AN ALGD-BIOLUMINESCENT REPORTER PLASMID TO MONITOR ALGINATE PRODUCTION IN BIOFILMS

A broad-host range algD-lux bioluminescent reporter plasmid was develo ped to examine the role of exopolymer production in biofilm function. The algD-lux reporter plasmid will allow rapid on-line in situ detecti on of environmental factors that induce alginate biosynthesis. The alg D promoter was stimulated by factors previously reported to induce alg inate production, including ethanol and NaCl, and differences were obs erved with different nitrogen sources. With growth on minimal media wi th either glucose or succinate as a carbon source, succinate had a gre ater inductive effect on the algD promoter. An increase in light outpu t of 1.3-fold and 1.7-fold was seen with cultures amended with 50 and 150 mM NaCl, respectively, compared to cultures with succinate alone. NaCl induction of the algD promoter was confirmed by algD RNA slot blo ts. Light output increased 2.0-fold and 1.7-fold with 0.25% and 0.5% e thanol, respectively, compared with controls grown with succinate only . While the rate of algD promoter response was initially similar when either NH4 or NO3 was used as a nitrogen source, NH4-grown cultures ma intained a higher light output during late log phase compared to NO3-g rown cultures.