Role of a Conserved Membrane Glycine Residue in a Dicarboxylate Transporter from Sinorhizobium meliloti

Abstract

Nitrogen-fixing rhizobial bacteroids import dicarboxylates by using the DctA transporter. G114 of DctA is highly conserved. A G114D mutant is inactive, but DctA with a small amino acid (G114A) or a helix disrupter (G114P) retains significant activity. G114 probably interacts with other membrane helices in stabilizing a substrate-binding pocket.

The rhizobia (including Sinorhizobium, Rhizobium, Bradyrhizobium, and Azorhizobium spp.) comprise several genera of soil bacteria that can enter into nitrogen-fixing symbioses with leguminous plants. The bacteria elicit the formation of specialized nodules on the roots of the host plant in which they undergo differentiation into the microaerophilic bacteroid state. Bacteroids reduce atmospheric dinitrogen to ammonia, which is supplied to the host plant in exchange for fixed carbon in the form of C₄-dicarboxylic acids (DCAs). The bacteroids are enclosed in a plant-derived, peribacteroid membrane (PBM), through which these metabolites must pass (17). DCAs cross the PBM by using a plant-supplied transporter (12) and then are pumped into the bacteroids by DctA (2, 4). DctA mutants are unable to import DCAs, and, as a result, the bacteria form ineffective, nonfixing nodules in symbiosis (reviewed in reference 17).

DctA is a member of the glutamate transporter family (10), which includes transporters critical to amino acid neurotransmitter uptake in mammalian neuronal, glial, and retinal cells, as well as bacterial proteins used in amino and organic uptake. Members of the glutamate transporter family have a high degree of sequence conservation, particularly in the C-terminal domain. The structure of a glutamate transporter homolog from the hyperthermophilic archaeon Pyrococcus horikoshii has recently been solved (15), revealing the active protein to be a unique, bowl-shaped trimer that inserts itself deep into the cell membrane. Each monomer possesses eight transmembrane alpha helices and two helical hairpins, an elegant structure built on features predicted by hydropathy profile analyses (10).

The substrate specificity of several bacterial DctA homologs has been investigated (16) and is known to include aspartate, fumarate, malate, oxaloacetate, and succinate. We have shown previously that not all substrates of DctA are inducers of dctA and that not all inducers of dctA are competitive inhibitors of DctA-mediated transport (16). That study also demonstrated that Sinorhizobium meliloti DctA could transport orotic acid, a monocarboxylic acid, and its toxic analog fluoroorotic acid (FOA) (1, 16), although with a much lower affinity than that for either malate or succinate.

Most of the mutations that altered dicarboxylate transport characteristics were clustered in the highly conserved C-terminal domains identified by others as being important in ion and substrate selection (10). However, one mutation, G114D, was located in the third transmembrane helix, which had not been previously implicated in transport (10). Multiple-sequence alignments presented previously (10, 18) found a high level of conservation in the region around G114, and the aligned sequences exhibit substrate-specific variation, suggesting a role in substrate recognition. A glycine corresponding to S. meliloti G114, G100, is present in the glutamate transporter of Pyrococcus horikoshii (15) in a region where several helices interact (Fig. 1). This study investigated additional substitutions at this site.

FIG. 1.

Predicted structure of DctA, showing the location of G114 in transmembrane helix 3 (modified from reference 15 with permission of the publisher). S. meliloti G114 corresponds to P. horikoshii G100 and is in the context VVGLVV. The structure of the P. horikoshii enzyme shows that the closest contacts to G100 (*) correspond to highly conserved amino acids in helix 6 and to amino acids on one face of motif C in helix 7.

Strains used here included the wild-type S. meliloti strain Rm1021 and WSUb20611-II, a dctA deletion strain (18); Escherichia coli DH5α was used for cloning purposes, and E. coli S17-1 was used to conjugate plasmid DNA into S. meliloti (9). E. coli strains were routinely grown using Luria-Bertani (LB) medium (8) or M9 minimal salts medium (8). Sinorhizobium meliloti strains were routinely grown at 30°C either in minimal mannitol medium containing NH₄ (11); in minimal medium containing NH₄ supplemented with 0.2% malate, fumarate, or succinate as the carbon source; or in modified M9 medium (16).

The dctA gene, including the entire dctA promoter, was amplified by PCR from pTH32 (14) as an 1,815-kb fragment that included 419 bp upstream of the DctA-coding region, using primers designed to incorporate a 21-bp FLAG epitope tag (13). This fragment was cloned into pCPP33 (5) to give pSM105. In vitro site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) to generate mutations G114A, G114D, G114F, and G114P in pSM105.

WSUb20611-II cells carrying the mutant dctA G114 alleles on plasmids were screened for substrate utilization phenotypes by plating serial dilutions onto plates containing defined carbon sources and scoring for growth (Table 1). The C₄-dicarboxylate utilization phenotype of the control strain, WSUb20611-II, carrying the mutant DctAs was completely complemented by the wild-type dctA gene in pSM105, with the strains growing well on all DCAs and demonstrating sensitivity to 1 μg/ml FOA. Both DctA G114A and DctA G114P could support growth on succinate, fumarate, or malate and were able to support nearly wild-type growth on mannitol medium in the presence of 1 μg/ml FOA. At FOA concentrations of above 1 μg/ml, growth of these mutants was severely compromised. DctA G114D and DctA G114F conferred completely Dct⁻ phenotypes in plate tests: no growth was recorded on any of the tested DCAs, and cells containing these alleles grew well using mannitol in the presence of 5 μg/ml FOA. This suggests that while each of the mutated G114 DctA species was impaired in FOA transport, as indicated by a partial capacity to tolerate the presence of FOA on solid media, the DctA G114 mutants differ significantly in their ability to transport DCAs.

TABLE 1.

Growth properties of G114 mutants on solid media

Strain	Growth on medium containinga:
	Succinate or fumarateb	Malate	FOA1	FOA2	FOA3 or FOA5b
Rm1021	3	3	0	0	0
WSUb20611-II	1	1	3	3	3
WSUb20611-II(pSM105-G114G)	3	3	0	0	0
WSUb20611-II(pSM105-G114A)	2	1.5	2.5	2	1
WSUb20611-II(pSM105-G114D)	1	1	3	3	3
WSUb20611-II(pSM105-G114F)	1	1	3	3	3
WSUb20611-II(pSM105-G114P)	2	2	2.5	2	1

^aFOA₁, FOA₂, FOA₃, and FOA₅, mannitol medium with fluoroorotate at 1 μg/ml, 2 μg/ml, 3 μg/ml, and 5 μg/ml, respectively; 3, growth equivalent to that of S. meliloti 1021 growing on equivalent medium or, for media containing FOA, to growth on media without FOA; 2, slightly less growth (diameter of single colonies was 75% of that formed by strains with a growth of 3); 1, visible but poor growth (colonies hardly visible); 0, no growth.

^bValues for the two different media were identical.

To more accurately characterize the ability of the DctA mutants to transport different substrates, growth curves were generated using a 96-well microtiter plate reader. Cells were grown in defined media in a 96-well plate at 30°C for 72 h. Absorbance readings at 600 nm were taken at 10-min intervals and plotted hourly (Fig. 2). The growth curves generally corroborate the plate assay data. WSUb20611-II carrying either DctA G114A or DctA G114P grew approximately as well on succinate as WSUb20611-II(pSM105) or Rm1021 (Fig. 2, left panel). This similarity was also seen with either l-malate or fumarate as the substrate (data not shown). No growth was seen for strains carrying either DctA G114D or DctA G114F, indicating that these mutant DctAs did not have sufficient dicarboxylate transport activity to support growth. The DctA deletion strain WSUb20611-II and WSUb20611-II cells carrying DctA G114A, DctA G114D, and DctA G114F grew in the presence of 1 μg/ml FOA (Fig. 2, right panel),. It is interesting to note that under these conditions, FOA transport by DctA G114A was apparently low enough to prevent FOA from accumulating to toxic levels, but succinate transport by DctA G114A was sufficient to support growth. Unlike their behavior on agar medium, cells expressing DctA G114P did not grow in mannitol broth containing 1 μg/ml FOA, indicating that FOA transport by this mutant DctA was sufficient to accumulate an inhibitory level of the toxic substrate.

FIG. 2.

Growth of strains with mutant G114 alleles. (Left) Growth in minimal salts medium supplemented with succinate. (Right) Growth on minimal mannitol medium containing NH₄ and supplemented with 1 μg/ml FOA. OD, optical density.

The uptake of radioactive succinate by the mutated DctA transporters at different substrate concentrations was measured as described previously (16), and V_max and K_m values were calculated from Lineweaver-Burk plots (Table 2). As predicted from the growth phenotypes discussed previously, DctA G114D and DctA G114F show no transport activity, with calculated K_m and V_max values similar to those of those of the deletion mutant WSUb20611-II. Cells with plasmids expressing DctA G114A and DctA G114P transport succinate at rates significantly lower than the rate of transport by pSM105, due primarily to lower V_max values. We were unable to detect the FLAG epitope in immunoblots of S. meliloti strains that carry pSM105 derivatives and so cannot normalize these values to DctA protein concentration, which might have been affected by the mutations. However, while the V_max values of the mutant DctAs are low, cells carrying either DctA G114A or DctA G114P grew as well as the wild type on the dicarboxylates tested. This suggests that DctA can operate well below its normal V_max while still supporting adequate nutrient import.

TABLE 2.

Kinetic parameters and symbiotic phenotypes of G114 mutants

Bacterial strain	Succinate Km (μM)	Vmax (nmol/μg/min)	Shoot dry massa (mg/plant)
Rm1021	91	161	13.5 ± 1.01
WSUb20611-II	1,891	4.4	4.5 ± 0.74
WSUb20611-II(pSM105-G114G)	170	132	10.5 ± 1.67
WSUb20611-II(pSM105-G114A)	225	12.7	7.3 ± 0.50
WSUb20611-II(pSM105-G114D)	2,116	5.85	4.9 ± 0.48
WSUb20611-II(pSM105-G114F)	1,898	5.12	4.9 ± 0.88
WSUb20611-II(pSM105-G114P)	94	33.6	9.0 ± 1.02
None (uninoculated)	NAb	NA	4.5 ± 0.58

^aPlant tests (18) used four boxes for each strain tested. Each box contained six plants. Values are means ± standard deviations.

^bNA, not applicable.

In symbiosis, WSUb20611-II carrying either DctA G114A or DctA G114P produced nodules that were heterogeneous, ranging from pink nodules of normal size to long nodules with a green tint at their base, similar to nodules formed by S. meliloti mutants with partial ability to transport DCAs (18). Because different nodule morphologies were observed on the same plant, these differences were probably not related to the genetic heterogeneity of alfalfa. Plants nodulated by the bacteria carrying DctA G114A or DctA G114P were slightly yellow and were smaller than plants inoculated with Rm1021 (Table 1). However, DctA G114A or DctA G114P led to partially functional symbioses, since plants inoculated with these two mutants were significantly larger than uninoculated plants and than plants inoculated with WSUb20611-II or with WSUb20611-II carrying either DctA G114F or DctA G114D (Table 1). This again suggests that DctA does not need to operate at its kinetic maximum in order to support a physiologically significant function. Indeed, the plant-derived PBM dicarboxylate transporter has a lower K_m and V_max than DctA (12), suggesting that DctA operates far below its V_max during symbiosis. The symbiotic performance of strains carrying partially active DctA proteins suggests that rhizobial nitrogen fixation was proportional to V_max, but the data set is obviously limited.

In the structure of the glutamate transporter from P. horikoshii, the G100 glycine corresponding to S. meliloti G114 is in transmembrane helix 3, which crosses the membrane obliquely (Fig. 1) (15). The model of Yernool et al. (15) suggests that the binding site for substrate is in a complex pocket formed by helices 6 and 7 and the hairpin bends HP1 and HP2, covering a region of helix 7 that contains two highly conserved regions, motif B and motif C (Fig. 1) (10). These motifs, together with sequences in helix 8, are thought to play a role in substrate selection and ion movement during transport (15). In this structure, the “other side” of helix 7 at this intersection crosses helix 3 at P. horikoshii G100, with P. horikoshii G100 on the face of the helix proximal to helix 7 and also near the start of helix 6. Thus, one can imagine that changing this glycine to other amino acids could affect the ability of the helix 6 and helix 7 regions to participate in the formation of the substrate binding pocket.

Analyses of the phenotypes of the point mutants that we have constructed are consistent with the model. Biochemically and physiologically, cells carrying DctA G114D and G114F were not discernibly different from the dctA deletion strain WSUb20622-II, suggesting that replacing S. meliloti G114 with either the negatively charged aspartate residue or the bulky and hydrophobic phenylalanine residue caused sufficient disruption of structure and microenvironment to prevent transport. In contrast, both DctA G114A and DctA G114P had activity, albeit at a lower levels than the wild type. This is corroborated by the ability of these mutant proteins to support both the growth of WSUb20611-II on dicarboxylates and the establishment of effective symbioses with the host plant alfalfa. Surprisingly, DctA G114P was the more active point mutant, with a V_max almost three times higher than that of DctA G114A and a relatively normal K_m. Proline is generally thought of as an amino acid that “breaks helices,” since an α-helix cannot incorporate the covalently constrained proline structure. However, recent papers on polytopic membrane proteins (3, 6, 7) argue that proline possesses a higher packing value in transmembrane helices, allowing it to stabilize interactions in membrane proteins by allowing helices to approach each other more closely, in much the same way that glycine stabilizes soluble proteins (6). In this way, glycine is thought to facilitate interhelical packing, and the available evidence suggests that proline can act in a similar way in membrane proteins (6). Thus, when packed into a membrane helix, the methyl side chain on alanine is potentially more disruptive than the kink formed by the constrained proline ring. Helix 3 in the P. horikoshii glutamate transporter is curved (15). The kink that G114P introduces into DctA may mimic this curvature enough to permit transport. Coupled with the high level of conservation of G114 in this class of transport protein, our data suggest that the functional significance of G114 is to facilitate packing of DctA helices into tight structures like those in the P. horikoshii enzyme.