Use of Tn5-gusA5to investigate environmental and nutritional effects on gene expression in the coronatine biosynthetic gene cluster ofPseudomonas syringaepv.glycinea

Abstract

Pseudomonas syringae pv. glycinea PG4180 produces coronatine (COR), a chlorosis-inducing phytotoxin that consists of the polyketide coronafacic 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 production of COR and its precursors remain unclear. In the present study, a λ delivery system for Tn5-gusA5 was developed and used to obtain transcriptional fusions in the COR gene cluster. Selected carbon (fructose and xylose) and amino acid (isoleucine and valine) sources significantly decreased COR biosynthesis at the transcriptional level. Transcriptional activity in the COR gene cluster was temperature dependent with maximal expression at 18–24 °C and significantly less expression at 14 and 30 °C. Interestingly, changes in osmolarity and the addition of complex carbon and nitrogen sources to the growth medium did not significantly affect COR gene expression, although both factors significantly impacted the quantity of COR produced. These results indicate that multiple factors impact COR production and only some of these directly affect transcription in the COR gene cluster.Key words: transcriptional fusion, glucuronidase, gene expression, reporter gene.