2 and 1.3 kb, respectively) were

observed in the agarose gel (Fig. 2b). The difference in size indicated that TPMA0004 was the mycF disruption mutant. For genetic complementation for the mycF disruption mutant TPMA0004, pMG508 including mycF was transferred to TPMA0004 buy EPZ5676 by intergeneric conjugation. The transconjugant TPMA0009 isolated from the conjugation plate containing apramycin and nalidixic acid produced M-II (8.29 μg mL−1) (Fig. 3). The amount of M-II produced by TPMA0009 was approximately 55% of that produced by the wild strain A11725. PCR was performed with several primer pairs to confirm the genetic condition of TPMA0009. As shown in Fig. 2b, the transconjugant TPMA0009 producing M-II was the homogenous mycF complementation strain in which the mycF gene was inserted into the chromosome by a site-specific recombination between the artificial attB site on the chromosome and the attP site on Trichostatin A cost pMG508. The disruption cassette FRT-neo-oriT-FRT-attB

was used to obtain the mycE disruption mutant TPMA0003 and the mycF disruption mutant TPMA0004 of M. griseorubida. In particular, PCR targeting with the phage λ-Red recombinase was performed to isolate the mycF disruption mutant. Furthermore, from these mutants, the homogenous complementation strains TPMA0003 and TPMA0004 were isolated by a site-specific recombination between the artificial attB site on the mutant chromosomes and the attP on pSET152 used as a vector. Recently, a simple and highly efficient

PCR-targeting system was developed for the gene targeting of Streptomyces strains (Gust et al., 2003). However, genetic engineering cannot be performed for actinomycete strains lacking the bacteriophage φC31 attB attachment site using vectors possessing a φC31 int gene and an Ribonucleotide reductase attP site. In this study, gene disruption and complementation studies could be performed for M. griseorubida, which lacked the bacteriophage φC31 attB site on the chromosome, using the disruption cassette FRT-neo-oriT-FRT-attB. A multiple gene disruption and complementation scheme using the disruption cassette is shown in Fig. 4. In this study, the complementation plasmid pMG508 possessing the int gene encoding integrase, the attP site, and the resistant marker aac(3)IV was inserted into the φC31 attB attachment site, which was flanked by the resistant marker neo and oriT on the mycF disruption mutant. For additional gene disruption and complementation studies of the complementation strain TPMA0009, resistant markers other than neo and aac(3)IV should be used. However, if a gene disruption mutant with the resistant marker eliminated was obtained by in-frame disruption, additional gene disruption studies can be performed with the same resistant marker.