Screening for a gene that activates the CpxR/CpxA system Chromosomal DNA prepared from an overnight culture of wild-type strain 14028s

was digested with Sau3AI (0.01 U/μl) for 4 h. The digested DNA was separated on a 0.8% agarose gel, and 0.5–5 kb fragments were collected and ligated to pUC19 plasmid DNA that had been digested with BamHI and dephosphorylated by alkaline phosphatase. The ligation mixture was transformed into E. coli DH5α, and ampicillin-resistant transformants were selected. Plasmid DNA was prepared from a pool of ~100,000 check details transformants and used to transform the strain AK1052. Transformants were serially diluted and spread onto LB plates containing ampicillin and 40 μg/ml X-gal to obtain 1,000 ~ 10,000 colonies per plate. Plasmids were isolated from colonies that developed a blue color on LB plates containing ampicillin and X-gal. These plasmids were

reintroduced into AK1052 by electroporation, and four transformants were selected on LB plates containing ampicillin and X-gal. A random single ML323 purchase white colony from the same plate was also selected as a negative control. Acknowledgements This work was supported, in part, by Grant-in-Aid for Young Scientists (Start-up) 19810025 and (A) 23688013 from the Japan Society for the Promotion of Science (JSPS), the Kato Memorial Bioscience Foundation, the Uehara Memorial Foundation, the Mochida Foundation,

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