USE OF TN5-GUSA5 TO INVESTIGATE ENVIRONMENTAL AND NUTRITIONAL EFFECTSON GENE-EXPRESSION IN THE CORONATINE BIOSYNTHETIC GENE-CLUSTER OF PSEUDOMONAS-SYRINGAE PV GLYCINEA

Pseudomonas syringae pv. glycinea PG4180 produces coronatine (COR), a chlorosis-inducing phytotoxin that consists of the polyketide coronafa cic acid (CFA) coupled via an amide bond to the ethylcyclopropyl amino acid coronamic acid (CMA). Both CFA and CMA function as intermediates in the pathway to coronatine, and genes encoding their synthesis have been localized; however, the precise factors that regulate the produc tion of COR and its precursors remain unclear. In the present study, a lambda delivery system for Tn5-gusA5 was developed and used to obtain transcriptional fusions in the COR gene cluster. Selected carbon (fru ctose and xylose) and amino acid (isoleucine and valine) sources signi ficantly decreased COR biosynthesis at the transcriptional level. Tran scriptional activity in the COR gene cluster was temperature dependent with maximal expression at 18-24 degrees C and significantly less exp ression at 14 and 30 degrees C. Interestingly, changes in osmolarity a nd the addition of complex carbon and nitrogen sources to the growth m edium did not significantly affect COR gene expression, although both factors significantly impacted the quantity of COR produced. These res ults indicate that multiple factors impact COR production and only som e of these directly affect transcription in the COR gene cluster.