Campylobacter fetus uses multiple loci for DNA inversion within the 5 ' conserved regions of sap homologs

Campylobacter fetus cells possess multiple promoterless sap homologs, each capable of expressing a surface layer protein (SLP) by utilizing a unique p romoter present on a 6.2-kb invertible element. Each sap homolog includes a 626-bp 5' conserved region (FCR) with 74 bp upstream and 552 bp within the open reading frame. After DNA inversion, the splice is seamless because th e FCRs are identical. In mutant strain 23D:ACA2K101, in which sapA and sapA 2 flanking the invertible element in opposite orientations were disrupted b y promoterless chloramphenicol resistance (Cm-r) and kanamycin resistance ( Km(r)) cassettes, respectively, the frequency of DNA inversion is 100-fold lower than that of wild-type strain 23D. To define the roles of a 15-bp inv erted repeat (IR) and a Chi-like site (CLS) in the FCR, we mutagenized each upstream of sapA2 in 23D:ACA2KI01 by introducing NotI and KpnI sites to cr eate strains 23D:ACA2K101N and 23D:ACA2K101K, respectively. Alternatively s electing colonies for Cm-r or Km(r) showed that mutagenizing the IR or CLS had no apparent effect on tl;e frequency of the DNA inversion. However, map ping the unique NotI or KpnI site in relation to the Cm-r or Km(r) cassette in the cells that changed phenotype showed that splices occurred both upst ream and downstream of the mutated sites. PCR and sequence analyses also sh owed that the splice could occur in the 425-bp portion of the FCR downstrea m of the cassettes. In total, these data indicate that C.fetus can use mult iple sites within the FCR for its sap-related DNA inversion.