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Applied and Environmental Microbiology, October 2008, p. 6239-6247, Vol. 74, No. 20
0099-2240/08/$08.00+0     doi:10.1128/AEM.02155-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Specific DNA Binding of a Potential Transcriptional Regulator, Inosine 5'-Monophosphate Dehydrogenase-Related Protein VII, to the Promoter Region of a Methyl Coenzyme M Reductase I-Encoding Operon Retrieved from Methanothermobacter thermautotrophicus Strain {Delta}H{triangledown}

Naoya Shinzato,1,2 Miho Enoki,2,3 Hiroaki Sato,4 Kohei Nakamura,2,5 Toru Matsui,1 and Yoichi Kamagata2,6*

Center of Molecular Biosciences, University of the Ryukyus, Nishihara-cho, Okinawa 903-0213, Japan,1 Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8566, Japan,2 Marine Biotechnology Institute, Kamaishi, Iwate 026-0001, Japan,3 Research Institute for Environmental Management Technology, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305-8569, Japan,4 Faculty of Applied Biological Sciences, Gifu University, Gifu, Gifu 501-1193, Japan,5 Research Institute of Genome-Based Biofactory, National Institute of Advanced Industrial Science and Technology, Sapporo, Hokkaido 062-8517, Japan6

Received 20 September 2007/ Accepted 20 August 2008

Two methyl coenzyme M reductases (MCRs) encoded by the mcr and mrt operons of the hydrogenotrophic methanogen Methanothermobacter thermautotrophicus {Delta}H are expressed in response to H2 availability. In the present study, cis elements and trans-acting factors responsible for the gene expression of MCRs were investigated by using electrophoretic mobility shift assay (EMSA) and affinity particle purification. A survey of their operator regions by EMSA with protein extracts from mrt-expressing cultures restricted them to 46- and 41-bp-long mcr and mrt upstream regions, respectively. Affinity particle purification of DNA-binding proteins conjugated with putative operator regions resulted in the retrieval of a protein attributed to IMP dehydrogenase-related protein VII (IMPDH VII). IMPDH VII is predicted to have a winged helix-turn-helix DNA-binding motif and two cystathionine β-synthase domains, and it has been suspected to be an energy-sensing module. EMSA with oligonucleotide probes with unusual sequences showed that the binding site of IMPDH VII mostly overlaps the factor B-responsible element-TATA box of the mcr operon. The results presented here suggest that IMPDH VII encoded by MTH126 is a plausible candidate for the transcriptional regulator of the mcr operon in this methanogen.

* Corresponding author. Mailing address: Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology, 2-17-2-1 Tsukisamu-Higashi, Toyohiraku, Sapporo, Hokkaido 062-8517, Japan. Phone: 81-11-857-8537. Fax: 81-11-857-8915. E-mail: y.kamagata{at}aist.go.jp

{triangledown} Published ahead of print on 29 August 2008.

Applied and Environmental Microbiology, October 2008, p. 6239-6247, Vol. 74, No. 20
0099-2240/08/$08.00+0     doi:10.1128/AEM.02155-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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