8 Genetic Methods for Bacteroides Species

Salyers, Abigail A.; Shoemaker, Nadja B.; Cooper, Andrew J.; D'Elia, John; Shipman, Joseph A.

doi:10.1016/s0580-9517(08)70119-3

Cited by 26 publications

(20 citation statements)

References 37 publications

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“…When both are present, however, digestion by exogenous protease was minor in the case of SusC and not detectable in the case of SusD. SusC has many homologues in the B. thetaiotaomicron genome and in the Porphyromonas gingivalis genome (6,11). The amino acid sequence of SusC, together with the fact that SusC is necessary and sufficient for utilization of intermediate-sized oligomers of glucose (9), suggests that SusC might be a porin.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Four Outer Membrane Proteins Involved in Binding Starch to the Cell Surface of Bacteroides thetaiotaomicron

2000

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Bacteroides thetaiotaomicron, a gram-negative obligate anaerobe, utilizes polysaccharides by binding them to its cell surface and allowing cell-associated enzymes to hydrolyze them into digestible fragments. We use the starch utilization system as a model to analyze the initial steps involved in polysaccharide binding and breakdown. In a recent paper, we reported that one of the outer membrane proteins involved, SusG, had starch-degrading activity but was not sufficient for growth on starch. Moreover, SusG alone did not have detectable starch binding activity. Previous studies have shown that starch binding is essential for starch utilization. In this paper, we report that four other outer membrane proteins, SusC through SusF, are responsible for starch binding. Results of 14 C-starch binding assays show that SusC and SusD both contribute a significant amount of starch binding. SusE also appears to contribute substantially to starch binding. Using affinity chromatography, we show in vitro that these Sus proteins interact to bind starch. Moreover, protease accessibility of either SusC or SusD greatly increased when one was expressed without the other. This finding supports the hypothesis that SusC and SusD interact in the outer membrane. Evidence from additional protease accessibility studies suggests that SusC, SusE, and SusF are exposed on the cell surface. Our results demonstrate that SusC and SusD act as the major starch binding proteins on the cell surface, with SusE enhancing binding. SusF's role in starch utilization has yet to be determined, although the fact that starch protected it from proteolytic attack suggests that it does bind starch.

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Section: Discussionmentioning

confidence: 99%

Characterization of Four Outer Membrane Proteins Involved in Binding Starch to the Cell Surface of Bacteroides thetaiotaomicron

2000

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“…However, in the transfer system [BT4001⍀QABC(pLYL72)] used in these studies, the CTn is essentially already in a circular form, since the region being transferred is located on a plasmid. This plasmid contains a pB8-51 replicon that is maintained at a copy number of approximately 8 to 10 per copy of the Bacteroides chromosome (19).…”

Section: Discussionmentioning

confidence: 99%

Characterization of Genes Involved in Modulation of Conjugal Transfer of theBacteroidesConjugative Transposon CTnDOT

2002

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In previous studies we identified an 18-kb region of the Bacteroides conjugative transposon CTnDOT that was sufficient for mobilization of coresident plasmids and unlinked integrated elements, as well as self-transfer from Bacteroides to Escherichia coli. When this 18-kb region was cloned on a plasmid (pLYL72), the plasmid transferred itself constitutively in the absence of a coresident conjugative transposon. However, when this plasmid was present in a Bacteroides strain containing a coresident conjugative transposon, conjugal transfer was repressed in the absence of tetracycline and enhanced in the presence of tetracycline. These results suggested that a negative and a positive regulator of conjugal transfer were encoded outside the transfer region of the CTnDOT element. In this work, a minimal and inducible transfer system was constructed and used in transfer and Western blot analyses to identify the differentially regulated genes from CTnDOT responsible for the enhancement and repression of pLYL72 conjugal transfer. Both of these regulatory functions have been localized to a region of the CTnDOT element that is essential for CTn excision. In the presence of tetracycline, the regulatory protein RteC activates the expression of a putative topoisomerase gene, exc, which in turn results in an increase in transfer protein expression and a concomitant 100-to 1,000-fold increase in the frequency of pLYL72 transfer. Our results also suggest that since exc alone cannot result in enhancement of transfer, other factors encoded upstream of exc are also required. Conversely, in the absence of tetracycline, a gene located near the 3 end of exc is responsible for the repression of transfer protein expression and also results in a 100-to 1,000-fold decrease in the frequency of pLYL72 transfer.Bacteroides spp. are obligate anaerobic bacteria and account for about 25 to 30% of the bacteria in the human intestinal tract (15). Although Bacteroides spp. play a number of roles as part of the normal intestinal microflora, some strains are also opportunistic pathogens. Bacteroides spp. are the anaerobes most frequently isolated from clinical specimens (9).Bacteroides spp. are hosts to a variety of transmissible elements, including plasmids, transposons, mobilizable transposons, and conjugative transposons (CTns) (18,20,21). The results of a recent survey suggest that it is this last group of elements, the CTns, that is responsible for the significant increase in tetracycline and macrolides-lincosamides-streptogramin-B-type resistance in the Bacteroides group (25).Bacteroides CTns range in size from 50 to 150 kb (1). The two best-characterized Bacteroides CTns are CTnERL and CTnDOT, which are almost identical except that CTnDOT contains a 13-kb insertion which contains an ermF resistance determinant (Fig. 1A) (34). A novel feature of the Bacteroides CTnDOT/CTnERL family of CTns is that self-transfer and the mobilization of coresident plasmids and mobilizable transposons are all stimulated 100-to 1,000-fold by pregrowth in a medium c...

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“…To generate transcriptional fusions, two different restriction fragments were fused to the uidA reporter gene (11,13,21). pGFK1 was constructed with a 1.3-kb BspEI-XmnI fragment, which starts upstream of orf2c and continues into orf3, cloned into the uidA vector pLYL02 (this laboratory).…”

Section: Methodsmentioning

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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8 Genetic Methods for Bacteroides Species

Cited by 26 publications

References 37 publications

Characterization of Four Outer Membrane Proteins Involved in Binding Starch to the Cell Surface of Bacteroides thetaiotaomicron

Characterization of Four Outer Membrane Proteins Involved in Binding Starch to the Cell Surface of Bacteroides thetaiotaomicron

Characterization of Genes Involved in Modulation of Conjugal Transfer of theBacteroidesConjugative Transposon CTnDOT

Regulation of Excision Genes of theBacteroidesConjugative Transposon CTnDOT

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