Growth of Pseudomonas aureofaciens PGS12 and the dynamics of HHL and phenazine production in liquid culture, on nutrient agar, and on plant roots

Abstract

The growth of Pseudomonas aureofaciens PGS12 was followed in nutrient broth (NB), on nutrient agar (NA), and on plant roots by monitoring cell numbers, the production of the autoinducer hexanoyl-homoserine lactone (HHL), and the antibiotic phenazine-1-carboxylic acid (PCA). In NB, as the growth rate declined in transition phase, HHL synthesis increased rapidly, shortly followed by PCA production. During stationary phase, HHL concentration declined rapidly while PCA concentration continued to increase slowly. The luxAB reporter genes were inserted in the phzB gene of the phenazine operon and phenazine transcriptional activity was monitored using measurement of luminescence. Levels and pattern of light output were similar to HHL accumulation and indicated that gene expression was maximal in transition phase and silenced in stationary phase. PCA production continued in stationary phase, suggesting that the protein products of the phenazine operon were maintained in the cell after down regulation. HHL accumulation was 60 times higher on NA than in NB per equivalent volume because of a 60-fold increase in cell density on NA. Higher levels of PCA per cell (6.8 times) and per equivalent volume (360-fold) accumulated in a colony compared to that found in broth. HHL remained at a high concentration in a colony for a longer period compared to a short burst in NB, and this may explain the increased PCA production. In contrast, on wheat seedlings and bean plant roots, bacterial growth was observed, but neither HHL nor PCA was detected; however, transcriptional activity of the phzB::luxAB reporter occurred on the bean plant roots.