Evolution of 2-Nitrotoluene 2,3-dioxygenase and Gene Manipulation of Catechol 2,3-dioxygenase in Acidovorax sp. Strain JS42

Student Name: Jessica Li

UCD Department: Microbiology and Molecular Genetics

UCD Mentor: Dr. Rebecca Parales

Acidovorax (formerly Pseudomonas) sp. JS42 is able to use the compounds 2-nitrotoluene (2NT) and nitrobenzene as carbon, nitrogen, and energy sources. In the first step of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase (2NTDO) converts 2NT to 3-methylcatechol with concomitant nitrite release. 2NTDO has three component proteins, and although it can break down all three mononitrotoluene isomers, JS42 cannot grow on 3-nitrotoluene (3NT) or 4-nitrotoluene (4NT). However, it is possible to force JS42 to adapt to 3NT and 4NT with resulting changes to 2NTDO. In this study, we evolved 4NT+ JS42 strains to grow on 3NT using long-term selection. Sequencing of the ntdAc gene encoding the α subunit of 2NTDO oxygenase revealed substitutions at the amino acid position 204 on the active site. Mutations at positions 238 and 248 were conserved from the parent 4NT+ strain and no other mutations were present, supporting the hypothesis that the amino acid position 204 is crucial for the evolution of JS42 to utilize new substrates. The next step of the 2NT degradation pathway involves catechol 2,3-dioxygenase (CDO). Both ctdE1 and ctdE2 encode CDO, and each has an associated regulatory gene (ctdR1 and ctdR2). Based on the phenotype of a mutant strain with deactivated ctdE1, ctdE1 seems critical to the degradation pathway. We will complement the ctdE1 mutants to verify the role of the enzyme in 2NT degradation. It is not yet known whether ctdR1 encodes an inducer or repressor, so ctdR1 inactivation will be used to determine the function of the regulatory protein.