Characterization of a
            <i>Bacteroides</i>
            Mobilizable Transposon, NBU2, Which Carries a Functional Lincomycin Resistance Gene

Wang, Jun; Shoemaker, Nadja B.; Wang, Guirong; Salyers, Abigail A.

doi:10.1128/jb.182.12.3559-3571.2000

Cited by 103 publications

(109 citation statements)

References 62 publications

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“…The acfA and luxI genes were previously cloned into pSUP102 (2). V. fischeri chromosomal DNA was purified as a template (37), and qsrP and ribB were amplified using the primers indicated in Table 2 and cloned into pSUP102. The promoter regions of a number of genes containing putative lux boxes (luxI, qsrP, ribB, acfA, ainS, qsrQ, qsrRST, and arcA) were PCR amplified using the primers indicated in Table 2, cloned into pGEM-T (Promega, Madison, WI), and sequenced to confirm their integrity (Virginia Bioinformatics Institute Core Laboratory, Virginia Tech, Blacksburg).…”

Section: Methodsmentioning

confidence: 99%

Analysis of LuxR Regulon Gene Expression during Quorum Sensing inVibrio fischeri

et al. 2007

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The regulation of the lux operon (luxICDABEG) of Vibrio fischeri has been intensively studied as a model for quorum sensing in proteobacteria. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis previously identified several non-Lux proteins in V. fischeri MJ-100 whose expression was dependent on LuxR and 3-oxo-hexanoyl-L-homoserine lactone (3-oxo-C6-HSL). To determine if the LuxR-dependent regulation of the genes encoding these proteins was due to direct transcriptional control by LuxR and 3-oxo-C6-HSL or instead was due to indirect control via an unidentified regulatory element, promoters of interest were cloned into a lacZ reporter and tested for their LuxR and 3-oxo-C6-HSL dependence in recombinant Escherichia coli. The promoters for qsrP, acfA, and ribB were found to be directly activated via LuxR-3-oxo-C6-HSL. The sites of transcription initiation were established via primer extension analysis. Based on this information and the position of the lux box-binding site near position ؊40, all three promoters appear to have a class II-type promoter structure. In order to more fully characterize the LuxR regulon in V. fischeri MJ-100, real-time reverse transcription-PCR was used to study the temporal expression of qsrP, acfA, and ribB during the exponential and stationary phases of growth, and electrophoretic mobility shift assays were used to compare the binding affinities of LuxR to the promoters under investigation. Taken together, the results demonstrate that regulation of the production of QsrP, RibB, and AcfA is controlled directly by LuxR at the level of transcription, thereby establishing that there is a LuxR regulon in V. fischeri MJ-100 whose genes are coordinately expressed during mid-exponential growth.

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Section: Methodsmentioning

confidence: 99%

Analysis of LuxR Regulon Gene Expression during Quorum Sensing inVibrio fischeri

et al. 2007

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“…The E. coli donor was BW19851, which has both the RP4 transfer functions and pir gene integrated in its chromosome carrying the mobilizable attDOT vector, pattDOT. The base plasmid, pEPE (25), is pir dependent and carries a chloramphenicol resistance marker and the oriT of RP4. The pir mutant E. coli EM24NR recipient strains carried pINT101 or pINT201.…”

Section: Methodsmentioning

confidence: 99%

“…The attB region (500 bp) was PCR amplified by using primers DLJ/U487F and DRJ/R2700R (3) and BT4001 chromosomal DNA. Another plasmid used in the in vitro integration assay, pattDOT, contained the 547-bp CTnDOT attachment site (attDOT) cloned into the ApaI and SstI sites of the pir-dependent plasmid pEPE (25) to generate pattDOT.…”

Section: Methodsmentioning

confidence: 99%

Development of an In Vitro Integration Assay for the Bacteroides Conjugative Transposon CTnDOT

et al. 2002

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Integrated self-transmissible elements called conjugative transposons (CTns) are responsible for the transfer of antibiotic resistance genes in many different species of bacteria. One of the best characterized of these newly recognized elements is the Bacteroides CTn, CTnDOT. CTnDOT is thought to have a circular transfer intermediate that transfers to and integrates into the genome of the recipient cell. Previous investigations of the mechanism of CTnDOT integration have been hindered by the lack of an in vitro system for checking this model of integration and determining whether the CTnDOT integrase alone was sufficient to catalyze the integration reaction or whether host factors might be involved. We report here the development of an in vitro system in which a plasmid containing the joined ends of CTnDOT integrates into a plasmid carrying a CTnDOT target site. To develop this in vitro system, a His-tagged version of the integrase gene of CTnDOT was cloned and shown to be active in vivo. The protein produced by this construct was partially purified and then added to a reaction mixture that contained the joined ends of the circular form of CTnDOT and a plasmid carrying one of the CTnDOT target sites. Integration was demonstrated by using a fairly simple mixture of components, but integration was stimulated by a Bacteroides extract or by purified Escherichia coli integration host factor. The results of this study demonstrate both that the circular form of CTnDOT is the form that integrates into the target site and that host factors are involved in the integration process.Bacteroides species are gram-negative anaerobes that account for about 25 to 30% of the bacterial population that normally resides in the human colon (14). Many Bacteroides strains carry large self-transmissible integrated elements that have been called conjugative transposons (CTns). In fact, a recent study has shown that over 80% of Bacteroides strains today contain at least one CTn and that the spread of resistance to tetracycline and erythromycin among Bacteroides strains has been mediated primarily by these elements (21). The best studied of the Bacteroides CTns is CTnDOT. Most of the CTns found in Bacteroides have proved to be closely related to this CTn.Previous studies have shown that CTnDOT integrates site selectively into at least seven different target sites (attB) in the Bacteroides chromosome (3). There is a conserved 10-bp sequence (GTANNTTTGC) found at one end of CTnDOT that shares identity with a 10-bp sequence in all of the chromosomal attB sites (3). The deduced amino acid sequence of the CTnDOT integrase gene has low but significant homology (30 to 40%) to some members of the tyrosine recombinase family, of which the integrase of bacteriophage lambda is also a member. However, unlike the lambda integrase protein that makes staggered cuts 7 bp apart within the identical core sequences found in attB and attP, the CTnDOT Int protein appears to use a different mechanism. It makes staggered cuts 5 bp apart adjacent to the 10-bp cons...

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“…Reactions were done in a 96-well microtiter PCR plate using 1 l of cDNA and (final concentrations) 0.4 M sense and antisense primers for amplifying 70 , rteC, and exc; 3 M MgCl 2 ; and 1ϫ iQ Data were analyzed using the iQcycler analysis software (Bio-Rad). Relative quantitation, which determines the changes in steady-state mRNA levels of a gene across multiple samples and provides a result relative to the levels of an internal control RNA, was used (36). The results were expressed as the difference (N) in the number of target gene copies relative to the number of 70 gene copies and were determined from N ϭ 2⌬⌬ Ct ϭ 2 (⌬Ct target Ϫ ⌬Ct 70 RNA)…”

Section: Real Time Rt-pcr Analysismentioning

confidence: 99%

Regulation of Excision Genes of theBacteroidesConjugative Transposon CTnDOT

et al. 2005

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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.Conjugative transposons (CTns) related to the Bacteroides CTn CTnDOT have been found in a number of Bacteorides species (22,26). Members of CTnDOT family appear to be contributing significantly to transfer of antibiotic resistance genes among Bacteroides species (22,26,40). Transfer of CTnDOT occurs in three steps: excision from the chromosome to form a double-stranded circular intermediate, conjugative transfer of the circular intermediate to the recipient, and integration of the transferred circular form into the recipient's chromosome (40).Excision is stimulated by tetracycline (7,8,30). In fact, no excision is detected unless the cells carrying CTnDOT are first exposed to tetracycline (8,30). A previous study identified four genes that were essential for excision (8,30). One was the integrase gene intDOT, which is located at one end of the CTn. The other genes were located in a cluster 13 kbp downstream of intDOT (Fig. 1). Single-crossover disruptions and deletions in three of these genes, orf2c, orf2d, and exc, abolished excision. A fourth gene located in this cluster, orf3, could be deleted without affecting excision. We report here that ge...

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Characterization of a Bacteroides Mobilizable Transposon, NBU2, Which Carries a Functional Lincomycin Resistance Gene

Cited by 103 publications

References 62 publications

Analysis of LuxR Regulon Gene Expression during Quorum Sensing inVibrio fischeri

Analysis of LuxR Regulon Gene Expression during Quorum Sensing inVibrio fischeri

Development of an In Vitro Integration Assay for the Bacteroides Conjugative Transposon CTnDOT

Regulation of Excision Genes of theBacteroidesConjugative Transposon CTnDOT

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