CLONING OF A SAPB HOMOLOG (SAPB2) ENCODING A PUTATIVE 112-KDA CAMPYLOBACTER-FETUS S-LAYER PROTEIN AND ITS USE FOR IDENTIFICATION AND MOLECULAR GENOTYPING

A sap gene encoding a surface layer protein was isolated from a Campyl obacter fetus ssp. fetus CIP 53.96T cosmid library. This sap gene, whi ch shows significant homology with the sapB conserved region, was name d sapB2. The complete ORF of 3339 nucleotides encodes a 1112-amino aci d polypeptide with a calculated molecular mass of 112 kDa. High homolo gy with the sapB gene was found in a region beginning 67 bp before the ORF and proceeding 546 bp into the ORF. Similarly, 98% homology with the sapA2 gene was observed in a 2038-bp region beginning 540 bp after the initiation codon. In the present study, we show that this sapB2 g ene has two main interesting features: the 5' end of the region which presents high homology with the sapA2 homologue was found to be presen t in every C. fetus strain, and the fragment (IG01) comprising the reg ion which presents homology with the sapB conserved region and the 5' end of the sapA2 homologue region, when used as a probe, can reveal ge nomic polymorphism among C. fetus strains. We exploited these features to develop a PCR assay for the specific detection of C, fetus and to set up a method for typing C. fetus isolates. The PCR assay was found to be species-specific. Oligonucleotide primers derived from the 5' en d of sapA2 homologue region were used in a polymerase chain reaction t est on genomic DNA extracted from 101 Campylobacter fetus, 18 Campylob acter non-fetus and seven non-Campylobacter strains. A 220-bp fragment was amplified only when C. fetus DNA was used as a target. In Souther n blot analysis, the IG01 probe was found to hybridize only with DNA e xtracted from C, fetus strains. Moreover, IG01 hybridized with several fragments of HindIII-digested DNA, giving a specific pattern for each strain. (C) 1998 Federation of European Microbiological Societies. Pu blished by Elsevier Science B.V. All rights reserved.