Nitrogen-fixation genes and nitrogenase activity in Clostridium acetobutylicum and Clostridium beijerinckii J-S Chen, J Toth and M Kasap Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 Several solvent - producing clostridia, including Clostridium acetobutylicum and C. beijerinckii, were previously shown to be nitrogen - fixing organisms based on the incorporation of 15 N 2 into cellular material. The key nitrogen - fixation ( nif ) genes, including nifH, nifD, and nifK for nitrogenase component proteins as well as nifE, nifN, nifB and nifV for synthesis of the iron – molybdenum cofactor ( FeMoco ) of nitrogenase, have now been identified in C. acetobutylicum or C. beijerinckii or both. The organization of these genes is similar to the distinctive pattern that was first observed in Clostridium pasteurianum, with the nifN and nifB genes fused into the nifN-B gene and with the nifV gene split into the nifV! and nifV genes. The corresponding nif genes of these three clostridial species are highly related to each other. However, in the two solvent - producing clostridia, the nifH and nifD genes are interspersed by two glnB - like genes, which are absent in the corresponding region in C. pasteurianum. However, the nifN-B and nifV! genes of C. pasteurianum are interspersed by the putative modA and modB genes ( for molybdate transport ) , which are absent in the corresponding region in C. acetobutylicum. C. acetobutylicum and C. beijerinckii grew well under nitrogen - fixing conditions, and the acetylene - reducing activity of nitrogenase was measured in the two species. Acetone, butanol, and isopropanol production occurred in nitrogen - fixing cultures, but the peak of nitrogen - fixing activity preceded the active solventogenic phase. Journal of Industrial Microbiology & Biotechnology (2001) 27, 281 – 286. Keywords: nif ; nitrogen fixation; solvent - producing clostridia Introduction Nitrogen fixation and alcohol production ( the production of butanol, ethanol, and isopropanol ) are two reductive metabolic processes that can conceivably compete against each other for the same source of reductant in the cell, if the two processes occur simultaneously. In the solvent -producing clostridia, active produc- tion of butanol and isopropanol normally occurs at a stage subsequent to the exponential phase of growth. If the solvent - producing clostridia could fix N 2 during the exponential phase of growth, nitrogen gas may become an inexpensive source of nitrogen for the growth of the solvent - producing clostridia. However, if the two processes overlap during solvent production, they may be utilized as an experimental system for the study of the relationship between the supply of reductants and the yield of nitrogen fixation or solvent production. Several solvent - producing clostridia, including Clostridium acetobutylicum and Clostridium beijerinckii, were previously found to be nitrogen - fixing organisms, when the incorporation of 15 N 2 was measured [10]. Clostridium butylicum, which has since been identified as C. beijerinckii was also reported as a nitrogen - fixing organism. Among the free - living nitrogen - fixing organisms, Clostridium pasteurianum was the first to be isolated [ 18 ] , and its nitrogen - fixation ( nif ) genes have been characterized [ 2 ] . Following the sequencing of the genome of C. acetobutylicum ATCC 824 ( http: // www.cric.com / genese- quences / clostridium / clospage.html ) , a region encompassing nucleotides 284,400 to 295,096 was found to contain open - reading frames ( ORFs ) that encode amino acid sequences highly similar to those encoded by the nif genes of C. pasteurianum. The identification of the nif - related genes in C. acetobutylicum is consistent with the previously observed nitrogen - fixing activity of this organism. In order to utilize the nitrogen - fixing activity as an experimental tool for study of the regulation of solvent production in the clostridia, we examined the nitrogen - fixing ability of the currently recognized strains of C. acetobutylicum and C. beijerinckii and also searched for the presence of nif genes in C. beijerinckii. In this paper, we report the organization of the nif genes in C. beijerinckii and the growth and solvent production of the clostridia under nitrogen - fixing conditions. Materials and methods Bacteria, plasmids, and growth conditions Spores of C. acetobutylicum ATCC 824, C. beijerinckii NRRL B593, and C. pasteurianum W5 were from laboratory stocks, and routine culture conditions were as reported [ 5,13 ] . Plasmids LITMUS 28 and 29 (New England BioLabs, Beverly, MA) and pUC19 (Life Technologies, Rockville, MD) were maintained as recommended by the manufacturers. Nitrogen - fixing cultures Nitrogen - fixing cultures were grown in a medium previously described [ 15 ] , except that sucrose was replaced by glucose. When needed, yeast extract was included at 0.5 or 1.0 g / l. Cultures were grown at 358C under N 2 in rubber - stoppered 160 - ml serum bottles containing 20 ml of medium. C. beijerinckii was also grown in a defined medium containing sucrose, 60 g / l; dibasic, potassium phosphate, 3.5 g / l; mineral solution [ 15 ] , 1 ml / l; cysteine, 0.5 g / l; alanine, 0.027 g / l; isoleucine, 0.016 g / l; leucine, 0.023 g / l; Correspondence: Dr J - S Chen, Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061, USA Received 2 September 2000; accepted 22 November 2000 Journal of Industrial Microbiology & Biotechnology (2001) 27, 281 – 286 D 2001 Nature Publishing Group 1367-5435/01 $17.00 www.nature.com/jim