REGULATION OF THE ALGINATE BIOSYNTHESIS GENE ALGC IN PSEUDOMONAS-AERUGINOSA DURING BIOFILM DEVELOPMENT IN CONTINUOUS-CULTURE

Reporter gene technology was used to observe the regulation of the alginate biosynthesis gene, algC in a mucoid strain of Pseudomonas aeruginosa in developing and mature biofilms in continuous culture on Teflon and glass substrata, The plasmid pNZ63, carrying an algC-lacZ transcriptional fusion, was shown to not be diluted in continuous culture over a period of 25 days in the absence of selection pressure, Biofilm cells under bulk phase steady-state conditions demonstrated fluctuations in algC expression over a 16-day period, but no trend of increased or decreased expression over the time interval was indicated, In vivo detection of algC up-expression in developing biofilms was performed with a fluorogenic substrate for the plasmid-borne lacZ gene product (beta-galactosidase) by using microscopy coupled with image analysis, By this technique, cells were tracked over time and analyzed for algC activity. During the initial stages of biofilm development, cells already attached to a glass surface for at least 15 min exhibited up-expression of algC, detectable as the development of whole-cell fluorescence, However, initial cell attachment to the substratum appeared to be independent of algC promoter activity, Furthermore, cells not exhibiting algC up-expression were shown to be less capable of remaining at a glass surface under flowing conditions than were cells in which algC up expression was detected.