Regulation of a
            <i>Bacteroides</i>
            Operon That Controls Excision and Transfer of the Conjugative Transposon CTnDOT

Wang, Yanping; Shoemaker, Nadja B.; Salyers, Abigail A.

doi:10.1128/jb.186.9.2548-2557.2004

Cited by 30 publications

(45 citation statements)

References 37 publications

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“…An RT-PCR experiment with primers flanking the presumed junction between nucB and tetQ mRNA confirmed that a single tetQ-nucB mRNA transcript exists. pRH3 is stable in P. bryantii TC1-1 in the absence of tetracycline (Accetto et al, 2005) and when tetracycline was omitted from the growth medium, the amount of nucB mRNA fell, which is in accordance with findings in Bacteroides thetaiotaomicron (Wang et al, 2004). Along with the drop of nucB mRNA, the DNase activity in the supernatants fell and His-tagged NucB was no longer detected on Western blots.…”

Section: Discussionsupporting

confidence: 72%

Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1

Accetto

Avguštin

2007

Microbiology

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Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1 Available tools for genetic analysis in the anaerobic rumen bacterium Prevotella bryantii are limited to only two known systems for gene delivery, and no genes, with the exception of plasmid maintenance and selection genes, have been successfully expressed from plasmids in any species of the genus Prevotella until now. It is shown here that nucB, a newly cloned nuclease gene from P. bryantii, can be controllably expressed from shuttle vector pRH3 in P. bryantii strain TC1-1, depending on the tetracycline concentration in the growth medium. nucB expression is also growth-medium dependent and this regulation presumably takes place at the translational level. His-tagged NucB was purified from P. bryantii TC1-1 culture supernatant and was shown to degrade DNA as well as RNA; it is most likely a minor 36 kDa P. bryantii non-specific nuclease. INTRODUCTIONMembers of the bacterial genus Prevotella have been found in the rumen as well as in the oral cavity and large intestine of man and animals (Edwards et al., 2004;Paster et al., 2001;Duncan et al., 2003;Eckburg et al., 2005; Leser et al., 2002). In the rumen they are symbionts contributing to the degradation of plant cell-wall polysaccharides (Miyazaki et al., 2003) and protein metabolism (Walker et al., 2003), whereas oral prevotellas are implicated in dental plaque establishment (Kolenbrander et al., 2002), periodontitis (Paster et al., 2001), advanced caries (Martin et al., 2002), oro-pharyngeal abscesses (Brook, 2004) and noma, a grotesque childhood orofacial gangrene, now confined mostly to sub-Saharan Africa (Enwonwu et al., 2006). The genetic tools that would make possible more thorough studies of prevotellas are still undeveloped despite their ecological and medical importance. This appears to be primarily due to the large phylogenetic distance between prevotellas and other well-characterized micro-organisms, and as a consequence of the rather specific genetic elements, exemplified by promoters, containing distinct 27/233 consensus sequences that are recognized by unusual primary s factor (Vingadassalom et al., 2005). Nevertheless, in two studies (Shoemaker et al., 1991;Accetto et al., 2005) the plasmid replicons and tetQ selectable marker derived from Prevotella and related colonic Bacteroides strains were used successfully for shuttle-vector introduction into the xylan-degrading ruminal species Prevotella bryantii, which forms a distinct phylogenetic lineage apparently somewhat closer to oral prevotellas than the other ruminal Prevotella species (Avguštin et al., 2001). Based on the conjugal transfer protocol developed in the first study, a P. bryantii B 1 4 carboxymethylcellulase gene, fused to a cellulose-binding domain of Thermomonospora fusca cellulase, and driven by a Prevotella ruminicola 23 xylanase promoter, was introduced into P. bryantii B 1 4. The gene was expressed in Bacteroides uniformis 1108, but not in P. bryantii B 1 4, possibly ...

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Section: Discussionsupporting

confidence: 72%

Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1

Accetto

Avguštin

2007

Microbiology

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“…RteA and RteB are members of a two-component regulatory system; however, recently it has been reported that neither RteA nor RteB affects expression of the tetQ-rteA-rteB operon (40). This operon may be regulated by TetQ by a translational attenuation mechanism in response to tetracycline (40). In contrast to CTnDOT, CTn9343 lacks tetQ, and rteA and rteB are in a putative operon that also includes satG and bexA.…”

Section: Discussionmentioning

confidence: 99%

“…The CTnDOT rteA and rteB genes together with the tetracycline resistance gene tetQ form an operon. RteA and RteB are members of a two-component regulatory system; however, recently it has been reported that neither RteA nor RteB affects expression of the tetQ-rteA-rteB operon (40). This operon may be regulated by TetQ by a translational attenuation mechanism in response to tetracycline (40).…”

Section: Discussionmentioning

confidence: 99%

The Bacteroides fragilis Pathogenicity Island Is Contained in a Putative Novel Conjugative Transposon

Franco

2004

J Bacteriol

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The genetic element flanking the Bacteroides fragilis pathogenicity island (BfPAI) in enterotoxigenic B. fragilis (ETBF) strain 86-5443-2-2 and a related genetic element in NCTC 9343 were characterized. The results suggested that these genetic elements are members of a new family of conjugative transposons (CTns) not described previously. These putative CTns, designated CTn86 and CTn9343 for ETBF 86-5443-2-2 and NCTC 9343, respectively, differ from previously described Bacteroides species CTns in a number of ways. These new transposons do not carry tetQ, and the excision from the chromosome to form a circular intermediate is not regulated by tetracycline; they are predicted to differ in their mechanism of transposition; and their sequences have very limited similarity with CTnDOT or other described CTns. CTn9343 is 64,229 bp in length, contains 61 potential open reading frames, and both ends contain IS21 transposases. Colony blot hybridization, PCR, and sequence analysis indicated that CTn86 has the same structure as CTn9343 except that CTn86 lacks a ϳ7-kb region containing truncated integrase (int2) and rteA genes and it contains the BfPAI integrated between the mob region and the bfmC gene. If these putative CTns were to be demonstrated to be transmissible, this would suggest that the bft gene can be transferred from ETBF to nontoxigenic B. fragilis strains by a mechanism similar to that for the spread of antibiotic resistance genes.

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“…BT4001⍀QAB has a single copy of tetQ-rteA-rteB without rteC (39). Bacteroides strains were grown in chopped meat (Remel) and then transferred to TYG (Trypticase-yeast extract-glucose) medium containing tetracycline (1 g/ml; induced) or no tetracycline (uninduced) (12,37). Cells were grown overnight.…”

Section: Methodsmentioning

confidence: 99%

“…Rather, exposure of cells to tetracycline brings into play a translational attenuation mechanism involving a leader region at the 5Ј end of the operon. Presumably the rate of ribosome movement along the mRNA, which is influenced by tetracycline, is responsible for the tetracycline-induced increase in production of TetQ, RteA, and RteB (37). Since RteA and RteB do not control the expression of the tetQ-rteA-rteB operon at the transcriptional level, their function might be to control the expression of the downstream gene, rteC.…”

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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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Regulation of a Bacteroides Operon That Controls Excision and Transfer of the Conjugative Transposon CTnDOT

Cited by 30 publications

References 37 publications

Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1

Studies on Prevotella nuclease using a system for the controlled expression of cloned genes in P. bryantii TC1-1

The Bacteroides fragilis Pathogenicity Island Is Contained in a Putative Novel Conjugative Transposon

Regulation of Excision Genes of theBacteroidesConjugative Transposon CTnDOT

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