title: Heterologous gene expression in Bacteroides fragilis.
abstract: Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.
authors.affiliation: Department of Microbiology and Immunology, East Carolina University, Greenville, North Carolina 27858.
authors.forename: C J
authors.initials: CJ
authors.lastname: Smith
authors.forename: M B
authors.initials: MB
authors.lastname: Rogers
authors.forename: M L
authors.initials: ML
authors.lastname: McKee
mesh_terms: D001441:Bacteroides fragilis / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003227:Conjugation, Genetic; D016384:Consensus Sequence / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D015183:Restriction Mapping; D014169:Transformation, Bacterial
chemical_list: D005819:Genetic Markers; D011994:Recombinant Proteins