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Journal of Bacteriology, January 2007, p. 160-168, Vol. 189, No. 1
0021-9193/07/$08.00+0     doi:10.1128/JB.01425-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Malic Enzyme Cofactor and Domain Requirements for Symbiotic N₂ Fixation by Sinorhizobium meliloti ^,

Michael J. Mitsch, Alison Cowie, and Turlough M. Finan^*

Center for Environmental Genomics, Department of Biology, McMaster University, 1280 Main Street West, Hamilton, Ontario, Canada L8S 4K1

Received 7 September 2006/ Accepted 17 October 2006

The NAD⁺-dependent malic enzyme (DME) and the NADP⁺-dependent malic enzyme (TME) of Sinorhizobium meliloti are representatives of a distinct class of malic enzymes that contain a 440-amino-acid N-terminal region homologous to other malic enzymes and a 330-amino-acid C-terminal region with similarity to phosphotransacetylase enzymes (PTA). We have shown previously that dme mutants of S. meliloti fail to fix N₂ (Fix^–) in alfalfa root nodules, whereas tme mutants are unimpaired in their N₂-fixing ability (Fix⁺). Here we report that the amount of DME protein in bacteroids is 10 times greater than that of TME. We therefore investigated whether increased TME activity in nodules would allow TME to function in place of DME. The tme gene was placed under the control of the dme promoter, and despite elevated levels of TME within bacteroids, no symbiotic nitrogen fixation occurred in dme mutant strains. Conversely, expression of dme from the tme promoter resulted in a large reduction in DME activity and symbiotic N₂ fixation. Hence, TME cannot replace the symbiotic requirement for DME. In further experiments we investigated the DME PTA-like domain and showed that it is not required for N₂ fixation. Thus, expression of a DME C-terminal deletion derivative or the Escherichia coli NAD⁺-dependent malic enzyme (sfcA), both of which lack the PTA-like region, restored wild-type N₂ fixation to a dme mutant. Our results have defined the symbiotic requirements for malic enzyme and raise the possibility that a constant high ratio of NADPH + H⁺ to NADP in nitrogen-fixing bacteroids prevents TME from functioning in N₂-fixing bacteroids.

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* Corresponding author. Mailing address: McMaster University, Department of Biology, 1280 Main St. West, Hamilton L8S 4K1, Canada. Phone: (905) 525-9140, ext. 24400. Fax: (905) 522-6066. E-mail: finan{at}mcmaster.ca.

Published ahead of print on 27 October 2006.

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

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Journal of Bacteriology, January 2007, p. 160-168, Vol. 189, No. 1
0021-9193/07/$08.00+0     doi:10.1128/JB.01425-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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