Genetic design of conditional D-glutamate auxotrophy for Bacillus subtilis: use of a vector-borne poly-gamma-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-gamma-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.
Ashiuchi
Makoto
M
Department of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502, Japan. ashiuchi@cc.kochi-u.ac.jp
Nishikawa
Yoshito
Y
Matsunaga
Ken'ichiro
K
Yamamoto
Masayoshi
M
Shimanouchi
Kazuya
K
Misono
Haruo
H
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001671:Biochemistry / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005821:Genetic Techniques; D005822:Genetic Vectors; D016680:Genome, Bacterial; D018698:Glutamic Acid / Q000737:chemistry*; D008957:Models, Genetic; D009154:Mutation; D010641:Phenotype; D011099:Polyglutamic Acid / Q000737:chemistry*; D019076:Transgenes
D011099:Polyglutamic Acid; D018698:Glutamic Acid