CitB mutation increases the alkaline protease productivity in Bacillus subtilis.

Bacillus is remarkable for its extracellular enzyme production (Priest, 1977). When Bacillus cells exhaust the nutrients of the environment and enter into the stationary phase, neutral and alkaline proteases encoded by nprE and aprE respectively are produced and secreted to the outside environment (Chaloupka and Kreckova, 1966; Din et al., 1969). The mechanism of aprE expression has been extensively studied and so for four DNA-binding proteins have been proved to directly regulate its expression, which include ScoC, SinR, AbrB, and DegU (Gaur et al., 1991; Kallio et al., 1991; Shimane and Ogura, 2004; Strauch et al., 1989). In addition to these four regulators, a number of proteins also infl uence the expression of aprE indirectly, such as DegQ, DegR, TenA, ProB, RapG, and RelA (Hata et al., 2001; Mukai et al., 1992; Ogura et al., 1994, 2003; Pang et al., 1991). Aconitase, an enzyme that catalyses the interconversion of citrate and isocitrate in the citric acid cycle, plays a great role in the cell metabolism and regulation of iron uptake (Alen and Sonenshein, 1999; Dingman and Sonenshein, 1987). A citB null mutation in B. subtilis has been reported to cause a blockage in Spo0Aphosphate-dependent gene expression (Craig et al., 1997; Ireton et al., 1995) and accumulation of citrate (Craig et al., 1997). It has also been shown that in some pathogenic bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, and Xanthomonas campestris pv. campestris, aconitase is involved in the regulation of pathogenicity factor production (Somerville et al., 1999, 2002; Wilson et al., 1998). Isolated from the natural environment of China, Strain NK1010 is a B. subtilis wild type strain with high protease productivity. To further increase the protease productivity of this strain, we carried out insertional mutagenesis of strain NK1010 using the transposon mini-Tn10 (Steinmetz and Richter, 1994) and isolated one strain with high production extracellular proteases. Surprisingly, in this strain, the citB gene encoding aconitase was inactivated. After the mutant strain was complemented with citB gene expressed from plasmid pHCMC02-citB, the phenotype of the mutant strain could be partially restored. Furthermore, a citB selective inactivation strain, constructed with pMUTIN4, exhibited increased extracellular protease productivity. In summary, our results indicate that alkaline protease production in B. subtilis may be infl uenced by aconitase activity. B. subtilis strain NK1010, B. subtilis strain 168, E. coli strain DH5α and plasmids pIC333, pDG1663, pHCMC02, and pMUTIN4 were stored in our laboratory. Strain citB(GWH1023), citB+ (GWH1024) and plasmids pDG1663-PaprE, pHCMC02-citB, and pMUTIN4citB were constructed in this study. Unless stated otherwise, the B. subtilis strains were routinely grown at J. Gen. Appl. Microbiol., 56, 403‒407 (2010)