Isolation, identification, and accumulation of 2-acetamidophenol in liquidcultures of the wheat take-all biocontrol agent Pseudomonas fluorescens 2-79

Pseudomonas fluorescens strain 2-79 (NRRL B-15132) is a classic biological control agent known to produce phenazine-1-carboxylic acid (PCA) as its pri mary means of suppressing take-all disease of wheat. In addition to PCA, an unknown metabolite was discovered in a liquid culture used to produce the biocontrol agent. The objective of the current study was to isolate, identi fy, and evaluate the accumulation of this compound in production cultures. Upon centrifugal fractionation of a production culture, thin-layer chromato graphy (TLC) analyses of extracts of the cells and cell-free supernatant in dicated the compound to be primarily in the supernatant. Purified compound was obtained by extraction of culture supernatant, followed by flash chroma tography of the extract and preparative TLC. The H-1 and C-13 nuclear magne tic resonance and electron impact mass spectra indicated the compound to be 2-acetamidophenol (AAP). Measured by reversed-phase HPLC, the accumulation s of AAP and PCA in cultures of strain 2-79 reached 0.05 g/l and 1 g/l, res pectively. The accumulations of AAP and PCA in liquid cultures were linearl y correlated (P < 0.001), as shown by studies of cultures stimulated to yie ld varying levels of PCA by controlling levels of oxygen transfer, pH, and growth medium composition. In this study, oxygen limitation, a defined amin o-acid-free medium, and neutral pH stimulated maximal production of both AA P and PCA. Furthermore, a transposon mutant of 2-79 [2A40 2-79 (phz-)] unab le to produce PCA did not accumulate AAP. These findings indicate that ABP and PCA are likely to share a common segment of biosynthetic pathway. This is the first report of BAP production by a strain of P. fluorescens. Possib le routes of AAP production are discussed relative to current knowledge of the phenazine biosynthetic pathway of strain 2-79. The pertinence of AAP to the design of commercial seed inoculants of phenazine-producing bacteria f or controlling wheat take-all is also considered.