Nitrogen metabolism was investigated during poly-gamma-glutamic acid (gamma-PGA) synthesis and phylogenetic analyses was used to explore glutamate dependency in Bacillus subtilis HSF1410. Bacillus subtilis HSF1410 was cultured with (15)NH4+ in the medium, and the gamma-PGA synthesized was then analyzed using N-15-NMR. The gamma-PGA framework was partially labeled with N-15, indicating that gamma-PGA was synthesized from inorganic nitrogen and carbohydrate. Assay of glutamate synthetase and glutamine synthetase activities also revealed that ammonium can be used to synthesize gamma-PGA in HSF1410. Phylogenetic trees based on gamma-PGA synthesis genes (pgsBCA) and 16S rRNA showed that HSF1410 falls within a cluster of glutamate-dependent strains, versus glutamate-independent strains, which is confirmed by HSF1410 being unable to produce gamma-PGA without glutamate in its medium we classify B. subtilis HSF1410 as a glutamate-dependent strain that can use exogenous inorganic nitrogen sources to synthesize gamma-PGA.