Degradation of 2,4,6-Trinitrotoluene (TNT): Involvement of Protocatechuate 3,4-Dioxygenase (P34O) in Buttiauxella sp. S19-1

Abstract

Extensive use and disposal of 2,4,6-trinitrotoluene (TNT), a primary constituent of explosives, pollutes the environment and causes severe damage to human health. Complete mineralization of TNT via bacterial degradation has recently gained research interest as an effective method for the restoration of contaminated sites. Here, screening for TNT degradation by six selected bacteria revealed that Buttiauxella sp. S19-1, possesses the strongest degrading ability. Moreover, BuP34O (a gene encoding for protocatechuate 3,4-dioxygenase-P34O, a key enzyme in the β-ketoadipate pathway) was upregulated during TNT degradation. A knockout of BuP34O in S19-1 to generate S-M1 mutant strain caused a marked reduction in TNT degradation efficiency compared to S19-1. Additionally, the EM1 mutant strain (Escherichia coli DH5α transfected with BuP34O) showed higher degradation efficiency than DH5α. Gas chromatography mass spectrometry (GC-MS) analysis of TNT degradation by S19-1 revealed 4-amino-2,6-dinitrotolune (ADNT) as the intermediate metabolite of TNT. Furthermore, the recombinant protein P34O (rP34O) expressed the activity of 2.46 µmol/min·mg. Our findings present the first report on the involvement of P34O in bacterial degradation of TNT and its metabolites, suggesting that P34O could catalyze downstream reactions in the TNT degradation pathway. In addition, the TNT-degrading ability of S19-1, a Gram-negative marine-derived bacterium, presents enormous potential for restoration of TNT-contaminated seas.

Keywords: 2,4,6-trinitrotoluene (TNT); 4-amino-2,6-dinitrotolunes (ADNT); Buttiauxella sp. S19-1; degradation; genetic manipulation; protocatechuate 3,4-dioxygenase (P34O).

Figure 1

TNT was degraded by six selected bacteria. Each bacteria culture at optical density (OD_600nm = 0.1) was exposed to TNT (15 μL of 0.1 mg/mL) and cultured for 9 h at 27 °C (see Mater als and Methods for a full description of bacterial strains). Each data point represents N = 4, $$\overline{\mathrm{x}}$$ ± SD. Statistical significance is denoted as p < 0.05 using a statistical program for social sciences (SPSS 24.0, IBM, USA, 2016).

Figure 2

TNT degradation by wild-type S19-1 (red) and the S-M1 (grey) mutant strain. TNT degradation was followed for 6 h or 9 h, respectively. Data are presented as mean of N = 4, $$\overline{\mathrm{x}}$$ ± SD. Statistical significance is denoted as p < 0.05 using SPSS.

Figure 3

TNT degradation by wild-type E. coli (pink) and the S-M1 (grey) mutant strain. TNT degradation was followed for 6 h or 9 h, respectively. Data are presented as mean of N = 4, $$\overline{\mathrm{x}}$$ ± SD. Statistical significance is denoted as p < 0.05 using SPSS.

Figure 4

GC-MS analysis of TNT metabolite following degradation by wild-type strain S19-1. The figure shows peaks corresponding to TNT and ADNT at time zero (short dashes), i.e., before incubation and after 9 h incubation with Buttiauxella sp. S19-1 (solid lines). Note the decline of TNT and formation of ADNT over time.

Figure 5

Degradation rates of TNT by Buttiauxella sp. S19-1 with or without rP34O. Data are presented as mean of N = 4, $$\overline{\mathrm{x}}$$ ± SD. Statistical significance is denoted as p < 0.05 using SPSS.

Figure 6

Degradation rates of ADNT by Buttiauxella sp. S19-1 with or without rP34O. Data are presented as mean of N = 4, $$\overline{\mathrm{x}}$$ ± SD. Statistical significance is denoted as p < 0.05 using SPSS.

Figure 7

Percentage degradation rates of ADNT by Buttiauxella sp. S19-1 over time. Buttiauxella sp. S19-1 cultures were exposed to 20 μL of 0.1 mg/mL ADNT and incubated for 2 h. rP34O was then added to cultures, and degradation rates were determined at the various time points (gray). S19-1 cultures without additional rP34O were treated as negative control (white). Values are presented as mean of N = 4, $$\overline{\mathrm{x}}$$ ± SD. Statistical significance is denoted as p < 0.05 using SPSS.

Figure 8

Proposed degradation pathway for TNT and ADNT in wild-type Buttiauxella sp. S19-1. TNT is sequentially reduced at its nitro moieties by nitroreductase via ADNT to 2,4,6-aminotoluene. Further de-amination leads to hydroxytoluenes and other unidentified metabolites that are the ring cleavage substrate(s) for P34O to yield TCA cycle metabolites and finally resulting in complete mineralization, adapted from [15], 2018, Elsevier.