The mutagenicity of 2,4-dinitrotoluene (24DNT), and 2,6-dinitrotoluene (26DNT), and their related transformation products such as hydroxylamine and amine derivatives, which are formed by Clostridium acetobutylicum, were tested in crude cell extracts using Salmonella typhimurium TA100. A previous publication already reported the mutagenic activities of 2,4,6-trinitrotoluene (TNT) and its related hydroxylamine derivatives in this test system. A time course of the mutagenicity during the anaerobic transformation of TNT, 24DNT, and 26DNT was also investigated under the same conditions to compare with the results from the pure compounds. The monohydroxylamino intermediates 2-hydroxylamino-4-nitrotoluene (2HA4NT), 4-hydroxylamino-2-nitrotoluene (4HA2NT) and 2-hydroxylamino-6-nitrotoluene (2HA6NT) formed during anaerobic transformation of dinitrotoluenes were proven to be mutagenic in the Ames test using Salmonella typhimurium TA100. This study reports that 4HA2NT is the most stable derivative, whereas 2HA4NT and 2HA6NT are less stable and these intermediates are mutagenic in the Ames test. Both 24DNT and 26DNT and their final metabolites 2,4-diaminotoluene (24DAT) and 2,6-aminotoluene (26DAT) appeared nonmutagenic. In a time-course study of TNT degradation, the temporal sample containing 85% of 2,4-dihydroxylamino-6-nitrotoluene (24HA6NT) is most mutagenic. These observations suggest that the bioremediation approach for treatment of 24DNT and 26DNT should be carried past the hydroxylamino intermediate.

The genes that encode oxygen-insensitive nitroreductases from Clostridium acetobutylicum possessing 2,4,6-Trinitrotoluene (TNT) transformation activity were cloned, sequenced and characterized. The gene products NitA (MW 31 kDa) and NitB (MW 23 kDa) were purified to homogeneity. The NitA and NitB are oxygen-insensitive nitroreductases comprised of a single nitroreductase domain. NitA and NitB enzymes show spectral characteristics similar to flavoproteins. The biochemical characteristics of NitA and NitB are highly similar to those of NfsA, the major nitroreductase from E. coli. NitA exhibited broad specificity similar to that of E. coli NfsA and displayed no flavin reductase activity. NitB showed broad substrate specificity toward nitrocompounds in a pattern similar to NfsA and NfsB of Escherichia coli. NitB has high sequence similarity to NAD(P)H nitroreductase from Archaeoglobus fulgidus. NitA could utilize only NADH as an electron donor, whereas NitB utilized both NADH and NADPH as electron donors with a preference for NADH. The activity of both nitroreductases was high toward 2,4-Dinitrotoluene (2,4-DNT) as a substrate. Both the nitroreductases were inhibited by dicoumarol and salicyl hydroxamate. The nitroreductases showed higher relative expression on induction with TNT, nitrofurazone and nitrofurantoin compared to the uninduced control.

The role of hydrogenase on the reduction of 2,4,6-trinitrotoluene (TNT) in Clostridium acetobutylicum was evaluated. An Fe-only hydrogenase was isolated and identified by using TNT reduction activity as the selection basis. The formation of hydroxylamino intermediates by the purified enzyme corresponded to expected products for this reaction, and saturation kinetics were determined with a K m of 152 M. Comparisons between the wild type and a mutant strain lacking the region encoding an alternative Fe-Ni hydrogenase determined that Fe-Ni hydrogenase activity did not significantly contribute to TNT reduction. Hydrogenase expression levels were altered in various strains, allowing study of the role of the enzyme in TNT reduction rates. The level of hydrogenase activity in a cell system correlated (R 2 ‫؍‬ 0.89) with the organism's ability to reduce TNT. A strain that overexpressed the hydrogenase activity resulted in maintained TNT reduction during late growth phases, which it is not typically observed in wild type strains. Strains exhibiting underexpression of hydrogenase produced slower TNT rates of reduction correlating with the determined level of expression. The isolated Fe-only hydrogenase is the primary catalyst for reducing TNT nitro substituents to the corresponding hydroxylamines in C. acetobutylicum in whole-cell systems. A mechanism for the reaction is proposed. Due to the prevalence of hydrogenase in soil microbes, this research may enhance the understanding of nitroaromatic compound transformation by common microbial communities.

Pseudomonas sp. strain PH1 was isolated from soil contaminated with pharmaceutical and dye industry waste. The isolate PH1 could use m-aminophenol as a sole source of carbon, nitrogen, and energy to support the growth. PH1 could degrade up to 0.32 mM m-aminophenol in 120 h, when provided as nitrogen source at 0.4 mM concentration with citrate (0.5 mM) as a carbon source in the growth medium. The presence of ammonium chloride as an additional nitrogen source repressed the degradation of m-aminophenol by PH1. To identify strain PH1, the 16S rDNA sequence was amplified by PCR using conserved eubacterial primers. The FASTA program was used to analyze the 16S rDNA sequence and the resulting homology patterns suggested that PH1 is a Pseudomonas.

The major enzyme in Clostridium acetobutylicum ATCC 824 leading to transformation of TNT has been reported to be the Fe-only hydrogenase. In this study, we examine the effect of inhibitors of hydrogenase on TNT reduction by Clostridial extracts. These experiments further demonstrate the major role of hydrogenase in TNT transformation. The C. acetobutylicum hydrogenase is closely related to that of C. pasteurianum; and can be fitted to the X-ray crystal structure with a root mean square deviation of 1.18 angstroms for the Calpha atoms of the generated 3D simulation model. The Hyd1, Hyd2, and Hyd3 antibodies generated against hydrogenase reacted with both the hydrogenase in cell extracts and with C. acetobutylicum hydrogenase expressed in Escherichia coli. Inhibition studies using antibodies against Fe-only hydrogenase from C. acetobutylicum indicated that the transformation of TNT by crude cell extracts was completely inhibited by Hyd2 antibody (to amino acid 415-428) whereas antibodies Hyd1 (to residues 1-16) and Hyd3 (to amino acid 424-448) inhibited less effectively. The TNT transforming activity of the cell extract was retained when Hyd2 antibody pretreated with purified but enzymatically inactive recombinant hydrogenase was added to the extract. Addition of the transition metal Cu2+ to extracts completely inhibited the transformation of TNT suggesting the destruction of [4Fe-4S] centers which are essential for transfer of electrons from the H2-activating site to TNT. Growth of C. acetobutylicum was also inhibited by 0.5 mM Cu2+ and Hg2+ ions. The triazine dye, procion red and the nitroimidazole drug, metronidazole inhibit TNT reduction. The inhibition studies using antibodies, procion red, metronidazole, and transition metals suggest that different portions of hydrogenase are required for effective TNT reduction.