Figure 2

Confirmation of the genome architecture of L. salivarius JCM1046. (A, C and D) PFGE gels of enzyme-treated gDNA of strains JCM1046, JCM1047 and AH43348. Corresponding Southern Hybridizations using replicon-specific probes are shown directly below each gel (B, D, and F). The probes used for the Southern Hybridizations targeted the following genes: the repB gene of pMP1046B (B), an endonuclease gene in pLMP1046 (D) and a region spanning the int-xis genes of pCTN1046 (F). None of the probes employed showed cross hybridisation with non-target replicons. S1 nuclease (+), SmaI (†), SphI (_*), PstI (‡) were used individually or in combination to determine the plasmid profiles of each strain. Untreated samples of gDNA are denoted by (−). Closed-black arrowheads indicate λ DNA concatamers used as size standards (H) (A-F). Chromosomal DNA bands of each strain are seen migrating to the equivalent of the 1 Mb marker (A, C and E). Open-black arrows indicate the S1 nuclease-linearised repA megaplasmids in each strain examined (A, C and E). A repB-type megaplasmid was found to be present in strain JCM1046 but absent from strains JCM1047 and AH43348 (A and B). Both S1-treated and untreated gDNA samples of JCM1046, JCM1047 and AH43348 show the presence of linear plasmids of 140 kb, 140 kb and 175 kb respectively (C), each of which hybridise to a pLMP1046-derived probe (D). S1-nuclease, SphI and PstI were independently used to linearise pCTN1046 (33 kb) (E). A probe based on the int and xis genes of pCTN1046 binds to the linear form of pCTN1046 (F). pCTN1046 does not have a SmaI site and is retained in the well in its circular form in the SmaI-digested sample.