Stringent response mutants of Pseudomonas chlororaphis PA23 exhibit enhanced antifungal activity against Sclerotinia sclerotiorum in vitro

Abstract

The stringent response (SR) is a regulatory mechanism that enables bacteria to adapt to nutrient stress through the production of the alarmone (p)ppGpp. The aim of the current study was to understand how the SR affects the antifungal (AF) activity of Pseudomonas chlororaphis PA23. Two SR mutants were generated, PA23relA and PA23relAspoT, that no longer produced (p)ppGpp. Both mutants exhibited increased inhibition of Sclerotinia sclerotiorum in vitro and elevated pyrrolnitrin (PRN), lipase and protease production. Phenazine (PHZ) levels, on the other hand, remained unchanged. Through transcriptional fusion analysis we discovered that prnA-lacZ (PRN) activity was increased in the SR mutants, whereas phzA-lacZ (PHZ) activity was equal to that of the wild-type. We also examined how the sigma factor RpoS impacts PA23-mediated antagonism. Similar to the SR mutants, an rpoS mutant of PA23, called PA23rpoS, exhibited enhanced AF activity in vitro and increased expression of PRN, protease and lipase. However, PHZ production and expression of phzA-lacZ were dramatically reduced. Consistent with what has been reported for other bacteria, the SR exerted positive control over rpoS expression. In addition, providing rpoS in trans restored the SR phenotype to that of the wild-type. Collectively, our findings indicate that this global stress response impacts production of PA23 AF compounds via regulation of rpoS transcription and has an overall negative influence on S. sclerotiorum antagonism.