Fig. 2

The construction of a polar mutation in pefC. a Schematic representation of the pefC::pMZ1 mutation in ZE4951 strain. The lines below the locus diagram denote the DNA fragments amplified by the analysis described in section B. The lines above the diagram denote the products the Q-PCR described in section C. Spec ^R signifies the spectinomycin resistance aad9 gene and ori represents pMZ1 origin of replication. The black regions in the pefC gene denote the internal fragment that was cloned into pMZ1 and is thus duplicated in the mutant chromosome. b PCR analysis of the pefRCD locus in the ZE4951 mutant. DNA was amplified by PCR and fractionated on 0.8 % agarose gel. The DNA ladder is shown in lane 1. For the analysis of the left chromosome/plasmid junction the reactions were done with the ZE553/SpecFw primer set and genomic DNA of NZ131 (lane 2), ZE4951 (lane 3), or no DNA (lane 4). For the right plasmid/chromosome junction, the reactions consist of the ZE554/SpecRev primer set and genomic DNA of NZ131 (lane 5), ZE4951 (lane 6), or no DNA (lane 7). For the spec ^R cassette, the reactions were preformed with the SpecFW/SpecRev primer set and genomic DNA of the NZ131 (lane 8), ZE4951 (lane 9), pMZ1 (lane 10, positive control), or no DNA (lane 11). c Relative expression of the pefC and pefD genes in ZE4951 and NZ131 strains. Total RNA was extracted and the relative expression of the pefC and pefD genes was evaluated by Q-PCR. The relative expression of the pefC and pefD genes was normalized to rpsL transcript levels. The asterisk (*) indicates P value of statistical significance (P <0.05) tested using student t-test (assuming equal variance) at 0.05 levels of significance