Preparation and characteristics of resting cells of bioluminescent Pseudomonas putida BLU

Abstract

A bioluminescent strain, Pseudomonas putida BLU, which was created by inserting the lux gene into the chromosome of P. putida mt-2 (TOL), a well-known degrader for benzene, toluene and xylene (BTX), produces luminescence depending on both cell concentration and intracellular metabolic activity, including the intracellular level of reduced riboflavin phosphate (FMNH 2). To estimate the cell concentration of a BTX degrader by the rapid detection of bioluminescence in a bioaugmentation system, the effect of metabolic activity on the output of bioluminescence needs to be considered. For this purpose, the preparation of resting cells of P. putida BLU, defined as incapable of growing but having metabolic activity, was tried using different surfactants. Resting cells formed on addition of n-dodecyltrimethylammonium bromide (DTAB, a cationic surfactant) at a concentration below the critical micellar concentration (CMC) to the bacterial culture, and exhibited morphological change. The P. putida BLU showed a decrease in bioluminescence output to a constant level and stopped growing immediately after the addition of DTAB, while maintaining activity for oxygen consumption. When the bacterial culture treated with DTAB was washed with a 0.9% saline solution, the growth and bioluminescence of the resting cells recovered to the normal level. The bioluminescence output (LUX) of the resting cells prepared at different time points from the bacterial culture correlated only with the cell concentration ( X), LUX∝ X 1.0, suggesting that generating resting cells enables the cell concentration to be simply and rapidly quantified by measuring the luminescence.