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Applied and Environmental Microbiology, March 2003, p. 1542-1547, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1542-1547.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

2,4,6-Trinitrotoluene Reduction by an Fe-Only Hydrogenase in Clostridium acetobutylicum

Mary M. Watrous,1 Sandra Clark,2 Razia Kutty,2 Shouqin Huang,2 Frederick B. Rudolph,2 Joseph B. Hughes,1 and George N. Bennett2*

Civil and Environmental Engineering,1 Department of Biochemistry and Cell Biology and the Institute of Biosciences and Bioengineering, Rice University, Houston, Texas 77005-18922

Received 26 August 2002/ Accepted 23 December 2002

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 Km of 152 µM. 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 (R2 = 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.

* Corresponding author. Mailing address: Department of Biochemistry and Cell Biology and the Institute of Biosciences and Bioengineering, Rice University, Houston, TX 77005-1892. Phone: (713) 348-4920. Fax: (713) 348-5154. E-mail: gbennett{at}bioc.rice.edu.

Applied and Environmental Microbiology, March 2003, p. 1542-1547, Vol. 69, No. 3
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.3.1542-1547.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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