Physiological considerations of environmental applications of lux reporter fusions

Abstract

The performance of the bioluminescent reporter bacterium Pseudomonas fluorescens HK44, which contains a nahG-luxCDABE fusion for naphthalene catabolism and bioavailability was investigated for environmental samples and mixed contaminants. For aqueous extracts from fuel hydrocarbon-contaminated soils, a reproducible bioluminescence response was obtained, which coincided with, but overestimated the presence of naphthalene. Therefore, the strain's bioluminescence response to mixed contaminants was investigated further. The strain showed a linear correlation between bioluminescence and the amount of JP-4 jet fuel present in an aqueous solution, representing a mixture of compounds including naphthalene. However, some non-inducing organic solvents such as toluene, p-xylene and acetone caused a significant bioluminescence increase as well. The analysis of nah-lux mRNA from cells exposed to toluene revealed that the bioluminescence response was not due to increased nahG-luxCDABE gene expression, whereas increased lux mRNA levels were found with exposure to naphthalene or JP-4 jet fuel. While different mixture combinations of solvents resulted in either additive or intermediate effects, the combination of naphthalene and solvent resulted in a synergistic effect on the bioluminescence response. The addition of n-decanal, a substrate for luciferase, showed that the cells were aldehyde-limited. If aldehyde was added, only the presence of naphthalene caused a significantly increased bioluminescence response over the control. The solvent effects were dependent on the physiological status of the reporter culture and were present in growing, but not in resting cell cultures. It was postulated that the increase in bioluminescence after exposure to solvents was due to changed fatty acid synthesis patterns affecting the aldehyde supply for the bioluminescence reaction. Exposure to toluene resulted in altered membrane fatty acid composition and release of fatty acids from the cells. Exposure to n-alkanes resulted in minor changes in the bioluminescence response, whereas, exposure to heavy metals or cyanide resulted in significant reductions in the overall bioluminescence. These results demonstrate the utility of bioluminescent reporter bacteria for environmental applications, as well as the need for adequate experimental controls in interpreting environmental sensing data.