The absence of CTXΦ or RS1 on chromosome 2 was established using primers chr2F/chr2R. Primers ctxAF/cepR were used to determine the presence Dabrafenib of CTX tandem arrays. Table 2 Primers used to determine CTX prophage array structure Primer Nucleotide sequence
(5′ to 3′) Position (GenBank Accession no. NC_002505-6) tlcF CCAAAACAACAGAAGCAACAGAGCAACG 1574460-1574487 rstCF GGCGCTTATACAGACGAAATCGCTC 1564180-1564201 rstCR AGCGCCTGAACGCAGATATAAA 1564290-1564311 rstAR CGACAAAAACAAACGGAGAAGCGT 1572748-1572771 ctxAF CTCAGACGGGATTTGTTAGGCACG 1567895-1567918 rtxR CAAGCTGCGATCAGCATGGCGTGGTC 1563652-1563671 cepR CAGTGTTTTGGTGACTTCCGT 1571101-1571121 chr2F CTCACGCTGAACAGCAAGTC 507564-507583 chr2R AAACCGGGAGAAGTGATTGC 509487-509506 Figure 1 ICE Vch Ang3 genetic structure. Schematic linear representation (adapted from Wozniak et al., 2009) of the genes amplified by PCR to define the molecular structure
of ICEVchAng3. The upper line represents the conserved backbone of the SXT/R391 family members. The black arrows indicate insertion sites for ICEVchInd5/ICEVchAng3 specific gene content. Genes in orange were tested with primer set A. Genes in blue were tested with primer set B. Genes not tested are shown in grey. VR: Variable Region; HS: Hotspot. GenBank accession no. of the full sequence of ICEVchInd5 is GQ463142. Three previously described primer sets were used to detect: (i) Classical, El Tor, or Kolkata type rstR gene , (ii) ctxB genotype sequencing , Ku0059436 (iii) and Classical or El Tor biotypes for tcpA . PCR results on organization and location of CTXΦ on chromosome 1 were further confirmed by Southern Blot hybridization assays. DNA probes were produced by PCR using the chromosomal
DNA of V. cholerae strain N16961 as template: ctxA gene (564 bp) with primers CTX-2 (CGGGCAGATTCTAGACCTCCTG) and CTX-3 (CGATGATCTTGGAGCATTCCCAC); rstA gene (789 bp) with primers rstA1F (AAACCTGCAAAATACCCCT) and rstA1R (ACAACTCGATACAAACGCT). Smoothened Probes for hybridization were labeled with alkaline phosphatase with AlkPhos Direct™ Labelling and Detection System with CDP-Star™ kit (Ge Healthcare), according to manufacturer’s instructions. Strains were cultured in Luria-Bertani medium and 1 ml of culture was used to extract and purify the genomic DNA using the DNeasy Blood & Tissue Kit (Qiagen). Aliquots of the extracted DNA (1,5 μg) were digested with EcoRV for CTXΦ element restriction fragment length polymorphism analysis. The digested fragments were separated by agarose gel electrophoresis (1% gel) and were blotted on nitrocellulose membranes using standard methods . Southern blots were hybridized O/N with ctxA or rstA labeled probes, and washed under stringent conditions, according to manufacturer’s instructions. Addition of CDP-Star Detection Reagent was followed by 10 min incubation, and autoradiography (20 min to 1 h) was performed to generate a signal.