Malic enzyme cofactor and domain requirements for symbiotic N2 fixation by Sinorhizobium meliloti

Abstract

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 N(2) (Fix(-)) in alfalfa root nodules, whereas tme mutants are unimpaired in their N(2)-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 N(2) 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 N(2) 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 N(2) 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 N(2)-fixing bacteroids.

FIG. 1.

Schematic outlining the integration of the various tme and dme constructs into the S. meliloti genome. A. Integration of the ptme-dme plasmid pTH597 into the tme::ΩSp locus. B. Integration of the pdme-tme plasmid pTH433 at the dme::Tn5 locus. C. Integration of the dmeΔc plasmid pTH458 at the dme::Tn5 locus. D. Integration of the pdme-sfcA plasmid pTH513 at the dme::Tn5 locus.

FIG. 2.

Western blot to detect TME protein present in S. meliloti extracts. Total bacteroid protein was loaded at 2 μg per sample, while 2 μg of free-living-cell extract from S. meliloti wild-type strain Rm1021 was run as a control. Lane 1, Rm1021 (wild-type free-living-cell extract); lane 2, RmG994 (Rm1021 dme-3::Tn5 tme-4::ΩSp); lane 3, Rm1021 (wild-type bacteroid extract); lane 4, RmG455 (Rm1021 dme-3::Tn5); lane 5, RmG994 (Rm1021, dme-3::Tn5 tme-4::ΩSp); lane 6, RmH897 (RmG994::pdme-tme); lane 7, RmH898 (RmG995::pdme-tme); lane 8, RmH899 (RmG994::pdme-tme); lane 9, RmH900 (RmG995::pdme-tme). Numbers on the left indicate molecular weight markers.

FIG. 3.

Western blot to detect DME in bacteroid extracts. Total bacteroid protein was loaded at 2 μg per sample, while 2 μg of free-living-cell extract from S. meliloti wild-type strain Rm1021 was run as a control. Numbers on the right indicate molecular weight markers. Lane 1, RmH979 (RmG454::ptme-dme); lane 2, RmH981 (RmG995::ptme-dme); lane 3, RmG995 (Rm1021 tme-4::ΩSp); lane 4, Rm1021 bacteroid extract (wild type); lane 5, RmH980 (RmG994::ptme-dme); lane 6, RmG994 (Rm1021 dme-3::Tn5 tme-4::ΩSp); lane 7, Rm1021 free-living-cell extract (wild type).

FIG. 4.

Western blot to detect DME and DMEΔC proteins in bacteroid extracts. Total bacteroid protein was loaded at 2 μg per sample. Lane 1, Rm1021 (wild type); lane 2, RmG454 (Rm1021 dme-2::Tn5); lane 3, RmH996 (RmG454::dmeΔc); lane 4, RmH998 (RmG454::dmeΔc); lane 5, RmG456 (Rm1021 dme-1::Tn5); lane 6, RmH999 (RmG456::dmeΔc); lane 7, RmH1000 (RmG456::dmeΔc). Numbers on the left indicate molecular weight markers.

FIG. 5.

Nondenaturing PAGE to show NAD⁺-dependent malic enzyme in bacteroid extracts of S. meliloti expressing sfcA. Protein was loaded at 30 μg per sample, and the gel was stained to detect NAD⁺-dependent malic enzyme activity. Lane 1, Rm1021 (wild type); lane 2, RmK219 (Rm1021::pdme-sfcA); lane 3, RmG995 (Rm1021 tme-4::ΩSp); lane 4, RmK218 (RmG995::pdme-sfcA); lane 5, RmG455 (Rm1021 dme-3::Tn5); lane 6, RmK215 (RmG455::pdme-sfcA); lane 7, RmG456 (Rm1021 dme-1::Tn5); lane 8, RmK216 (RmG456::pdme-sfcA); lane 9, RmG994 (Rm1021 dme-3::Tn5 tme-4::ΩSp); lane 10, RmK217 (RmG994::pdme-sfcA).