Molecular analysis of one of multiple protease-encoding genes from the prototype virulent strain of Bacteroides nodosus.
The aim of these studies was to examine the organization of the Bacteroides nodosus protease-encoding gene(s). The extracellular serine proteases (38 kDa) from the prototype virulent strain of B. nodosus were purified and used to raise a specific antiserum in rabbits. This antiserum was used in a colony immunoassay to screen a genomic DNA library constructed in Escherichia coli using BamHI-digested B. nodosus DNA and the plasmid pBR322. An E. coli clone expressing a 50-kDa immunoreactive polypeptide was identified. No protease activity was detected in the culture media, or in crude soluble and membrane fractions prepared from this clone. Restriction mapping and deletion analysis of the recombinant plasmid, pEKM2, was used to locate the coding region to a 1.4-kb EcoRI-BamHI fragment which was subsequently sequenced. A large open reading frame was found to extend through the BamHI site from a putative start codon just downstream from the EcoRI site, which indicated that the complete gene was not isolated. Southern blotting demonstrated that there were at least three B. nodosus BamHI fragments which were homologous to the 0.4-kb PstI-BamHI fragment of pEKM2. Based on these results the existence of multiple protease genes in B. nodosus was postulated.
Moses
E K
EK
Department of Agriculture and Rural Affairs, Regional Veterinary Laboratory, Hamilton, Victoria, Australia.
Rood
J I
JI
Yong
W K
WK
Riffkin
G G
GG
D000595:Amino Acid Sequence; D001439:Bacteroides / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D007158:Immunologic Techniques; D008969:Molecular Sequence Data; D005810:Multigene Family; D015183:Restriction Mapping; D012697:Serine Endopeptidases / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification
D004269:DNA, Bacterial; D012697:Serine Endopeptidases