Critical Role of Monooxygenase in Biodegradation of 2,4,6-Trinitrotoluene by Buttiauxella sp. S19-1.

2,4,6-Trinitrotoluene (TNT) is an aromatic pollutant that is difficult to be degraded in the natural environment. The screening of efficient degrading bacteria for bioremediation of TNT has received much attention from scholars. In this paper, transcriptome analysis of the efficient degrading bacterium Buttiauxella sp. S19-1 revealed that the monooxygenase gene (BuMO) was significantly up-regulated during TNT degradation. S-ΔMO (absence of BuMO gene in S19-1 mutant) degraded TNT 1.66-fold less efficiently than strain S19-1 (from 71.2% to 42.9%), and E-MO mutant (Escherichia coli BuMO-expressing strain) increased the efficiency of TNT degradation 1.33-fold (from 52.1% to 69.5%) for 9 h at 180 rpm at 27 °C in LB medium with 1.4 µg·mL-1 TNT. We predicted the structure of BuMO and purified recombinant BuMO (rBuMO). Its specific activity was 1.81 µmol·min-1·mg-1 protein at pH 7.5 and 35 °C. The results of gas chromatography mass spectrometry (GC-MS) analysis indicated that 4-amino-2,6-dinitrotoluene (ADNT) is a metabolite of TNT biodegradation. We speculate that MO is involved in catalysis in the bacterial degradation pathway of TNT in TNT-polluted environment.

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

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 ß-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.