Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

Abstract

Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3-or 4-nitrotoluene. Using both long-and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the ␣ subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome. N itroaromatic compounds are toxic and stable in the environment and are important components in the manufacture of dyes, polymers, explosives, and pesticides (1). The synthesis and widespread use of these products have caused environmental contamination of soil and groundwater (2, 3). In addition, nitroaromatic compounds tend to undergo reduction, resulting in the formation of mutagenic aromatic amines (4). Due to the stability, toxicity, and mutagenicity of these chemicals, the U.S. Environmental Protection Agency has designated several nitroaromatic compounds as priority pollutants (5).Although the majority of nitroarene compounds are anthropogenic, a number of microorganisms have developed the ability to utilize compounds of this chemical class as sources of carbon, nitrogen, and energy (6). Bacterial strains capable of growth on nitrobenzene (7-9), mononitrotoluenes (10-16), and dinitrotoluenes (17-19) have been isolated from various contaminated environments. For example, Acidovorax sp. strain JS42 (formerly Pseudomonas sp. strain JS42) uses nitrobenzene (NB) and 2-nitrotoluene (2NT) as sole sources of carbon, nitrogen, and energy (15,20), while Comamonas sp. strain JS765 is capable of growth on NB and 3-nitrotoluene (3NT) (9, 21). The first enzymes of each degradation pathway, 2-nitro...

Application of Aromatic Hydrocarbon Dioxygenases

“…Interestingly, the 4nitrotoluene + mutants retained their ability to grow on 2-nitrotoluene [80], while the 2-nitrotoluene-degrading ability of most of the 3-nitrotoluene + variants was reduced. In contrast to the 4-nitrotoluene + dioxygenases, which were sufficient to confer the ability to grow on 4-nitrotoluene to a JS42 mutant lacking a functional 2-nitrotoluene dioxygenase, introduction of the mutant dioxygenase genes from the evolved 3-nitrotoluene + strains did not confer the ability to grow on all substrates [81]. Together, these results indicate that additional beneficial mutation(s) elsewhere in the genome must have been acquired during selection on 3-nitrotoluene [81].…”

“…In contrast to the 4-nitrotoluene + dioxygenases, which were sufficient to confer the ability to grow on 4-nitrotoluene to a JS42 mutant lacking a functional 2-nitrotoluene dioxygenase, introduction of the mutant dioxygenase genes from the evolved 3-nitrotoluene + strains did not confer the ability to grow on all substrates [81]. Together, these results indicate that additional beneficial mutation(s) elsewhere in the genome must have been acquired during selection on 3-nitrotoluene [81]. These types of laboratory selections have allowed the isolation of bacterial variants that are capable of degrading multiple pollutants and provide insight into the selection processes that occur in nature.…”

“…strain JS42 that are able to grow on and degrade alternative nitroarene substrates [80,81]. Wild-type strain JS42 is capable of using 2nitrotoluene as a sole carbon, nitrogen, and energy source [82].…”

“…In two different studies, Ju and Parales [80] and Mahan et al [81] evolved variants of Acidovorax sp. strain JS42 that are able to degrade various mononitrotoluene iso mers by forcing the wild-type strain to grow on 3-nitrotoluene or 4-nitrotoluene.…”

3,6-Dichlorosalicylate Catabolism Is Initiated by the DsmABC Cytochrome P450 Monooxygenase System in Rhizorhabdus dicambivorans Ndbn-20

“…The reason might be that the transformation of 3,6-DCSA was the rate-limiting step in dicamba catabolism and that other metabolites were quickly transformed to an undetectable level. In previous reports, many genes or gene clusters responsible for the degradation of xenobiotic compounds were located on mobile genetic elements or catabolic plasmids and were thus prone to be lost without selective pressure from substrates (24)(25)(26)(27)(28)(29). Therefore, in this study, we attempted to obtain a 3,6-DCSA degradation-deficient mutant by growing Ndbn-20 cells on 1/5 Luria-Bertani (LB) agar without the addition of 3,6-DCSA.…”