CLONING AND EXPRESSION OF GENES REQUIRED FOR CORONAMIC ACID (2-ETHYL-1-AMINOCYCLOPROPANE 1-CARBOXYLIC ACID), AN INTERMEDIATE IN THE BIOSYNTHESIS OF THE PHYTOTOXIN CORONATINE

Coronamic acid (CMA; 2-ethyl-1-aminocyclopropane 1-carboxylic acid) is an intermediate in the biosynthesis of coronatine (COR), a chlorosis- inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG41 80. Tn5 mutagenesis and substrate feeding studies were previously used to characterize regions of the COR biosynthetic gene cluster required for synthesis of coronafacic acid and CMA, which are the only two cha racterized intermediates in the COR biosynthetic pathway. In the prese nt study, additional Tn5 insertions were generated to more precisely d efine the region required for CMA biosynthesis. A new analytical metho d for CMA detection which involves derivatization with phenylisothiocy anate and detection by high-performance liquid chromatography (HPLC) w as developed. This method was used to analyze and quantify the product ion of CMA by selected derivatives of P. syringae pv. glycinea which c ontained mutagenized or cloned regions from the CMA biosynthetic regio n. pMU2, a clone containing a 6.45-kb insert from the CMA region, gene tically complemented mutants which required CMA for COR production. Wh en pMU2 was introduced into P. syringae pv. glycinea 18a/90 (a strain which does not synthesize COR or its intermediates), CMA was not produ ced, indicating that pMU2 does not contain the complete CMA biosynthet ic gene cluster. However, when two plasmid constructs designated pMU23 4 (12.5 kb) and pKTX30 (3.0 kb) were cointroduced into 18a/90, CMA was detected in culture supernatants by thin-layer chromatography and HPL C. The biological activity of the CMA produced by P. syringae pv. glyc inea 18a/90 derivatives was demonstrated by the production of COR in c osynthesis experiments in which 18a/90 transconjugants were cocultivat ed with CMA-requiring mutants of P. syringae pv. glycinea PG4180. CMA production was also obtained when pMU234 and pKTX30 were cointroduced into P. syringae pv. syringae B1; however, these two constructs did no t enable Escherichia coli K-12 to synthesize CMA. The production of CM A In P. syringae strains which lack the COR biosynthetic gene cluster indicates that CMA production can occur independently of coronafacic a cid biosynthesis and raises interesting questions regarding the evolut ionary origin of the COR biosynthetic pathway.