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).