Growth phase and temperature influence promoter activity, transcript abundance, and protein stability during biosynthesis of the Pseudomonas syringae phytotoxin coronatine

The plant-pathogenic bacterium Pseudomonas syringae pv. glycinea PC4180.N9 synthesizes high le, cls of the polyketide phytotoxin coronatine (COR) at 18 degrees C, whereas no detectable toxin is produced at 28 degrees C. Previously, we reported that the temperature sensitive activation of three promoters within the COR biosynthetic gene cluster might explain thermoregulation of COR biosynthesis. The present study was aimed at furthering our understanding of the transcriptional as web as the posttranslational effects of temperature on expression of cmaB, which encodes an enzyme involved in COR biosynthesis. Transcriptional fusions using a promoterless glucuronidase gene and Northern blot analyses were used to monitor promoter activities and transcript abundance for the cmaABT operon during bacterial growth at 18 and 28 degrees C. Promoter activity and transcription rates were maximal when cells mere incubated at 18 degrees C and sampled at mid-logarithmic phase. Transcription declined moderately during the transition to stationary phase but remained higher at 18 degrees C than at 28 degrees C. Western blot analysis indicated that CmaB accumulated in the late stationary phase of P. syringae cultures grown at 18 degrees C but not in cultures incubated at 28 degrees C. Temperature shift experiments indicated that CmaB stability was more pronounced at 18 degrees C than at 28 degrees C. Although temperature has a strong impact on transcription of COR biosynthetic genes, we propose that thermoregulation of protein stability might also control COR synthesis.