Fig. 4

Confirmation of the mutation in PA14ΔhepP. PA14 and PA14ΔhepP were grown in LB broth and the chromosomal DNA was extracted. a PCR analysis to detect the presence of MAR2xT7 within hepP . PCR reactions were run using the chromosomal DNA from each strain as a template and primers corresponding to the DNA sequences 94 bp upstream and 179 bp downstream of the hepP structural gene (zbdP-For3/hepP-Rev3, Table 2). The expected 1926-bp fragment from PA14 (lane 1) and the 2920-bp fragment (the additional 994 bp from MAR2xT7) from PA14ΔhepP (lane 2) were detected. Lane 3 is a no-template control and the molecular size standards are in lane 4. b Restriction analysis of the PCR products. The coding sequence for hepP does not contain an EcoRV restriction enzyme site, while MAR2xT7 contains a single EcoRV site. Digestion of the PCR products with EcoRV failed to reduce the size of the 1926-bp fragment obtained from PA14 (lane 3) but resulted in the cleavage of the product obtained from PA14ΔhepP into the expected 800 bp and 2120 bp fragments (lane 4). Lane 1 contains the molecular size standards; lane 2 was left empty