THE PEPTIDE SYNTHETASE GENE PHSA FROM STREPTOMYCES VIRIDOCHROMOGENES IS NOT JUXTAPOSED WITH OTHER GENES INVOLVED IN NONRIBOSOMAL BIOSYNTHESIS OF PEPTIDES

By complementation of a previously described non-phosphinothricin trip eptide (PTT)-producing mutant, NTG1, which is blocked in nonribosomal synthesis of the peptide, a DNA fragment including the putative peptid e synthetase gene phsA was isolated (MT, Wohlleben, R, Alijah, J, Dore ndorf, D, Hillemann, B. Nu beta baumer, and S, Pelzer, Gene 115:127-13 2, 1992), Sequence analysis of phsA revealed that it encodes a protein of 622 amino acids with regions which are highly similar to core moti fs characteristic for peptide synthetases. PhsA represents one functio nal domain of a peptide synthetase which is necessary for activation a nd condensation of one amino acid, probably N-acetyl-demethyl-phosphin othricin. With regard to the arrangement of the flanking genes, phsA i s the first peptide synthetase gene which is not in the direct neighbo rhood of additional peptide synthetase genes involved in the formation of peptide antibiotics, Gene disruption mutants with internal fragmen ts of phsA subcloned in temperature-sensitive pGM vectors were generat ed, Integration occurred either into the chromosomal copy of phsA or i nto a gene outside the known phsA locus, resulting in two classes of n on-PTT-producing mutants, In cofeeding experiments the former phsA mut ants showed the same phenotype as did NTG1, which confirmed participat ion of phsA in nonribosomal synthesis of PTT, A truncated phsA gene wa s overexpressed in Escherichia coli, and the resulting protein of 593 amino acids was purified for raising antibodies, By performing immunob lotting experiments, the expression of phsA could be detected in Strep tomyces viridochromogenes Tu494 in the stationary-growth phase after 4 days of incubation.