Reconstructing the evolutionary history of nitrotoluene detection in the transcriptional regulator NtdR

Abstract

Many toxic man-made compounds have been introduced into the environment, and bacterial strains that are able to grow on them are ideal model systems for studying the evolution of metabolic pathways and regulatory systems. Acidovorax sp. strain JS42 is unique in its ability to use 2-nitrotoluene as a sole carbon, nitrogen, and energy source for growth. The LysR-type transcriptional regulator NtdR activates expression of the 2-nitrotoluene degradation genes not only when nitroaromatic compounds are present, but also in the presence of a wide range of aromatic acids and analogues. The molecular determinants of inducer specificity were identified through comparative analysis with NagR, the activator of the naphthalene degradation pathway genes in Ralstonia sp. strain U2. Although NagR is 98% identical to NtdR, it does not respond to nitrotoluenes. Exchange of residues that differ between NagR and NtdR revealed that residues at positions 227 and 232 were key for the recognition of nitroaromatic compounds, while the amino acid at position 169 determined the range of aromatic acids recognized. Structural modelling of NtdR suggests that these residues are near the predicted inducer binding pocket. Based on these results, an evolutionary model is presented that depicts the stepwise evolution of NtdR.

Fig. 1.

2-Nitrotoluene degradation pathway in Acidovorax sp. strain JS42.

Fig. 2.

Timing of ntd gene expression during growth of JS42 derivatives. Growth as measured by culture turbidity at 660 nm (A, B), β-galactosidase activity measured from the chromosomal ntdA-lacZ fusion (C, D), and 2NTDO activity (E, F) were followed over a 24-hour period for JS42–1 and JS42R-1(pNtd1). Cultures were grown in MSB with 10 mM succinate (open diamonds, dashed lines), or 10 mM succinate supplemented with 500 μM of salicylate (squares), 2-nitrotoluene (triangles), or 2-nitrobenzyl alcohol (circles) as inducers. n = 6. Error bars indicate standard deviations.

Fig. 3.

Expression of the ntd genes in wild type and catabolic and regulatory mutants of Acidovorax sp. JS42. β-Galactosidase activity measured from the chromosomal ntdA-lacZ fusion (A) and 2NTDO activity (B) in cultures of JS42–1 (wild type; white bars), JS42R-1 (ntdR::Km; light grey bars), JS42Ac-1 (ntdAc::Km; dark grey bars), and JS42E1–1 (ctdE1::Sm; black bars) grown on succinate alone (none) or with 500 μM of salicylate (2HBen), 2-nitrobenzyl alcohol (2NBa) or 2-nitrotoluene (2NT) provided as inducers. n = 6. Error bars indicate standard deviations.

Fig. 4.

Inducing compounds detected by NagR and NtdR in Acidovorax sp. JS42R-1. (A) β-Galactosidase activity measured from the chromosomal ntdA-lacZ fusion in response to aromatic acids and related compounds detected by NagR (white bars) and NtdR (black bars). None: no inducer added; Ben: benzoate; HBen: 2-, 4-hydroxybenzoate; NBen: 2-, 3-, 4-nitrobenzoate; ABen: 2-, 4-aminobenzoate; ClBen: 2-, 3-, 4-chlorobenzoate; 25HBen: 2,5-dihydroxybenzoate (gentisate); 3MBen: 3-methylbenzoate; 4IPBen: 4-isopropylbenzoate; MSal: methyl salicylate; PAA: phenylacetic acid; NA: nicotinic acid; NBa: 2-, 3-, 4-nitrobenzyl alcohol; 3NBzl: 3-nitrobenzaldehyde. (B) β-Galactosidase activity in response to nitrobenzenes, nitrotoluenes, nitrophenols, and related compounds by NagR (white bars) and NtdR (black bars). None: no inducer added; NB: nitrobenzene; TNB: 1,3,5-trinitrobenzene; NT: 2-, 3-, 4-nitrotoluene; DNT: 2,4-, 2,6-dinitrotoluene; TNT: 2,4,6-trinitrotoluene; 2ADNT: 2-amino-4,6-dinitrotoluene; 4ADNT: 4-amino-2,6-dinitrotoluene; CNB: 2-, 3-, 4-chloronitrobenzene; NP: 2-, 3-, 4-nitrophenol; 24DNP: 2,4-dinitrophenol; 1NNap: 1-nitronaphthalene; AT: 3-aminotoluene; Atz: atrazine. Compounds not detected by either protein were 3-hydroxybenzoate, 3-aminobenzoate, benzyl alcohol, 2-hydroxybenzylalcohol, 2-aminobenzyl alcohol, benzaldehyde, 3-hydroxybenzaldehyde, benzene, aminobenzene, chlorobenzene, toluene, 2- and 4-chloroaminobenzene, phenol, 2-aminophenol, catechol, 3- and 4-methylcatechol, cyanuric acid, and naphthalene. N = 6. Error bars indicate standard deviations.

Fig. 5.

Uninduced levels of expression from the ntdA promoter mediated by wild-type NagR and NtdR and the 13 active derivatives. β-Galactosidase activities from the chromosomal ntdA-lacZ fusion were measured in cultures of JS42R-1 expressing NtdR-NagR variants from plasmid pBBR1-MCS after growth in minimal medium containing succinate and chloramphenicol. N = 6. Error bars indicate standard deviations. No activity was detected with NagR H169L (data not shown). The amino acid substitutions in each NagR and NtdR variant are indicated below the graph. Residues native to NagR and NtdR are indicated in white and black boxes, respectively.

Fig. 6.

Effect of selected inducers on activity from the ntdA promoter in JS42R-1 expressing NtdR-NagR variants. β-Galactosidase activities from the chromosomal ntdA-lacZ fusion were measured in cultures grown as described in the legend to Fig. 5 in response to benzoate (white bars); salicylate (grey bars); 2NT, 2-nitrotoluene (black bars); TNT, 2,4,6-trinitrotoluene (striped bars). Activities are reported as fold induction over background for each strain. N = 6. Standard deviations are reported in Tables S1-S8. No activity was detected with NagR H169L (data not shown). The amino acid substitutions in each NagR and NtdR variant are indicated below the graph. Residues native to NagR and NtdR are indicated in white and black boxes, respectively.

Fig. 7.

Inducing compounds detected by NagR, NtdR, and NtdR L169H in Acidovorax sp. JS42R-1. (A) β-Galactosidase activity measured from the chromosomal ntdA-lacZ fusion in response to aromatic acids and related compounds. (B) β-Galactosidase activity in response to nitrobenzenes, nitrotoluenes, nitrophenols, and related compounds. Abbreviations are as in the legend to Fig. 4. NagR (white bars) NtdR L169H (grey bars) and NtdR (black bars). N = 6. Standard deviations are reported in Tables S1-S8.

Fig. 8.

Crystal structure of the effector binding domain of BenM (tan) and model of NtdR (blue) based on the crystal structure of DntR. The natural inducers recognized by BenM, cis,cis-muconate (CCM) in pocket 1 (P1) and benzoate (Ben) in pocket 2 (P2), are shown in green. The amino acid residues 169 (red), 189 (green), 227 (yellow), and 232 (light blue) that were changed in this study (corresponding to 162, 185, 225, 230 in BenM), are indicated in space fill format. The root mean-square deviation of the modeled NtdR was 0.06 Å using 227 C _α atoms from DntR and NtdR (residues 75–301).

Fig. 9.

Model depicting the possible pathway by which mutations could have accumulated during the evolution of NtdR from an ancestral NagR based on measured levels of β-galactosidase activity during induction with 2-nitrotoluene. For clarity, all of the NtdR-variants are indicated in the boxes as mutants of NagR (i.e. NtdR = NagR I74V H169L K189R P227S I232V; for reference, the mutant names that are used in the text are indicated below each boxed protein). The increased shading of the boxed NagR proteins (bottom to top of the figure) represents increasing levels of gene expression in response to 2-nitrotoluene; boxed NtdR variants shown in white do not respond to 2-nitrotoluene. Solid arrows indicate steps that lead to increased induction, and white arrows indicate other possible routes to improved induction that were not explored in this study. Asterisks (*) indicate changes that do not increase activity in response to 2-nitrotoluene, but increase the total number of inducers recognized. Dashed arrows labeled with an “X” indicate routes to regulators with decreased or no activity that would result in a loss in fitness for the host strain during evolution for growth on 2-nitrotoluene. The total number of inducers that elicited significant induction (minimum 2-fold induction with standard deviations less than 20–25%) for each NtdR-NagR-variant is indicated next to the box.