isolated before 1970 and after 1990 suggests that the CTnGERM1 type of CTn entered Bacteroides species relatively recently. One of the genes located upstream of ermG encoded a protein that had 85% amino acid sequence identity with a macrolide efflux pump, MefA, from Streptococcus pyogenes. Our having found >90% sequence identity of two upstream genes, including mefA, and the remnants of two transposon-carried genes downstream of ermG with genes found previously only in gram-positive bacteria raises the possibility that gram-positive bacteria could have been the origin of CTnGERM1.

Dataset Information

Regulation of excision genes of the Bacteroides conjugative transposon CTnDOT.

Publications

Regulation of excision genes of the Bacteroides conjugative transposon CTnDOT.

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