Cloning of a Putative Peptide Synthetase Gene Involved in the Synthesis of Phaseolotoxin

Abstract

Phaseolotoxin [ N^δ-(N’-sulfo-diaminophosphinyl)-L-ornithyl-L-alanyl-L-homo-arginine ] is a chlorosis-inducing toxin produced by Pseudomonas syringae pv. phaseolicola. Our aim is to clone the gene involved in the synthesis of the peptide moiety of phaseolotoxin, which probably encodes a peptide synthetase similar to those involved in the synthesis of bioactive peptides produced by a variety of organisms. The primary structure of peptide synthetase genes is highly conserved, and consists of distinct homologous domains. A domain consists of at least six highly conserved core sequences of about three to eight amino acids whose order and location within all known domains is very similar. Based on this knowledge we synthesized degenerate primers whose sequences were derived from the core sequences “TGD” and “LGGXS”. Using these primers we amplified and cloned, from the P.s. pv. phaseolicola chromosomal DNA, a fragment of expected length (500 bp). We sequenced this fragment which revealed the expected domain structure with the core sequences “KIRGRIELGEIE” and “NGK” separated by the correct number of nucleotides. This shows that the P.s. pv. phaseolicola chromosome contains a peptide synthetase gene. This 500 bp fragment was used to screen the genomic cosmid library of P.s. pv. phaseolicola which yielded a clone containing a 23 kb insert. A 9.3 kb sublcone is being used in genetic experiments to determine if this gene encodes a product that is involved in the synthesis of the peptide moiety of phaseolotoxin as weil as in sequencing.