Evolution of a new bacterial pathway for 4-nitrotoluene degradation

Abstract

Bacteria that assimilate synthetic nitroarene compounds represent unique evolutionary models, as their metabolic pathways are in the process of adaptation and optimization for the consumption of these toxic chemicals. We used Acidovorax sp. strain JS42, which is capable of growth on nitrobenzene and 2-nitrotoluene, in experiments to examine how a nitroarene degradation pathway evolves when its host strain is challenged with direct selective pressure to assimilate non-native substrates. Although the same enzyme that initiates the degradation of nitrobenzene and 2-nitrotoluene also oxidizes 4-nitrotoluene to 4-methylcatechol, which is a growth substrate for JS42, the strain is incapable of growth on 4-nitrotoluene. Using long-term laboratory evolution experiments, we obtained JS42 mutants that gained the ability to grow on 4-nitrotoluene via a new degradation pathway. The underlying basis for this new activity resulted from the accumulation of specific mutations in the gene encoding the dioxygenase that catalyses the initial oxidation of nitroarene substrates, but at positions distal to the active site and previously unknown to affect activity in this or related enzymes. We constructed additional mutant dioxygenases to identify the order of mutations that led to the improved enzymes. Biochemical analyses revealed a defined, step-wise pathway for the evolution of the improved dioxygenases.

Fig. 1.

Nitroarene dioxygenase activity in Acidovorax sp. JS42 and 4NT⁺ evolved strains induced with salicylate. Substrates: nitrobenzene (white), 2NT (light grey), 3NT (dark grey), or 4NT (black) were provided at 1 mM and nitrite in culture supernatants was measured after 30 min. Fold increases in activity with 4NT are indicated. Error bars indicate standard deviations (N = 3).

Fig. 2.

Nitrite produced by E. coli cultures expressing the 2NTDO variants in 6 h whole-cell reactions with nitrobenzene (white), 2NT (light grey), 3NT (dark grey), or 4NT (black) as substrates. The wild-type and evolved dioxygenases are labeled with the names of the parental strains; the other enzymes were generated by site-directed mutagenesis. The S242N and L238V S242N mutants did not produce detectable amounts of nitrite from any of the substrates. Fold increases in activity with 4NT are indicated. Error bars indicate standard deviations (N = 3).

Fig. 3.

Comparison of 4NT degradation pathways in Pseudomonas sp. strain 4NT (Haigler and Spain, 1993), P. putida TW3 (Hughes and Williams, 2001; James and Williams, 1998; James et al., 2000; Rhys-Williams et al., 1993), Mycobacterium sp. strain HL4-NT-1 (Spiess et al., 1998) and evolved variants of Acidovorax sp. strain JS42.

Fig. 4.

Proposed pathway for the evolution of 2NTDO to variant enzymes with improved 4NT oxidation activity. Dioxygenases with higher relative activities with 4NT are indicated with darker shading; those with no detectable activity are shown with dashed-borders. Solid black arrows indicate steps that lead to enzymes with improved activity (enzymes identified in this study indicated by asterisks), and solid white arrows indicate another possible route to enzymes with improved activity. Dashed arrows indicate routes to enzymes with decreased activity.