Isolation of <i>B. subtilis</i> mutants that have inactivated the <i>gudB</i> gene.

<p>(A) Lack of exogenous glutamate is the driving force allowing the selection of mutants with inactivated <i>gudB</i> alleles. (B) Prior to growth in the absence of glutamate, the <i>B. subtilis rocG^–</i> mutant strain GP801 (<i>ΔrocG gudB⁺</i>) expressing only the active GDH, GudB was grown in C minimal medium supplemented with glucose and glutamate as carbon and nitrogen sources (plus glutamate), respectively. During growth in minimal medium lacking glutamate (no glutamate) samples were taken at indicated time points. (C) 5 µl were plated from serial dilutions (from non-diluted till 10⁻⁶) of cell suspensions of the <i>gudB^CR</i> and <i>gudB⁺</i> control strains GP754 (<i>ΔrocG gudB^CR</i>) and GP801 (<i>ΔrocG gudB⁺</i>), respectively, and the isolated <i>gudB^–</i> mutants for phenotypic analyses. The dilutions were spotted on minimal medium agar plates supplemented either with glucose and ammonium or with glutamate and ammonium. The plates were incubated for 48 h at 37°C. (D) Western blot analysis to monitor synthesis of the GDH, GudB in the <i>gudB^–</i> isolates using GDH-specific antibodies. Results of the sequence analysis of the <i>gudB^–</i> alleles are summarized below (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066120#pone-0066120-t001" target="_blank">Table 1</a>).