Genetic design of conditional d-glutamate auxotrophy for Bacillus subtilis: Use of a vector-borne poly-γ-glutamate synthetic system

Bacillus subtilis possesses two glutamate racemase isozymes, RacE and YrpC. For the first time, we succeeded in constructing glutamate racemase-gene disruptants of B. subtilis. Phenotypic analysis of their d-glutamate auxotrophy indicated that the RacE-type glutamate racemase is important for ensuring maximum growth rate but dispensable. The YrpC-type glutamate racemase probably operates as an anaplerotic enzyme for RacE, especially under liquid culture conditions. We found novel applicability of RacE-less mutants inheriting only a marginal activity for endogenous d-glutamate supply, viz. the employment for the in vivo identification of d-glutamate-consuming systems. In fact, the genetic induction of a poly-γ-glutamate synthetic system led a RacE-less mutant to severe growth suppression, which was overcome in the presence of a high concentration of exogenous d-glutamate. The results indicate that a significant amount of d-glutamate is consumed during poly-glutamate biosynthesis. To our knowledge, this is the first report of conditional d-glutamate auxotrophy for B. subtilis.