S1). In our previous work, we developed the ‘CRS cassette method’ to construct and combine markerless deletion mutations

(Hashimoto et al., 2005). The CRS cassette has a positive-selection marker (CmR) and two negative-selection markers (sacB+ and rpsL+) (Hashimoto et al., 2005; Kato & Hashimoto, 2008). First, the chromosome region to be deleted was replaced with the CRS cassette using a positive-selection marker and lambda red homologous recombination (Murphy, 1998). Next, the CRS cassette was removed using negative-selection markers and red recombination (Hashimoto et al., 2005). Two types of deletion mutants with and without the CRS cassette were constructed and transferred to recipient cells by transduction selleckchem with P1 phage. To avoid creating strains with synthetic growth defects and circumvent the complications associated with the use of a long CRS cassette fragment, a convenient method (ApR-415S Sm system) for introducing the new deletions was developed (Fig. 1). First, the ApR deletion units were constructed by replacing them with an ApR fragment that was shorter than the CRS cassette. After confirming that there was no synthetic growth defect, the ApR fragment was removed using ‘the 415S Sm system’ (Kato & Hashimoto, 2008), in which OSI-906 order the chromosomal regions flanking the region to be deleted were cloned into a ts replication plasmid 415S Sm to yield ‘the deletion plasmid.’ The 415S Sm plasmid was constructed by inserting

a negative-selection marker, the wild-type rpsL allele, into the ts plasmid pHSG415S that harbored a positive-selection ADAMTS5 marker CmR (Hashimoto-Gotoh et al., 1981). The deletion plasmid was then introduced into the

rpsL (SmR) mutant with an ApR deletion unit and the CmR transformants were selected at 42 °C. The transformants were incubated at 30 °C to obtain the SmR ApS strains. It was sometimes difficult to isolate ApS strains from the SmR strains using the ApR-415S Sm system. To make this easier, the FRT4 system was used (Fig. 2), in which a CmR fragment containing an FRT site, a recombination site for the FLP site-specific recombinase, was replaced with the deleted region to construct the CmR–FRT deletion unit. The deletion plasmid was constructed by inserting the fragment with the wild-type rpsL allele and the joined chromosomal regions that flanked the deleted regions into the plasmid pSG76A (ApR), which is an R6k derivative plasmid that lacks the pir gene required for replication (Posfai et al., 1997; Kato & Hashimoto, 2008). The deletion plasmid was introduced into the rpsL (SmR) mutant that harbored a CmR–FRT deletion unit and ApR transformants were selected. The FLP-containing plasmid was introduced into the ApR recombinant and, after adding tetracycline to the culture media to induce FLP recombinase, SmR strains were obtained (Posfai et al., 1997). Previously, a series of large-scale chromosome deletion mutants (Δ1–Δ16) were constructed.