Construction of an S. islandicus mutant with unmarked in-frame apt and amyA deletions.

S. islandicus RJW004 (ΔargD ΔpyrEF ΔlacS) (12) was used as the host strain to construct the Δapt mutant (RJW009). The linearized knockout plasmid pMID-apt was introduced into RJW004 by an electroporation-mediated transformation procedure that was described earlier (41) and modified as described in reference 12. The transformants with the hybrid marker cassette argD-pyrEF-lacS integrated into the RJW004 chromosome were screened on selective plates without agmatine. The sequence of one candidate strain (pMID-apt-T) was further confirmed by PCR using the primers apt-flankP-F/R, which were specific for sequences located outside the apt flanking region (Table 2). To obtain unmarked apt deletion strains, colonies in a 6-methylpurine-resistant culture were first enriched in liquid medium containing uracil (20 μg/ml), agmatine (1 mg/ml), GMP (0.5 mM), and 6-MP (150 to 300 μM) and then plated on solid medium. Single colonies were screened by colony PCR using two distinct primer sets, apt-flankP-F/R and apt-intP-F/R, which annealed outside the flanking regions and internal sequences of apt, respectively (Table 2). The sequences of the resulting PCR fragments amplified by primer set apt-flankP-F/R were further confirmed by sequencing of the apt locus.

The ΔamyA mutant (RJW010) was constructed by using RJW009 (RJW004 Δapt) as a parent strain. Again, the transformant (pMID-amyA-T) with a hybrid maker cassette argD-apt-lacS integrated into the RJW009 chromosome was selected without agmatine, and successful integration was confirmed by PCR using the proper primer sets shown in Table 2. Cell cultures positive for pMID-amyA-T were then directly subjected to counterselection in DT medium containing uracil (20 μg/ml), agmatine (1 mg/ml), GMP (0.5 mM), and 6-MP (150 to 300 μM). The in-frame deletion of amyA was confirmed by PCR and sequencing analysis at the amyA locus with appropriate primers (Table 2).