Cloning and expression in Escherichia coli of the gene encoding the structural subunit of Bacteroides nodosus fimbriae

Abstract

Bacteroides nodosus is the primary causative agent of ovine foot rot. Virulent isolates of this bacterium contain fimbriae which appear to play a major role in both infectivity and protective immunity. This paper presents the cloning and expression in Escherichia coli of the gene encoding the structural subunit of the fimbriae of B. nodosus. Total DNA was isolated from B. nodosus VCS 1001 (serogroup A), digested with HindIII, and inserted into the positive-selection vector pTR262. Recombinant E. coli clones were screened directly with anti-fimbrial antiserum by using a colony immunoassay. Several positive colonies were identified, each of which contained the same 5.5-kilobase HindIII insert. The prototype has been designated pBA101. Some clones also contained additional flanking sequences from the B. nodosus genome. Western transfer analyses verified that the positive clones were producing the B. nodosus fimbrial structural subunit, molecular weight ca. 17,500. The level of expression of the antigen in E. coli was comparable to that in B. nodosus itself and was unaffected by the insertion site or orientation of the cloned fragment, indicating that synthesis was being directed from an internal promoter. Restriction mapping and deletion analyses localized the fimbrial subunit gene to the vicinity of a PvuII site near the central region of the original HindIII insert. The expressed antigen was located in the membrane-cell wall fraction and may be exposed on the surface of the recombinant E. coli cells.