2.2. Construction of Mutant Strains

B. subtilis strains carrying single, double and triple mutations were generated as indicated in Table 1, employing standard techniques.

The pBT and pTRG vectors containing mutY (PERM1084) and mfd (PERM1072) genes were generated as mentioned below. The mutY and mfd ORFs were first amplified by PCR using chromosomal DNA purified from B. subtilis YB955 with the following set of oligonucleotide primers: for mutY, 5′-CGGGATCCATGAACGTACTTGAAGAC-3′ (forward) and 5′-CGCTCGAGCGGAGCAGCCGAGATGGC-3′ (reverse); for mfd, 5′-CGGGATCCATGGACAACATTCAAACC-3′ (forward) and 5′-CGCTCGAGCGTTGATGAAATGGTTTG-3′ (reverse). Primers were designed to insert BamHI/XhoII sites in both cases (sequences underlined). Amplifications were performed with Vent DNA polymerase (New England BioLabs, Beverly, MA, USA). PCR products purified from a low-melting-point agarose gel were ligated into PCR-BluntII-TOPO (Invitrogen, Carlsbad, CA, USA). The resulting constructs were treated with NotI/XhoI and BamHI/XhoI to release mutY and mfd ORFs. The purified fragments were ligated into pBT and pTRG to generate the constructs PERM1084 and PERM1072. The correct orientation of the inserted genes was verified by restriction analysis.