Construction and characterization of a lux-marked phenanthrene degrading bacterium

Construction of a luminescent microbial biosensor as a sensitive and rapid biotechnological tool for monitoring survival and activity of genetically engineered microorganisms (GEMs) in the contaminated environment has been the main focus of this study. Four rifampicin resistant phenanthrene-degrading bacteria viz., Commamonas testosteroni GZ38A, C. testosteroni GZ39, Pseudomonas putida GZ44 and P. stutzeri P16 were made using gradient plate techniques. All resistant strains were transformed with the plasmid harboring the mini-Tn5-tet transposon cassette, containing the luxAB genes to confer stability. C. testosteroni GZ38A and P. stutzeri P16 were successfully lux-marked in this manner. It was found that lux-marked strains of C. testosteroni GZ38A were not able to degrade phenanthrene. Although the maximum growth rate of lux-marked strain was significantly lower (P < 0.05) than that of the wild type P. stutzeri P16, the P. stutzeri P16-luxAB4 was selected because it has the following criteria: the stability of expression of tet- resistance over 180 generations, phenanthrene -degradability and high level of luminescence light output in the absence and presence of phenanthrene. The addition of 1 litter (0.5 % v/v) n-decyl aldehyde produced consistently high levels of luminescence at various stages of selected strain. Results clearly indicated that lux-marked strain was appropriately constructed and it is a novel biodegradative luminescent biosensor which enables us to monitor the fate of a phenathrene bacterial degrader genetically or non-genetically made within a polluted environment.