Growth Phase and Temperature Influence Promoter Activity, Transcript Abundance, and Protein Stability during Biosynthesis of thePseudomonas syringaePhytotoxin Coronatine

Abstract

The plant-pathogenic bacterium Pseudomonas syringae pv. glycinea PG4180.N9 synthesizes high levels of the polyketide phytotoxin coronatine (COR) at 18°C, whereas no detectable toxin is produced at 28°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 well as the posttranslational effects of temperature on expression ofcmaB, 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°C. Promoter activity and transcription rates were maximal when cells were incubated at 18°C and sampled at mid-logarithmic phase. Transcription declined moderately during the transition to stationary phase but remained higher at 18°C than at 28°C. Western blot analysis indicated that CmaB accumulated in the late stationary phase of P. syringae cultures grown at 18°C but not in cultures incubated at 28°C. Temperature shift experiments indicated that CmaB stability was more pronounced at 18°C than at 28°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.

Pseudomonas syringae pv. aesculi: foliar infection of Aesculus species and temperature–growth relationships

“…Pathovar aesculi has been found to produce coronatine (Bultreys et al 2006(Bultreys et al , 2008Janse et al 2006). Budde et al (1998) found that coronatine production at 28°C, the optimum temperature for growth of P. syringae, was negligible, whereas it peaked at 18°C in pv. glycinea.…”

Extraction of high‐quality bacterial RNA from infected leaf tissue for bacterial in planta gene expression analysis by multiplexed fluorescent Northern hybridization

“…Validation of the method was addressed by in planta gene expression analysis of the P. syringae genes mucD , cmaA , cfl , corR , corS and corP , which we had investigated in previous studies (Budde et al ., 1998; Schenk et al ., 2006; Weingart et al ., 2004). Sensitivity was assessed by monitoring the moderately expressed lsc gene, indicating that gene expression from ~10 6 CFU per leaf disc can be detected.…”

“…As a result, PG4180.muc is able to produce large amounts of the exopolysaccharide alginate, whereas alginate production is negligible in PG4180 (Schenk et al ., 2006). We investigated the in vitro and in planta gene expression of mucD , coding for a periplasmic serine protease previously implicated in alginate regulation (Schenk et al ., 2006), expression of lsc , coding for levansucrase required for synthesis of the exopolysaccharide levan (Li et al ., 2001), expression of cmaA and cfl , genes involved in the biosynthesis of the phytotoxin coronatine (Budde et al ., 1998), and expression of corR , corS and corP , genes coding for regulators of coronatine biosynthetic genes (Weingart et al ., 2004). Several of these genes were previously reported to exhibit a thermoresponsive pattern of expression in vitro (Budde et al ., 1998).…”

How microbes utilize host ubiquitination

“…In turn, coronatine induces re-opening of the stomata and enables bacterial entry (Melotto et al, 2006). The biosynthetic pathways for coronatine in bacteria and jasmonyl-isoleucine in plants are fundamentally different indicative of true structural mimicry (Creelman and Mullet, 1997;Budde et al, 1998). By contrast, structural mimicry of proteins might also be derived from bacterial proteins with an enzymatic mechanism or fold that resemble partly the requirements of the novel function.…”