Construction and Characterization of a Lux-Marked Phenanthrene Degrading Bacterium

Abstract

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