Isolation and characterization of alanine mutants in Bacillus subtilis SB 19

Mutants of a wild-type B. subtilis strain, SB 19, were selected for their inability to metabolize L-alanine as a sole carbon and nitrogen source. One isolate, strain MH 312, was deficient in alanine racemase (E.C.5.1.1.1) and required small amounts of sc D-alanine to synthesize an osmotically stable cell wall in complex media. Exogenous glycine in complex media or basal salts minimal media inhibited stable cell wall formation resulting in lysis. sc D-Alanine prevented glycine-induced lysis. Up to 99% lysis occurred in both dilute and dense cell suspensions after addition of 1% glycine. No amino acid tested other than glycine induced lysis. Accumulation of four nucleotide-activated peptidoglycan precursors by mutant cultures was associated with lysis. No accumulation of precursors was observed in the absence of exogenous glycine, and sc D-alanine prevented accumulation. Characteristics of SB 19 cultures with alanine racemase inhibitors are similar to MH 312 cultures. $\beta$-Chloro- sc D-alanine addition to SB 19 caused a slight inhibition of growth but added in combination with 1% glycine initiated rapid cell lysis. sc D-cycloserine behaved similarly, and sc D-alanine prevented lysis in both cases. Addition of glycine or $\beta$-chloro- sc D-alanine separately to SB 19 cultures did not cause precursor accumulation or cell lysis, but added in combination caused accumulation of the same precursors observed with MH 312 cultures. Racemase inhibitors in combination with glycine exhibit strong antibacterial action. Ultrastructural studies revealed a fundamental difference between parent and mutant cell morphologies. Glycine-induced damage was not localized in specific regions and occurred at sites of new cell wall synthesis. Some protoplasts had remnants of cell wall closely associated to the membrane surface. In plasmid DNA transformation studies using polyethylene glycol, protoplasts formed with lysozyme, autolysins, and glycine transformed equally well (10$\sp5/\mu$g DNA) however regeneration frequencies differed. Another alanine mutant (strain WS 610) required no sc D-alanine and had normal levels of alanine racemase but generally much higher levels (20 fold) of alanine and lactate dehydrogenase activities