Clostridium scindens VPI 12708 (previously known as Eubacterium sp. VPI 12708) is a bile-acid dehydroxylating bacterium originally isolated from the feces of a colon cancer patient. Many genes required for bile acid 7-a dehydroxylation are found on a large bile acid inducible operon (bai) that has been extensively studied. However the bai promoter, which directs expression of the bai operon, has yet to be characterized due, in part, to a lack of a functional genetic transfer system for this strain. A spontaneous rifampinresistant Clostridium scindens VPI 12708 mutant was used as a recipient to determine the efficacy of conjugation as a method of DNA transfer. The Clostridium perfringens plasmid pECU-001, containing a chloramphenicol acetyl transferase marker, and the Birmingham IncP-a oriT was transformed into Escherichia coli S17-1 which was used as a donor for conjugation. E. coli EM24 (Rif R) served as a recipient to ensure the donor was conjugal. All bacterial cultures were grown to mid-log phase and harvested by centrifugation. Matings between the donor and recipient were carried out on the surface of anaerobic tryptic soy agar slants. hloramphenicol-resistant E. coli EM24 colonies were isolated suggesting E. coli S17-1 was conjugal under the conditions tested. Chloramphenicol-resistant Clostridium scindens VPI 12708 (RifR) colonies were isolated using both 4:1 and 1:4 (vol:vol) donor: recipient ratios. Plasmid preparations with subsequent restriction map analysis from putative Clostridium scindens VPI 12708 transconjugants confirmed the presence of pECU-001. Conjugal transfer of pWKU-001 (which is pECU-001 with the bai promoter insert and a downstream b-glucuronidase reporter gene) was attempted using a similar protocol, but with erythromycin as selection for recipient. The plasmid was successfully introduced into C. scindens, however pWKU-001 was not maintained and lost from the culture. These data suggest DNA can be introduced into Clostridium scindens VPI 12708 via E. coli S17-1-mediated conjugal transfer, which may prove useful for the study of the genetic regulation of the bai promoter. Unfortunately, pWKU-001 appears to be unstable and unsuitable in present form for extensive studies.

Bile acids in the small intestines of animals serve to breakdown fats and fatsoluble vitamins. Most of the bile acids are reabsorbed into the enterohepatic circulation, but approximately five percent of these bile acids pass into the large intestine. These bile acids are swiftly deconjugated by the bacterial population, and then subjected to further intestinal bacterial chemical modifications. The most significant of these modifications are 7α-dehydroxylations which form secondary bile acids (deoxycholate and lithocholate). Much research has illuminated the 7α-dehydroxylation pathway: of particular interest is the bile acid inducible operon, for which Clostridium scindens VPI 12708 serves as the model organism. There is a lack of knowledge on how this operon is regulated, so the goal of this project was to create a genetic construct consisting of upstream regulatory elements, a bile acid inducible promoter, and a ϐ- glucuronidase reporter. Cloning strategies utilized PCR to amplify desired DNA fragments and sewing methodology to combine DNA fragments. DNA fragments were ligated into plasmids and transformed into competent E. coli. Transformants were evaluated for the desired reporter gene fusion by blue/white screening, additional PCR, and/or restriction digestion. The bile acid inducible promoter was successfully amplified, and the upstream sequence and uidA (ϐ- glucuronidase) reporter was demonstrated. However, no E. colitransformants were demonstrated to possess the baiP-uidA gene fusion. The project strategy is plausible and data regarding the bile acid inducible promoter are greatly needed.