Characterization of Acidovoraxspecies Strain JS42 Taxis Towards 2-Nitrotoluene

Abstract

2-Nitrotoluene (2NT) is a toxic man-made nitroarene compound generated in the production and use of products such as industrial solvents, pesticides and explosives. Acidovorax sp. strain JS42 was isolated for its ability to utilize 2NT as its sole carbon, nitrogen and energy source. The key enzyme in the 2NT-degradation pathway, 2NT-dioxygenase (2NTDO; encoded by the ntd operon) catalyzes the oxidation of 2NT to 3-methylcatechol and nitrite. Motile bacteria like strain JS42 have the ability to direct their movement either towards or away from specific chemicals in the environment using a behavior called chemotaxis. This ability has been shown to increase the efficiency of biodegradation. The goal of this research was to determine whether strain JS42, which has recently evolved proteins for degradation and regulation of 2NT mineralization, has also evolved the ability to detect and respond to 2NT using chemotaxis, and if so, how that system works. Qualitative capillary assays were used to evaluate the chemotactic responses of strain JS42 to 2NT and 2NT metabolites. 2NT elicited a constitutive response that was strongly increased when the cells were grown with 2NT. I also determined that induction of ntd operon expression correlated with the induced taxis response. Salicylate, a gratuitous inducer of the ntd operon, also induced taxis towards 2NT, while intermediates of the 2NT degradation pathway that do not induce ntd operon expression (such as 3-methylcatechol and nitrite) did not induce taxis towards 2NT. Metabolism of 2NT was required for the induced response, but not for part of the constitutive response. Mutants unable to catalyze the first step in 2NT degradation (those lacking genes encoding 2NTDO or ntdR, which codes for the transcriptional activator required for expression the ntd operon) did not show the induced response, but did show a weak constitutive response lower than that of wild type, indicating that 2NT is sensed directly. I also found that the 2NT metabolite nitrite is a strong attractant for strain JS42. I quantified the amount of nitrite produced during taxis assays with 2NT as the attractant, and results suggest that strain JS42 cells produce sufficient nitrite during the taxis assay to account for a significant part of the induced response to 2NT. A mutant strain JS42 defective for ntdY, which codes for a putative chemoreceptor, and is located adjacent to ntdR, showed a defect in taxis towards 2NT. NtdY appears to detect 2NT directly but further experiments will be needed to confirm this result. Finally, soft agar swarm plates were used to evaluate the tactic response towards 2NT of a mutant defective for the energy-taxis receptor, Aer. Results indicated that a functional aer is required for a substantial part of the wild-type induced response to 2NT. In summary, my results show that strain JS42 utilizes three types of taxis to sense and respond to 2NT: constitutive metabolism-independent 2NT-specific chemotaxis, most likely mediated by NtdY, metabolism-dependent nitrite-specific chemotaxis, and energy taxis mediated by Aer.