Control of Gluconate Utilization in Sinorhizobium meliloti

Abstract

The Sinorhizobium meliloti megaplasmid pSymA has previously been implicated in gluconate utilization. We report a locus on pSymA encoding a putative tripartite ATP-independent periplasmic (TRAP) transporter that is required for gluconate utilization. The expression of this locus is negatively regulated by a GntR family regulator encoded adjacent to the transporter operon.

Sinorhizobium meliloti is a soil bacterium known for its metabolic versatility and its ability to engage in a nitrogen-fixing symbiosis with alfalfa and its relatives. Consistent with its oligotrophic character, S. meliloti harbors numerous solute uptake pathways, including 146 predicted ATP-binding cassette (ABC) uptake systems and 15 predicted tripartite ATP-independent periplasmic (TRAP) uptake systems (6). The ability of S. meliloti to use gluconate as sole carbon source is widely accepted, though its uptake and catabolism have not been well characterized. Multiple studies suggest that the periplasmic conversion of glucose to gluconate may be a significant route for carbon flux in glucose-grown S. meliloti cells (3, 9). It has been recognized that the symbiotic megaplasmid pSymA is somehow required for gluconate utilization (8). It has also been noted that S. meliloti might transport gluconate by a unique mechanism, since homologues of previously characterized gluconate permeases are not found in S. meliloti (2).

When S. meliloti strain Rm1021 is patched at high density on SMM agar (5) supplemented with succinate, a visible lawn emerges within two days at 30°C (Fig. 1A, panel A1). When succinate is replaced with gluconate as the sole carbon source, the resulting lawn is nearly invisible after two days, but after five days of growth, rare gluconate-utilizing mutant colonies arise (Fig. 1, panels A2 and A6). One such colony (strain B124) was streaked to purity and found to form lawns equally well on both succinate and gluconate (Fig. 1, panels A3 and A4). The mutation conferring enhanced gluconate utilization in B124 was mapped by cotransduction of random transposon insertions by using transducing phage N3. The gluconate utilization trait mapped to pSymA gene SMa0246, which encodes a previously uncharacterized GntR family regulator. Strain B124 harbored a missense mutation (C607G) near the 3′ end of SMa0246. We also isolated gluconate-utilizing mutants after mutagenesis with Tn5-110. In this series of mutants, the associated transposon insertions again mapped to SMa0246. To confirm that loss of SMa0246 function gives rise to the gluconate utilization phenotype, we created an in-frame deletion of SMa0246 using the same technique as described previously (4, 5). The ΔSMa0246 allele gave rise to a gluconate utilization phenotype identical to that observed for the spontaneous and transposon-induced alleles. Gluconate nonutilization was restored to the ΔSMa0246 strain upon the addition of a plasmid (pJG229) expressing a wild-type copy of SMa0246. These observations allow us to conclude that SMa0246 encodes a factor that negatively regulates gluconate utilization, and it has consequently been renamed gntR. Several alleles of gntR that lead to the utilization phenotype are shown in Fig. 1B, and growth curves in liquid SMM-succinate and SMM-gluconate media show that loss of gntR results in exponential-phase growth rates in gluconate that are comparable to those observed when cells are grown in succinate (Fig. 1C).

FIG. 1.

Isolation and characterization of gluconate-utilizing mutants of S. meliloti. (A) Patches of S. meliloti (strain Rm1021 or B124) were grown on SMM agar supplemented with either 0.3% sodium succinate or 0.3% sodium gluconate. Representative patches are shown after growth for either 2 days or 5 days, as indicated. (B) Map of gntR alleles giving rise to the gluconate utilization phenotype. (C) Quantitative growth measurements of gntR⁺, gntR1, and ΔgntR cultures grown in liquid SMM medium supplemented with either 0.3% sodium succinate (Suc) or 0.3% sodium gluconate (Gnt). OD₆₀₀, optical density at 600 nm.

The results presented above show that S. meliloti is normally incapable of growth on gluconate as sole carbon source but that simple loss-of-function mutations in gntR lead to strong gluconate-dependent growth. To determine whether this characteristic is unique to strain Rm1021, we tested the growth of 12 other wild isolates of S. meliloti (see USDA strains in Table 1) on SMM-succinate and SMM-gluconate plates. In all cases, the strains readily formed lawns on SMM-succinate and grew very poorly on SMM-gluconate until a few spontaneous gluconate-utilizing colonies formed after several days, mirroring what we observed for Rm1021.

TABLE 1.

Strains and plasmids used in this study

Strain(s) or plasmid	Relevant characteristic(s)a	Source or reference
Strains
Rm1021	S. meliloti SU47 Smr (progenitor to all strains below)	7
B124	gntR1	This study
B138	gntR2	This study
B140	gntR4	This study
B217	gntR6::Tn5-110	This study
B219	gntR8::Tn5-110	This study
B250	ΔgntR (deletion created using pJG230)	This study
B322	ΔSMa0244 (deletion created using pJG252)	This study
B324	ΔSMa0244 gntR4	This study
B343	ΔSMa0247 (deletion created using pJG268)	This study
B377	ΔSMa0247 gntR2	This study
B492	gntA::pJG299	This study
B494	ΔgntR gntA::pJG299	This study
B256	gntB::pJG241	This study
B254	ΔgntR gntB::pJG241	This study
B258	ΔgntR SMa0247::pJG244	This study
B260	SMa0247::pJG244	This study
USDA set	S. meliloti wild isolates USDA1941, USDA1946, USDA1959, USDA1965, USDA6509, USDA6511, USDA6563, USDA6620, USDA6636, USDA6646, USDA6649, and USDA6655	USDA
Plasmids
pJG220	Suicide vector with promoterless lacZ (Kmr/Nmr)	4
pJG263	RK2-derived lacZ reporter plasmid	4
pJQ200sk	Vector for construction of deletions (Gmr)	11
pRF771	RK2-derived Ptrp expression plasmid (Tcr)	12
pJG110	Mini-Tn5 delivery vector (Apr, Kmr/Nmr)	5
pJG229	pRF771, Ptrp::gntR (primers oJG606 and -607)	This study
pJG230	pJQ200sk, ΔgntR (primers oJG608, -609, -610, and -611)	This study
pJG252	pJQ200sk, ΔSMa0244 (primers oJG650, -651, -652, and -653)	This study
pJG268	pJQ200sk, ΔSMa0247 (primers oJG672, -673, -674, and -675)	This study
pJG241	pJG220, IF(gntB::lacZ) (primers oJG623 and -624)	This study
pJG244	pJG220, IF(kanR::SMa0247) (primers oJG625 and -626)	This study
pJG299	pJG220, IF(gntA::lacZ) (primers oJG724 and -725)	This study
pJG282	pJG263, fragment 1 (primers: oJG684 and -685)	This study
pJG272	pJG263, fragment 2 (primers oJG684 and -686)	This study
pJG301	pJG263, fragment 3 (primers oJG685 and -759)	This study
pJG304	pJG263, fragment 4 (primers oJG756 and -759)	This study
pJG305	pJG263, fragment 5 (primers oJG757 and -759)	This study
pJG306	pJG263, fragment 6 (primers oJG758 and -759)	This study
pJG303	pJG263, fragment 7 (primers oJG685 and -761)	This study

^aAbbreviations: Ap, ampicillin; Gm, gentamicin; Km, kanamycin; Nm, neomycin; Sm, streptomycin; Tc, tetracycline; IF, integration fragment. Sequences for primers indicated are given in Table S1 in the supplemental material.

gntR is flanked upstream by a gene encoding a putative dehydrogenase (SMa0244) and downstream by a cluster of genes encoding a putative hydrolase (SMa0247) and a putative TRAP transporter (SMa0249, SMa0250, and SMa0252) (see Fig. 2A). To characterize the possible roles of these genes in gluconate utilization, they were either deleted or disrupted by integration of a lacZ-containing plasmid. These mutations were made in both gntR⁺ and ΔgntR backgrounds, and the resulting gluconate utilization phenotypes are shown in Fig. 2A. While SMa0244 and SMa0247 are not required for gluconate utilization, disruption of SMa0249 or SMa0250 leads to an inability to utilize gluconate, even when gntR is deleted. Due to the gluconate nonutilization phenotype arising from mutations in SMa0249 and SMa0250, they have been renamed gntA and gntB, respectively. Since SMa0252 is predicted to encode the third (periplasmic solute binding protein) component of this tripartite transport system, it is designated gntC. The lacZ insertions in gntA and gntB were designed to create transcriptional fusions, allowing us to monitor the expression of these regions in the presence or absence of gntR. Both fusions revealed an approximately eightfold increase in expression in the absence of gntR function, suggesting that gntR negatively regulates the transcription of gntA and gntB (Fig. 2B).

FIG. 2.

gntR controls the expression of a putative TRAP transporter required for gluconate utilization. (A) A map of gntR and nearby genes depicts the location of deletions, point mutations, and insertions/disruptions analyzed. Growth on SMM agar plates supplemented with either 0.3% sodium succinate (Suc) or 0.3% sodium gluconate (Gnt) is indicated as “+” (robust growth in two days) or “−” (no growth in two days). For more precise descriptions, strain names shown in the left column can be cross-referenced to Table 1. (B) Strains containing lacZ transcriptional fusions to TRAP transporter genes gntA and gntB were assayed for β-galactosidase activity after growth in LB medium to late exponential phase. Error bars show standard deviations.

The results presented above suggest that the expression of the putative TRAP transporter encoded by the consecutive genes gntA, gntB, and gntC is required for gluconate utilization but is repressed by GntR in wild-type cells. gntABC, along with gene SMa0247, appear to be cotranscribed, and we hypothesized that the cis-acting control region for this putative operon is situated in the intergenic region between gntR and SMa0247. Based on this model, we predicted that the repression of operon expression could be overridden by the insertion of a constitutive promoter just upstream of SMa0247 (see strains B258 and B260 in Fig. 2A). This was accomplished by integrating a plasmid such that the constitutively expressed Km^r gene would be transcriptionally fused to SMa0247-gntABC. This manipulation was sufficient to allow robust growth on gluconate plates, independent of gntR functionality. This result, combined with the data presented above, provides compelling evidence that wild-type S. meliloti is limited for growth on gluconate specifically due to GntR-mediated repression of the SMa0247-gntABC operon.

To further define the cis-acting region controlling the expression of the SMa0247-gntABC operon, we performed cDNA end amplification, as described previously (4). This analysis identified a transcription start site upstream of SMa0247, as indicated in Fig. 3A. This start site is appropriately positioned downstream of near-consensus −35 and −10 promoter elements and 6 nucleotides upstream of a palindromic sequence that could possibly serve as a GntR repressor binding site. To test whether transcription from this promoter (referred to hereinafter as P_gnt) is modulated by GntR, various fragments were fused to lacZ on a low-copy-number plasmid, as shown in Fig. 3A. Only fragments encompassing this entire 60-bp P_gnt region proved to be transcriptionally modulated by GntR. Interestingly, partial removal of the palindromic sequence directly downstream of the transcription start site (see fragment 7 in Fig. 3A) stimulated very high reporter gene expression that was not significantly affected by gntR function, consistent with this element functioning as the GntR repressor binding site.

FIG. 3.

Dissection of a GntR-controlled promoter. (A) The sequence of the core promoter upstream of SMa0247 is shown, with the transcription start site (+1) inferred from cDNA end amplification. −35 and −10 sites are labeled, and a palindromic element is indicated by inverted arrows. These specified elements are in boldface. Fragments 1 to 7 were amplified by PCR and cloned into the low-copy-number reporter plasmid pJG263. Primers are indicated in Table 1, with specific primer sequences given in Table S1 in the supplemental material. (B) These constructs were transferred to either strain Rm1021 (gntR⁺, indicated by “+”) or B250 (ΔgntR, indicated by “−”), and β-galactosidase activity was measured as described for Fig. 2. Error bars show standard deviations.

Our observations suggest that this TRAP transporter operon, while required for growth on gluconate, is not transcriptionally induced by gluconate. This transporter may be adapted for the uptake of some other naturally encountered compound while exhibiting cross-specificity for gluconate. To explore this idea, strain Rm1021 harboring pJG301 (P_gnt-lacZ) was grown in LB with and without 10 mM gluconate, followed by measurement of β-galactosidase activity. As expected, we observed no difference in expression between the two treatments (data not shown). Since S. meliloti is reported to convert glucose to gluconate by a periplasmic glucose dehydrogenase (3), we speculated that transcription from P_gnt may be induced by glucose, but we observed no induction by glucose in the same assay system. Likewise, the related compounds galactose, glucuronic acid, galacturonic acid, lactose, and d-arabinose were ineffective inducers. l-Arabinose, on the other hand, induced marked reporter expression, with cells being 70% induced compared to the level in a “fully induced” isogenic strain lacking gntR. This observation is consistent with the results of a study in which an independently constructed reporter fusion containing the P_gnt region was also found to be induced specifically by l-arabinose (6). It should be noted, however, that a transporter locus required for l-arabinose utilization has previously been characterized elsewhere in the S. meliloti genome (10). The gntABC solute uptake operon is therefore atypical in that it is not induced by a compound whose utilization it facilitates, reminiscent of the lactose utilization system recently described in Vibrio vulnificus (1). Further studies of the inducibility of this operon by l-arabinose and possible roles in the utilization of other carbon sources are currently under way.