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 cohtain 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 HindIlI, and inserted into the positive-selection vector pTR262. Recoimbinant E. coli clones were screened directly with anti-fimbrial antiserum by using a colony immunoassay. Several positive colonies were identified, each of which con'tained the same 5.5-kilobase Hindlll 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, molecuilar weight ca. 17,500. The level of expression of the antigen in E. eoli was comparable to that in B. nodqsus 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 originai 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.

Expression of the pilin gene from Bacteroides nodosus in Escherichia coli

“…This result suggested that the copurification of pilin with the inner membrane was not due to cosedimentation of an overproduced insoluble complex. Although Anderson et al (1) suggested that recombinant E. coli may express pilin on the outer cell surface, our findings argue against this premise and against the suggestion that immunoprotection is due to a small quantity of highly immunogenic material in the outer cell membrane. Firstly, recombinant cells, although eliciting specific agglutinating antibody in sheep, are not themselves agglutinated by such sera.…”

Electroporation-mediated transformation of the ovine footrot pathogen Dichelobacter nodosus

“…DNA was prepared as before [14] from D. nodosus cells grown in EYE broth. HpaI-digested genomic DNA was fractionated by electrophoresis through 0.8% agarose gels and the DNA transferred to a nylon membrane by the method of Southern [15].…”

Cloning and expression in Escherichia coli of a gene coding for a chondroitin lyase from Bacteroides thetaiotaomicron

“…The fact that a chondroitin lyase from B. thetaiotaomicron is produced in E. coli from a promoter that lies within the cloned Bacteroides DNA indicates that a least some Bacteroides genes can be expressed in E. coli. Recently, Anderson et al (1) reported that a cloned gene that codes for a fimbral subunit from Bacteroides nodusus, a Bacteroides species that is distantly related to the human colonic Bacteroides species, is also expressed in E. coli.…”