We isolated a novel gram-positive bacterium, Brevibacillus texasporus, that produces an antibiotic, BT. BT is a group of related peptides that are produced by B. texasporus cells in response to nutrient limitation. We report here purification and determination of the structure of the most abundant BT isomer, BT1583. Amino acid composition and tandem mass spectrometry experiments yielded a partial BT1583 structure. The presence of ornithine and d-form residues in the partial BT1583 structure indicated that the peptide is synthesized by a nonribosomal peptide synthetase (NRPS). The BT NRPS operon was rapidly and accurately identified by using a novel in silico NRPS operon hunting strategy that involved direct shotgun genomic sequencing rather than the unreliable cosmid library hybridization scheme. Sequence analysis of the BT NRPS operon indicated that it encodes a colinear modular NRPS with a strict correlation between the NRPS modules and the amino acid residues in the peptide. The colinear nature of the BT NRPS enabled us to utilize the genomic information to refine the BT1583 peptide sequence to Me(2)-4-methyl-4-[(E)-2-butenyl]-4,N-methyl-threonine-L-dO-I-V-V-dK-V-dL-K-dY-L-V-CH2OH. In addition, we report the discovery of novel NRPS codons (sets of the substrate specificity-conferring residues in NRPS modules) for valine, lysine, ornithine, and tyrosine.