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Journal of Bacteriology, November 2009, p. 6928-6935, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00947-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Physiology and Posttranscriptional Regulation of Methanol:Coenzyme M Methyltransferase Isozymes in Methanosarcina acetivorans C2A ^,

Rina B. Opulencia, Arpita Bose, and William W. Metcalf^*

Microbiology Department, University of Illinois at Urbana-Champaign, B103 CLSL, 601 S. Goodwin Ave., Urbana, Illinois 61801

Received 20 July 2009/ Accepted 11 September 2009

Methanosarcina species possess three operons (mtaCB1, mtaCB2, and mtaCB3) encoding methanol-specific methyltransferase 1 (MT1) isozymes and two genes (mtaA1 and mtaA2) with the potential to encode a methanol-specific methyltransferase 2 (MT2). Previous genetic studies showed that these genes are differentially regulated and encode enzymes with distinct levels of methyltransferase activity. Here, the effects of promoter strength on growth and on the rate of methane production were examined by constructing strains in which the mtaCB promoters were exchanged. When expressed from the strong PmtaC1 or PmtaC2 promoter, each of the MtaC and MtaB proteins supported growth and methane production at wild-type levels. In contrast, all mtaCB operons exhibited poorer growth and lower rates of methane production when PmtaC3 controlled their expression. Thus, previously observed phenotypic differences can be attributed largely to differences in promoter activity. Strains carrying various combinations of mtaC, mtaB, and mtaA expressed from the strong, tetracycline-regulated PmcrB(tetO1) promoter exhibited similar growth characteristics on methanol, showing that all combinations of MtaC, MtaB, and MtaA can form functional MT1/MT2 complexes. However, an in vitro assay of coupled MT1/MT2 activity showed significant variation between the strains. Surprisingly, these variations in activity correlated with differences in protein abundance, despite the fact that all the encoding genes were expressed from the same promoter. Quantitative reverse transcriptase PCR and reporter gene fusion data suggest that the mtaCBA transcripts show different stabilities, which are strongly influenced by the growth substrate.

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* Corresponding author. Mailing address: Microbiology Department, University of Illinois at Urbana-Champaign, B103 CLSL, 601 S. Goodwin Ave., Urbana, IL 61801. Phone: (217) 244-1943. Fax: (217) 244-6697. E-mail: metcalf{at}illinois.edu

Published ahead of print on 18 September 2009.

Supplemental material for this article may be found at http://jb.asm.org/.

Present address: Microbiology Division, Institute of Biological Sciences, University of the Philippines at Los Baños, College, Laguna, Philippines 4031.

Present address: Department of Biology, Massachusetts Institute of Technology, 31 Ames Street, 68-380 Koch Biology Building, Cambridge, MA 02139.

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Journal of Bacteriology, November 2009, p. 6928-6935, Vol. 191, No. 22
0021-9193/09/$08.00+0     doi:10.1128/JB.00947-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.