Cloning and expression of the Bacteroides fragilis TAL2480 neuraminidase gene, nanH, in Escherichia coli

Abstract

We have cloned the Bacteroides fragilis TAL2480 neuraminidase (NANase) structural gene, nanH, in Escherichia coli. This was accomplished by using the cloning shuttle vector pJST61 and a partial Sau3A library of TAL2480 chromosomal inserts created in E. coli. The library was mobilized into the NANase-deficient B. fragilis TM4000 derivative TC2. NANase-producing colonies were enriched by taking advantage of the inability of TC2, but not the wild-type or NANase+ revertant, to grow in vitro in fluid aspirated from the rat granuloma pouch. Plasmids pJST61-TCN1 and pJST61-TCN3, containing inserts of 9.1 and 4.5 kilobases (kb), respectively, were found in the TC2 derivatives that grew in the rat pouch medium. In B. fragilis, NANase production from the two plasmids was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates, just as in the parental TAL2480 strain. However, when these plasmids were transferred back to E. coli, NANase activity was barely detectable. A 3.5-kb portion of the insert in pJST61-TCN3 was subcloned in pJST61 to give plasmid pJST61-SC3C; NANase was produced from this plasmid both in E. coli and in B. fragilis. In E. coli, NANase expression was under the control of the vector promoter lambda PR and was therefore completely abolished by the presence of a lambda prophage. In B. fragilis, NANase production was inducible by free N-acetylneuraminic acid or sialic acid-containing substrates. By using deletion analysis and TnlOOO mutagenesis, the NANase structural gene and control region that functions in B. fragUis were localized to a 1.5-to 2.0-kb region of the insert. A partial nucleotide sequence of the NANase-deficient Tnl000 insertion mutants allowed us to identify the nanH gene and deduce the amino acid sequence of a portion of the NANase protein. We identified five regions showing great similarity to the Asp boxes, -Ser-X-Asp-X-Gly-X-Thr-Trp-, of other bacterial and viral NANase proteins.

Sialic Acid ( N -Acetyl Neuraminic Acid) Utilization by Bacteroides fragilis Requires a Novel N -Acetyl Mannosamine Epimerase

“…We hypothesized that these genes would be regulated by the presence of NANA in the medium and might share upstream regulatory sequences. Thus, we used the DNA sequences upstream of the nanH gene (25) to search the B. fragilis NCTC 9343 and the unfinished B. fragilis 638R genome databases for matches. Indeed, a matching region was found just upstream of a gene cluster whose protein products showed similarities to proteins involved in NANA catabolism in other organisms.…”

Bacteroides: the Good, the Bad, and the Nitty-Gritty

“…This enzyme is found in many pathogenic bacteria and is generally considered a virulence factor (223), and many strains of B. fragilis produce neuraminidase (22,274). Neuraminidase can catalyze the removal of the sialic acid from host cell surfaces and from important immunoactive proteins such as IgG and some components of complement and may consequently disrupt important host functions (237).…”

Identification of two genetic groups in Bacteroides fragilis by multilocus enzyme electrophoresis: distribution of antibiotic resistance (cfiA, cepA) and enterotoxin (bft) encoding genes The GenBank accession numbers for the sequences determined in this work are AF197508–AF197534.

“…pili promoting adherence to epithelial cells and mucus, neuraminidase, and an enterotoxin (Akimoto et al, 1994 ;Brook & Mihal, 1991 ;Brook et al, 1992 ;Duimstra et al, 1991 ;Kleivdal & Hofstad, 1995 ;Moncrief et al, 1995 ;Myers et al, 1989 ;Russo et al, 1990 ;Sack et al, 1992 ;Smith & Callihan, 1992). Previous studies have suggested a role of enterotoxinproducing Bacteroides fragilis (ETBF) in diarrhoeal disease of children (Sack et al, 1994 ;San Joaquin et al, 1995).…”