Bacterial cell growth requires the coordinated synthesis of all the metabolites necessary for the enlargement of the cell. One essential component of the bacterial cell wall that is unique to bacteria is D-alanine. This amino acid isomer is universally used by bacteria in the synthesis of the peptidoglycan (PG) where it acts as a substrate for penicillin binding protein-mediated crosslinking of the glycan strands (Hernandez & Cava, 2016;Lam et al., 2009). Also, in Grampositive bacteria, D-alanine is used to modify the teichoic acids, which are present as a second major polymeric component of the cell wall and membrane (Perego et al., 1995;Wecke et al., 1996). The other isomeric form, L-alanine, is an essential precursor for protein synthesis.Bacteria obtain D-alanine primarily by converting L-alanine by racemization. L-alanine, on the other hand, can be biosynthesized by the cell, or assimilated by the cell if present in the extracellular environment. Alanine synthesis and metabolism is increasingly being exploited for antibacterial and industrial applications. For example, exogenous L-alanine has been shown to increase the susceptibility of some pathogens to antibiotics (Peng et al., 2015); D-alanine can

Despite a long history of genetic manipulation of Bacillus subtilis using auxotrophic markers in genetic manipulation, the genes involved in alanine metabolism have not been characterised fully. Here we show that B. subtilis expresses an alanine uptake permease, YtnA (DatA), that has a major role in the assimilation of D-alanine from the environment. Since this isomer of the amino acid is not normally abundant it likely source is form the cells own cell wall probably through the action of carboxypeptidases and/or the spontaneous release of D-alanine from the teichoic acids. Also in this work we clarify the synthetic pathways acting in the biosynthesis of alanine. Genetically we show that, unlike E. coli where multiple enzymes have a biochemical activity that can generate alanine, in B. subtilis the primary synthetic enzyme for alanine is encoded by alaT, although a second gene, dat, is present that can support slow growth of an alanine auxotroph however our data suggests that this enzyme probably synthesises D-alanine. In summary this work has provided an explanation of the observation that growth of B. subtilis is linked with an efficient recycling system for D-alanine that is released from the cell as the cell envelope is processed to permit cell enlargement. The results also suggest that the relative abundance of D- and L-alanine that might be linked with cytosolic pool of D and L-glutamate, and so enabling tight coupling protein and cell envelope synthesis with the metabolic status of the cell.