Cloning of a sapB homologue (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 Campylobacter fetus ssp. fetus CIP 53.96T cosmid library. This sap gene, which shows significant homology with the sapB conserved region, was named sapB2. The complete ORF of 3339 nucleotides encodes a 1112-amino acid polypeptide with a calculated molecular mass of 112 kDa. High homology 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 gene has two main interesting features: the 5' end of the region which presents high homology with the sapA2 homologue was found to be present in every C. fetus strain, and the fragment (IG01) comprising the region which presents homology with the sapB conserved region and the 5' end of the sapA2 homologue region, when used as a probe, can reveal genomic 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' end of sapA2 homologue region were used in a polymerase chain reaction test on genomic DNA extracted from 101 Campylobacter fetus, 18 Campylobacter non-fetus and seven non-Campylobacter strains. A 220-bp fragment was amplified only when C. fetus DNA was used as a target. In Southern blot analysis, the IG01 probe was found to hybridize only with DNA extracted from C. fetus strains. Moreover, IG01 hybridized with several fragments of HindIII-digested DNA, giving a specific pattern for each strain.
Casadémont
I
I
Laboratoire de Prédéveloppement des Sondes, Centre de Biologie Médicale Spécialisée, Institut Pasteur, Paris, France.
Chevrier
D
D
Guesdon
J L
JL
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics*; D001426:Bacterial Proteins; D001483:Base Sequence; D002168:Campylobacter fetus / Q000145:classification / Q000235:genetics*; D003001:Cloning, Molecular; D017931:DNA Primers; D004269:DNA, Bacterial; D005838:Genotype; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D008562:Membrane Glycoproteins; C055764:S-layer proteins