| An unusual class of pectolytic fluorescent Pseudomonas spp. was associated with rapid decay of celery and reduction of storage-life of fresh cut vegetables under refrigerated conditions. The objective of our research was to characterize and evaluate the potential role of surfactant products produced by this fluorescent Pseudomonas as a colonization and virulence factor. The minimum genetic elements required for biosurfactant biosynthesis were identified and near-isogenic strains, phenotypically biosurfactant-negative, were selected for comparative studies. To determine the potential for random isolation of phenotypic negative clones, the frequency of spontaneous loss of biosurfactant activity of a celery isolate, designated 123 RifR , identified as Pseudomonas fluorescens, was determined in liquid culture. A gradual population shift of 123 RifR was demonstrated; the capacity to reduce the surface tension of water (STW) was lost. When spontaneous biosurfactant-minus clones were inoculated in celery, all of them caused discoloration, as did the wild type and, the population reverted to a biosurfactant-positive phenotype. A group of six biosurfactant-minus mutants, induced by Tn5 mutagenesis, could not be differentiated in growth kinetics in culture, or on broccoli florets or lettuce leaves from the parental 123 RifR. They could be differentiated by their inability to reduce the STW, to grow in minimal medium supplemented with hexadecane and to rapidly detach from a lettuce leaf surface. Although these mutants retained their ability to cause decay on celery, broccoli and cauliflower, the extent of decay and re-distribution to non-inoculated parts was variable. One of these mutants caused a very low degree of decay in broccoli and cauliflower supporting the hypothesis that biosurfactant production has a role in virulence of the parental P. fluorescens. A 7.1 kb figment was isolated from a 123 RifR gene library using a cloned fragment from one mutant as probe. This fragment restored the ability of the mutant to reduce the STW. No biosurfactant compounds were obtained from cell-free supernatants of 123 RifR liquid cultures; however, bacterial pellets reduced the STW when they were tested, suggesting that the surfactant activity of 123 RifR is not released to the medium or must be associated with the intact membrane. |
