Unité Mixte de Recherches INRA UHP 1136 Interaction Arbres Microorganismes, IFR 110 Ecosystèmes Forestiers, Agroressources, Bioprocédés et Alimentation, Faculté des Sciences et Technologies, Nancy Université BP 70239, Vandoeuvre-lès-Nancy Cedex, France
The SLC26/SulP (solute carrier/sulphate transporter) proteins are a ubiquitous superfamily of secondary anion transporters. Prior studies have focused almost exclusively on eukaryotic members and bacterial members are frequently classified as sulphate transporters based on their homology with SulP proteins from plants and fungi. In this study we have examined the function and physiological role of the Escherichia coli Slc26 homologue, YchM. We show that there is a clear YchM-dependent growth defect when succinate is used as the sole carbon source. Using an in vivo succinate transport assay, we show that YchM is the sole aerobic succinate transporter active at acidic pH. We demonstrate that YchM can also transport other C4-dicarboxylic acids and that its substrate specificity differs from the well-characterized succinate transporter, DctA. Accordingly ychM was re-designated dauA (dicarboxylic acid uptake system A). Finally, our data suggest that DauA is a protein with transport and regulation activities. This is the first report that a SLC26/SulP protein acts as a C4-dicarboxylic acid transporter and an unexpected new function for a prokaryotic member of this transporter family.
Under aerobic conditions, facultative anaerobic bacteria such as Escherichia coli can take up succinate from the environment for use as a carbon and energy source. However, under anaerobic conditions, succinate represents the end-product of fumarate respiration and is thus excreted into the growth medium. E. coli possesses a battery of five import/export systems for succinate transport, namely DctA, DcuA, DcuB, DcuC, DcuD, each of which is expressed differently under aerobic or anaerobic conditions (Janausch et al., 2002).
Aerobically, exogenous succinate is transported into the cell by the well-studied C4-dicarboxylate transporter system, DctA. DctA belongs to the dicarboxylate/amino acid:cation symporter (DAACS, TC2.A.23) family, which act as proton symporters. DctA can also transport fumarate, malate, aspartate, tartrate and orotate (Kay and Kornberg, 1971; Baker et al., 1996). Expression of dctA in response to C4-dicarboxylic acid is induced by the DcuSR two-component system (Zientz et al., 1998; Davies et al., 1999; Golby et al., 1999). The membrane-bound sensor kinase (DcuS) binds C4-dicarboxylates and stimulates phosphorylation of the cytoplasmic response regulator (DcuR) which activates expression of genes involved in C4-dicarboxylate metabolism, including DctA (Zientz et al., 1998; Davies et al., 1999; Golby et al., 1999). A recent study demonstrated that there is a direct interaction between DcuS and helix VIIIb of DctA, mediated by the cytoplasmic PAS domain of DcuS, and it has been proposed that the transporter acts as co-sensor by directly modulating the activity of DcuS (Davies et al., 1999; Witan et al., 2012).
Anaerobically, succinate is excreted into the growth medium by DcuA and DcuB, which are carriers from the C4-dicarboxylate uptake family (Dcu, TC2.A.61). These transporters are capable of C4-dicarboxylate exchange and uptake but operate preferentially as fumarate/succinate antiporters, excreting the latter. Interestingly, like DctA, DcuB also interacts with DcuS, and has been proposed to act as co-sensor by directly modulating the activity of DcuS in a similar manner to DctA but under anaerobic conditions (Davies et al., 1999; Kleefeld et al., 2009; Witan et al., 2012). The DcuC and DcuD transporters belong to a separate family of C4-dicarboxylate efflux systems (DcuC, TC2.A.61), DcuC acts as a proton/succinate co-exporter, while the function of DcuD is still unclear (Janausch et al., 2002).
DctA is the most active carrier under aerobic conditions with a dctA mutant presenting a clear phenotypic growth defect when C4-dicarboxylates are used as sole carbon source (Davies et al., 1999). However, it has been shown that a single dctA and a quintuple dctA, dcuA, dcuB, dcuC, dcuD mutant retained aerobic growth on succinate at acidic pH, indicating the presence in E. coli of an additional, unknown succinate transporter (Janausch et al., 2001). Attempts to identify this extra transporter have, to date, been unsuccessful.
The Solute Carrier 26 (SLC26; animals) and sulphate transporter (SulP; plants and fungi) family is a ubiquitous superfamily of secondary anion transporters conserved from bacteria to man (Saier et al., 1999). These proteins comprise an integral membrane domain containing 10–12 transmembrane helices followed by a C-terminal cytoplasmic Sulphate Transporter and Anti-Sigma factor antagonist (STAS) domain (Shelden et al., 2010). Using small angle neutron scattering combined with contrast variation, we have recently shown that a Yersinia enterocolitica Slc26 homologue forms a dimer stabilized via its transmembrane core; the cytoplasmic STAS domain projects away from the transmembrane domain and is not involved in dimerization (Compton et al., 2011). Proteins within this family exhibit a wide variety of functions, transporting anions ranging from halides to bicarbonate. The human genome encodes at least 10 SLC26 proteins that play critical roles in cell physiology and are medically important, being implicated in genetic diseases such as diastrophic dysplasia, congenital chloride diarrhoea, Pendred syndrome and nonosyndromic deafness (Mount and Romero, 2004; Dorwart et al., 2008). In plants and fungi, SulP proteins are primarily sulphate uptake transporters, with mutations in the encoding genes leading to auxotrophic phenotypes in fungi (Marzluf, 1997; Hawkesford and De Kok, 2006).
Although these proteins are present ubiquitously among bacteria (Fig. 1), their physiological functions are almost completely unknown. The only comprehensive physiological and topological analysis of a bacterial Slc26/SulP protein concerned the Synechococcus Slc26 homologue BicA, which has been reported to act as a Na+-dependent bicarbonate transporter (Price et al., 2004; 2011; Price and Howitt, 2011). Bacterial Slc26/SulP proteins are frequently classified as sulphate transporters based on their homology with SulP proteins from plants and fungi. The only evidence to support this view comes from a study of the Mycobacterium tuberculosis SLC26 homologue Rc1739c which when overproduced in E. coli stimulated sulphate transport (Zolotarev et al., 2007). However, given the wide variety of substrates transported and the diverse physiological roles of the SLC26/SulP proteins, particularly in humans, it is impossible to predict their function based solely on sequence similarity. Thus there is clearly a need for the functional characterization of additional members of the bacterial Slc26 protein family.
Recently, a crystal structure of the isolated STAS domain from the E. coli Slc26 homologue YchM was reported (Babu et al., 2010). Surprisingly, the YchM STAS domain co-crystallized in an apparent complex with acyl-carrier protein (ACP), leading the authors to propose that YchM, like the Synechococcus SLC26 homologue BicA, was a bicarbonate transporter and that it was involved in fatty acid metabolism. However, an exhaustive phylogenetic analysis of bacterial SLC26 homologues clearly shows that E. coli YchM clusters independently from the BicA/BicA-like proteins (Fig. 1). We therefore took an unbiased approach to elucidate the substrate and physiological role of E. coli YchM. In this study we show that there is a clear YchM-dependent growth defect when succinate is used as the sole carbon source. Using an in vivo succinate transport assay, we show that YchM is the sole aerobic succinate transporter active at acidic pH and that although it can also transport other C4-dicarboxylic acids it has a different substrate spectrum to DctA. Therefore we have re-named ychM as dauA (for dicarboxylic acid uptake system A) and this designation is used from here on in. Finally, our data suggest that DauA may impact upon DctA expression and/or activity. Taken together, these results demonstrate that DauA is the previously uncharacterized succinate transporter identified in E. coli and point towards a role for DauA in the regulation of C4-dicarboxylate metabolism. This is the first report that a SLC26/SulP protein acts as a C4-dicarboxylic acid transporter, an unexpected new function for a prokaryotic member of this transporter family.
A ΔdauA mutant presents a clear growth defect phenotype on succinate
As a first step towards identifying the physiological role of DauA we constructed an in-frame deletion of dauA in the E. coli BW25113 strain background to give strain EK1 and used phenotypic microarray (PM) technology to compare the metabolic activity of the wild-type versus the ΔdauA mutant under a wide range of metabolic conditions. To assay phenotypes during aerobic metabolism, a tetrazolium salt was used that is reduced by dehydrogenases and reductases produced by cells to yield a formazan dye, which indicates that the cells are actively metabolizing a substrate. No colour change implies that the cells are not metabolically active and the substrate of interest, for example a specific carbon source, is not capable of being metabolized (Bochner, 2009).
The first substrates we tested were different sulphur sources. However, no significant difference in the metabolic activity between the wild-type and ΔdauA strains was observed when either sulphate or a range of other sulphur sources were tested (Fig. S1), indicating that DauA does not play an essential role in sulphur assimilation. We therefore next switched our attention to testing different carbon sources as substrates. Unexpectedly, and in contrast to the wild-type strain, the ΔdauA strain was metabolically inactive when succinate was used as sole carbon source (Fig. 2; open symbols). However both strains were equally metabolically active when other carbon sources such as glucose, fructose, maltose and glycerol were tested (Fig. 2; open symbols). This indicates that the difference in phenotype observed when succinate was present as the sole carbon source is due to the inability of the ΔdauA strain to specifically metabolize succinate and does not result from a general problem of carbon metabolism. In these experiments citrate was use as a negative control since E. coli is not able to use citrate as carbon source under aerobic conditions.
The dauA-dependent metabolic phenotype was next confirmed by measuring growth rates with succinate as sole carbon source. Using MOPS minimal growth medium, the ΔdauA mutant was unable to grow with added succinate, whereas normal growth was observed for all other added carbon sources tested for both strains (Fig. 2; closed symbols). Taken together these results indicate that DauA is involved in succinate metabolism.
DauA is involved in succinate metabolism
During aerobic growth, exogenous succinate is transported into E. coli by the well-studied C4-dicarboxylate transporter system DctA. A ΔdctA mutant shows poor growth on minimal medium containing succinate as the sole carbon source at pH 7; however, the strain shows almost wild-type growth using succinate at acidic pH (≤6) (Janausch et al., 2001). This suggests that E. coli possesses an additional succinate transporter that is active at acidic pH and we therefore hypothesized that DauA may be involved in this transport process.
To assess whether this was the case, we constructed an in-frame deletion of dctA in the E. coli BW25113 and EK1 (ΔdauA) strain backgrounds to give strains EK2 (BW25113, ΔdctA) and EK3 (BW25113, ΔdauA/ΔdctA) respectively. We then tested the growth phenotype of the wild-type and the isogenic ΔdauA, ΔdctA and ΔdauA/ΔdctA strains on agar plates made using MOPS minimal medium and supplemented with 50 mM of either glucose or succinate as the sole carbon source. After 24 h of aerobic growth at 37°C, only the wild-type strain grew on the succinate-supplemented plates, although all four strains grew equally well in the presence of glucose (Fig. 3A, left panel). After 48 h, the individual ΔdctA and ΔdauA mutant strains showed some growth on the succinate-supplemented plates, but very weak growth was observed for the ΔdctA/ΔdauA strain (Fig. 3A, right panel).
Next we compared aerobic growth of the same four strains in liquid eM9 medium (which is M9 minimal medium containing 0.1% hydrolysed casein) containing 50 mM glucose or 50 mM succinate as the carbon source, at pH 7 and pH 5. The strains grew equally well on glucose at pH 7, while all grew poorly on glucose at pH 5 (Fig. 3B). The poor growth on glucose at acidic pH is most likely due to glucose repression of the acid resistance system (Castanie-Cornet et al., 1999) since all of the strains grew normally on glycerol at pH 5 (Fig. S2). In media containing succinate, at pH 7, the ΔdauA strain did not grow quite as well as the wild-type strain, while the ΔdctA strain grew less well than the ΔdauA strain and the double mutant showed the poorest growth. Very interestingly, at pH 5, the wild-type and ΔdctA strains showed a similar growth rate whereas ΔdauA and ΔdauA/ΔdctA were devoid of any growth. Taken together these results indicate that DauA plays a role in succinate transport and/or metabolism at pH 5.
DauA is produced at pH 5 and pH 7, whereas DctA is produced primarily at pH 7
The results presented above indicate that DauA is required to support growth on succinate, primarily at pH 5. We therefore sought to examine the production of DauA and DctA at pH 5 and pH 7, when succinate was supplied as the sole carbon source. To this end we introduced DNA encoding a C-terminal 6xHis epitope tag onto the chromosomally encoded dauA in the BW25113 (wild-type) and EK2 (ΔdctA) strain backgrounds to give strains EK4 and EK5 respectively. Likewise we similarly introduced DNA encoding a C-terminal 6xHis epitope tag onto the chromosomally encoded dctA in the BW25113 (wild-type) and EK1 (ΔdauA) strains, producing strains EK6 and EK7 respectively.
We first confirmed that the introduction of this epitope tag onto either DauA or DctA did not grossly affect protein function by assessing the growth of these strains in eM9 medium supplemented with succinate at pH 5 or pH 7 (Fig. S2). At pH 7, strains EK4 (DauA-H6) and EK6 (DctA-H6) showed similar growth to the analogous strains producing the native forms of DauA and DctA, and likewise strains EK5 (DauA-H6, ΔdctA) and EK7 (DctA-H6, ΔdauA) behaved similarly to the strains producing untagged proteins. Again at pH 5, the EK4 (DauA-H6), EK5 (DauA-H6, ΔdctA) and EK6 (DctA-H6) strains grew normally whereas the EK7 (DctA-H6, ΔdauA) strain was unable to grow, giving similar growth phenotypes to the analogous strains producing the native forms of DauA and DctA. These results indicate that the introduction of the histidine tag fusions at the C-terminus of DctA and DauA does not appear to grossly impair their functions.
In order to investigate the presence of DctA and DauA in succinate-grown cells at pH 5 and pH 7, strains EK4 (DauA-H6), EK6 (DctA-H6), EK5 (DauA-H6, ΔdctA) and EK7 (DctA-H6, ΔdauA) were grown overnight in M9 medium containing glucose as carbon source, washed twice and subsequently cultured for 6 h on M9 minimal medium containing succinate at pH 7 or pH 5. We monitored whole cell (WC), soluble fraction (SF) and membrane (M) fractions for the presence of DctA or DauA by Western blot using anti-His antibodies. TatC is a membrane protein known to be constitutively produced (Jack et al., 2001) and was used as a loading control. In all of the strains tested, signals for DctA-H6, DauA-H6 and TatC were only detected in the membrane fraction, indicating that the proteins were correctly targeted to the membrane (Fig. 4A). DauA-H6 (predicted mass, 59.2 kDa) and DctA-H6 (predicted mass, 45.3 kDa) were detected at approximate molecular masses of ∼40 kDa and ∼35 kDa respectively. It should be noted that anomalous migration of membrane protein on SDS-PAGE is common and is likely due to the anomalous SDS-loading capacity and partial unfolding of hydrophobic proteins (Rath et al., 2009).
Interestingly it was clear that DauA-H6 was present in membranes of cells grown both at pHs and in the dctA+ and ΔdctA strain backgrounds (Fig. 4B). Conversely, while DctA-H6 was detected when the strains were grown at pH 7, no protein was detected when the cells were grown in the presence of succinate at pH 5. Unexpectedly, it also appeared that less DctA-H6 was present at pH 7 in the strain where dauA was deleted. This latter observation might suggest that the presence and/or activity of DauA can affect the expression of dctA. To investigate this further, we constructed an in-frame deletion of dauA in E. coli strain IMW385 [As MC4100, λ(ΦdctA'–'lacZ)hyb, bla; A. Kleefeld and G. Unden, unpublished] to give strain EK8 and assessed the activity of the single copy dctA'–'lacZ fusion after growth of the strains in an identical manner to those used for the Western blot analysis above. Consistent with the lack of detectable DctA-H6 at pH 5, activity of the dctA'–'lacZ fusion was not detectable above the background level at pH 5 (Fig. S3). At pH 7, expression of dctA'–'lacZ was clearly observed and this activity was approximately twofold lower in the ΔdauA mutant background (Fig. S3). This is entirely consistent with the results of the Western blot analysis of DctA-H6, and indicates that there is some interplay between the presence/activity of DauA and the production of DctA.
DauA acts as a succinate transporter
The results presented above are consistent with the idea that DauA transports succinate. In order to obtain firm evidence that this is indeed the case, we developed an in vivo succinate uptake assay. Uptake and cellular accumulation of [14C]-succinate were measured by incubating cells with [14C]-succinate for up to 6 min, samples were removed periodically and cells were captured by vacuum filtration onto polycarbonate filters. The filter-trapped cells were subsequently washed thoroughly and the level of radioactivity present on the filter, and thus associated with the cells, was then measured.
We have shown in the previous section that after growth in the presence of succinate at pH 5 only DauA, and not DctA, is present (Fig. 4B and Fig. S3). Therefore, these accumulation assays were initially performed following growth of cells for 6 h in the presence of succinate at pH 5 using equal-sized inocula of the wild-type, ΔdctA, ΔdauA and ΔdctA/ΔdauA strains. As shown in Fig. 5A, the [14C]-succinate uptake activity was linear over the time period of measurement for all of the strains tested. The uptake activity for the wild-type strain was 0.043 ± 0.007 μmol−1 min−1 g−1 dry weight and was 1.4 times lower in ΔdctA strain. A negligible uptake rate was measured for the ΔdauA and ΔdauA/ΔdctA cells (0.008 ± 0.003 and 0.008 ± 0.001 μmol−1 min−1 g−1 dry weight respectively). The activity measured in the absence of DctA and the negligible uptake in the absence of DauA is strong evidence that DauA acts as a succinate transporter and that it is the major route for succinate import under these growth conditions.
We have also shown that in the wild-type and ΔdctA strains DauA was present when cells were grown in the presence of succinate at pH 7 (Fig. 4B). Therefore, in order to determine whether DauA plays a role in succinate uptake at pH 7, the wild-type, ΔdctA, ΔdauA and ΔdauA/ΔdctA strains were cultured in the presence of succinate at pH 7 and [14C]-succinate uptake was measured. Again, as observed previously, the uptake activity was linear over the time period of measurement for all strains (Fig. 5A). The succinate uptake rate in the wild-type cells was 0.546 ± 0.084 μmol−1 min−1 g−1 dry weight and was almost negligible in the ΔdctA and ΔdauA/ΔdctA cells. This shows that under these growth conditions, DauA is largely inactive and DctA is the main succinate transporter. Although DauA appears not to be active in succinate transport under these growth conditions, the [14C]-succinate uptake activity measured in the ΔdauA cells was six times lower than in the wild-type. This result is consistent with our previous findings that in the absence of DauA, dctA is not fully expressed (Fig. 4B; Fig. S3).
Comparison of the [14C]-succinate uptake rates measured in wild-type cells grown in the presence of succinate at pH 5 (when DauA is the main transporter) and pH 7 (when DctA is the main transporter) shows that the total rate of uptake is around 10 times lower at pH 5. This suggests that DauA may transport succinate at a lower rate than DctA. Although an in vitro assay using purified protein reconstituted into liposomes would be necessary to precisely compare the uptake rates of DctA and DauA, we explored the kinetic parameters of DauA-dependent uptake compared with DctA-dependent uptake in whole cells by measuring the rates of [14C]-succinate accumulation for each of the four strains, grown at pH 7 or pH 5, over a substrate concentration range of 40 μM to 5 mM. For each pH growth condition, the background rate of uptake measured in the ΔdauA/ΔdctA strain was subtracted from the rates obtained for the other strains and the results presented in Fig. 5B.
At pH 5, [14C]-succinate accumulation in the wild-type and ΔdctA strains follows Michaelis–Menten-type kinetics, consistent with carrier-mediated transport (Fig. 5B). The very low residual transport activity in the ΔdauA strain shows linear kinetics with increasing succinate concentrations (R2 = 0.96), which may result from either a diffusion process, the activity of a very low-affinity, uncharacterized transporter, or non-specific binding of the substrate to the membrane. The measurements obtained for the wild-type and ΔdctA strains were fitted to Michaelis–Menten kinetics and the apparent half-saturation constant (Km) and maximum uptake rate (Vm), reflecting DauA activity, were derived from a Hanes–Woolf plot of the data (Table 1).
Table 1. Kinetics parameters for DauA and DctA
(mmol−1 min−1 g−1 DW)
0.025 ± 0.005
1.05 ± 0.02
0.040 ± 0.019
0.45 ± 0.03
0.56 ± 0.15
0.34 ± 0.02
1.19 ± 0.84
0.38 ± 0.09
In order to compare the apparent kinetic parameters of DauA with those of DctA, the same uptake assays were performed after growth of cells on succinate at pH 7, conditions under which DctA is the main active succinate transporter. In the wild-type and ΔdauA strains, succinate accumulation also follows Michaelis–Menten-type kinetics, again consistent with carrier-mediated transport (Fig. 5B). The apparent kinetic parameters, derived from a Hanes–Woolf plot of the data, are presented Table 1. In the ΔdctA mutant, the uptake rate was very low and increased linearly with increasing succinate concentrations (R2 = 0.92) (Fig. 5B), again consistent with either of a diffusion process, the activity of a very low-affinity uncharacterized transporter, or non-specific binding of the substrate to the membrane, and thus confirming that under these growth conditions DctA is the major succinate transporter.
The analysis of the apparent kinetic parameters for DauA and DctA in the whole cell assay reveal that, first, the Km values measured after growth at pH 7, reflecting DctA activity, are very similar to those determined for succinate transport in vivo by others (20–30 μM; Lo et al., 1972), validating our transport assay. Second, comparison of the kinetic parameters obtained for the wild-type strain after growth at pH 5 (reflecting DauA activity) and pH 7 (reflecting DctA activity) suggests that DauA has a weaker affinity for succinate. The results also indicate that DauA has a lower apparent maximal transport rate than DctA in this whole cell assay system. Finally, the significantly reduced Vm measured in the ΔdauA strain compared with the wild-type strain after growth with succinate at pH 7 confirms the reduction of dctA expression that we have observed in the absence of DauA (Fig. 4 and Fig. S3). This indicates that DauA is necessary at pH 7 for optimum DctA production and activity.
DauA can transport other C4-dicarboxylic acids
DctA has been shown to transport a broad spectrum of substrates, including succinate, fumarate, malate, aspartate and oxaloacetate (Janausch et al., 2002). Therefore, in order to gain information about DauA specificity, the uptake of [14C]-succinate (40 μM final concentration) in wild-type and ΔdctA strains was measured in the presence of various unlabelled carboxylic acids (2 mM final concentration) after growth of cells in the presence of succinate at pH 5. The rate of uptake measured for the negative control strain ΔdauA/ΔdctA under the same conditions was subtracted in each case. We reasoned that if DauA is able to transport any of the other carboxylic acids tested, we would expect to observe severe inhibition of succinate accumulation in the presence of the large excess of competitor. As shown in Fig. 6A, succinate uptake in both strains was severely inhibited by the presence of excess aspartate or fumarate. Oxaloacetate and butyrate gave a weak inhibition of succinate transport (≈40%) and no significant inhibition was observed in the presence of malate (Fig. 6A, Table S1). These results indicate that although DauA is able to transport aspartate and fumarate the inability of malate to inhibit succinate uptake suggests that DauA has differing substrate specificity to DctA.
In order to confirm previous observations that DctA can indeed transport succinate, fumarate, malate, aspartate and oxaloacetate (Janausch et al., 2002), the same experiment was repeated using the wild-type and ΔdauA strains after growth with succinate at pH 7. As expected, succinate uptake in both strains was inhibited by aspartate, fumarate, malate and oxaloacetate, in agreement with the previously reported specificity of DctA (Fig. 6B and Table S1) (Kay and Kornberg, 1971). The weak inhibition measured in the presence of butyrate has been previously reported (Janausch et al., 2001).
To validate our inhibition studies and confirm that DauA is indeed able to transport fumarate and aspartate, we directly measured [14C]-fumarate and [14C]-aspartate accumulation in the wild-type, ΔdctA and ΔdauA strains after growth in the presence of succinate at pH 5 (where we have already shown that DauA is the major transporter). As before, the background rate measured for the ΔdauA/ΔdctA strain under the same conditions was subtracted from the rate we measured for each of the other strains. The [14C]-fumarate and [14C]-aspartate accumulation rates measured in the wild-type and ΔdctA cells, shown in Fig. 7, were comparable and again the radioactive accumulation measured in the ΔdauA cells was negligible for fumarate and very low for aspartate. These results confirm that DauA is able to transport fumarate and aspartate at pH 5.
Finally, in order to confirm that when the cells are grown in the presence of succinate at pH 7 DauA is necessary for correct DctA expression and/or activity, [14C]-fumarate accumulation was measured. Similar to our previous findings for succinate transport, the [14C]-fumarate accumulation rate measured in the wild-type was also approximately four times higher than the rate measured in the ΔdauA cells. This result confirms that DctA expression and/or activity is impaired in the absence of DauA. In contrast, aspartate accumulation in the three strains was comparable. This result was expected as E. coli possesses at least two additional aspartate transport systems that are active at pH 7 (Schellenberg and Furlong, 1977).
DauA is able to transport mono- and di-carboxylates
The net charge of a C4-dicarboxylate varies from −2 (i.e. the di-carboxylate form) to 0 (i.e. the dicarboxylic acid form) according to the pKa of the two carboxylic acid groups (Table S1). Succinate is present mainly as a di-carboxylate at pH 7 and mono-carboxylate at pH 5 (Fig. S4). Since our results show that DauA is only active when the cells have been grown in the presence of succinate at pH 5, this would be consistent with the explanation that DauA can only transport the mono-carboxylate form of succinate. Inversely, the succinate transporters from the DAACS family characterized so far, including E. coli DctA, appear to recognize and transport only di-carboxylates (Gutowski and Rosenberg, 1975).
We therefore developed an assay to test whether DauA could transport the mono-carboxylate form of succinate. We first used the assay to confirm that DctA transports the di-carboxylate form of succinate. To this end we grew the BW25113 (wild-type), EK1 (ΔdauA) and EK3 (ΔdauA/ΔdctA) strains for 6 h in the presence of succinate at pH 7 (where we have already shown that DctA is the major transporter), washed them and then performed the [14C]-succinate transport assay (10 μM final concentration) in a buffer of pH 5 or pH 7. As before, the rate measured for the EK3 (ΔdauA/ΔdctA) strain under the same conditions was subtracted from the rates for the other strains. When the external pH shifts from 7 to 5, the concentration of succinate di-carboxylate decreases seven times, therefore, if DctA transports only succinate di-carboxylate, and because the total succinate concentration (10 μM) is below the Km we have measured (Table 1), we expect that the uptake activity at pH 7 would be significantly higher than at pH 5. The results in Fig. 8A clearly show that in the wild-type and the ΔdauA cells grown in the presence of succinate at pH 7, the [14C]-succinate uptake activity is seven and four times higher, respectively, if measured at pH 7 compared with pH 5. This result confirms that DctA is able to transport only succinate di-carboxylate.
We next used this approach to elucidate the protonation state of succinate that can be transported by DauA. To this end we grew the BW25113 (wild-type), EK2 (ΔdctA) and EK3 (ΔdauA/ΔdctA) strains for 6 h in the presence of succinate at pH 5 (where we have already shown that DauA is the major transporter), washed them and then performed the [14C]-succinate transport assay in a buffer of pH 5 or pH 7. As before, the rate measured for the EK3 (ΔdauA/ΔdctA) strain under the same conditions was subtracted from the rates for the other strains. When the external pH shifts from 7 to 5, the concentration of succinate di-carboxylate decreases seven times while the concentration of succinate mono-carboxylate increases 15 times (Fig. S4). Therefore, if DauA transports only succinate mono-carboxylate, and because the total succinate concentration (10 μM) is below the Km we have measured (Table 1), we expect that the uptake activity at pH 5 would be higher than at pH 7. If DauA transports only succinate di-carboxylate, we expect that the uptake activity at pH 5 would be lower than at pH 7, and if DauA transports both succinate mono- and di-carboxylates, we expect to see the same succinate uptake activity at both pHs. The results in Fig. 8B clearly show that in wild-type and ΔdctA cells grown in the presence of succinate at pH 5, the [14C]-succinate uptake activity is the same if measured at pH 7 or pH 5 (ratio = 1 for both strains in Fig. 8B). This result indicates that DauA is able to transport both mono- and di-carboxylate forms of succinate.
We have shown in Fig. 7 that DauA is able to transport fumarate. To confirm that DauA can also transport the mono- and di-carboxylate form of fumarate, we measured the [14C]-fumarate uptake activity of cells grown in the presence of succinate at pH 5. When the external pH shifts from 7 to 5, the concentration of fumarate di-carboxylate decreases 1.4 times while the concentration of fumarate mono-carboxylate increases 64 times (Fig. S4). However, in close agreement with what we observed for succinate transport, the [14C]-fumarate uptake activity of the wild-type and ΔdctA strains was the same if measured in buffer at pH 7 or pH 5 (since the ratio is very close to 1 for both strains in Fig. 8C), again confirming transport of both mono- and di-carboxylate forms.
DauA production is not dependent on the DcuSR activity
Expression of dctA is induced by the DcuSR two-component system in response to C4-dicarboxylic acid (Zientz et al., 1998; Davies et al., 1999; Golby et al., 1999). To examine whether DauA production is also dependent on the activity of DcuSR, we introduced DNA encoding a C-terminal FLAG epitope tag onto the chromosomally encoded dctA in strain EK4 (DauA-H6), to give strain EK9 (DauA-H6, DctA-FLAG). We then constructed an in-frame deletion of dcuS in the EK9 (DauA-H6, DctA-FLAG) strain background, giving strain EK10 (DauA-H6, DctA-FLAG, ΔdcuS). To investigate the presence of DctA and DauA in succinate-grown cells at pH 5 and pH 7, strains EK9 (DauA-H6, DctA-FLAG) and EK10 (DauA-H6, DctA-FLAG, ΔdcuS) were grown overnight in M9 medium containing glucose as the carbon source, washed twice with M9 medium lacking carbon source and subsequently cultured for 6 h in M9 minimal medium containing succinate at pH 7 or pH 5. We monitored membrane fractions for the presence of DctA or DauA by Western blot using anti-FLAG or anti-His antibodies respectively. Again TatC was used as a loading control. The Western blot analysis (Fig. 9) shows that under these conditions, DctA is not produced in a ΔdcuS background or at pH 5, and we can therefore conclude that under these conditions dctA expression is totally dependent on the activity of the DcuSR two-component system. However, DauA is produced at the same level in the wild-type or ΔdcuS strains, which indicates that dauA expression is not dependent on DcuSR under the conditions we have used in these experiments.
DauA is a protein with transport and regulation activity
The SLC26/SulP (solute carrier/sulphate transporter) proteins are a ubiquitous superfamily of anion transporters. Prior studies on this family of transporters have focused overwhelmingly on eukaryotic members, particularly as some of the human members are implicated in genetic diseases (Mount and Romero, 2004; Dorwart et al., 2008). Despite the fact that these proteins are present in almost all bacteria (Fig. 1), their physiological functions are almost completely unknown. In this study we have identified E. coli DauA as a previously undiscovered class of C4-dicarboxylic acid transporters. Although this is the first report that a member of the SLC26/SulP family acts as a C4-dicarboxylic acid uptake system, it should be noted that from the 10 SLC26A proteins expressed in humans, six have been shown to transport oxalate, a C2-dicarboxylic acid (Mount and Romero, 2004). These transporters (Slc26A1, 3, 6, 7, 8 and 9) act as oxalate/chloride or oxalate/sulphate exchangers (Mount and Romero, 2004; Dorwart et al., 2008), it is therefore possible that these transporters share common features with the bacterial DauA-like proteins, paving the way to establish E. coli DauA as a paradigm for the ubiquitous SLC26/SulP transporter family. Further studies using an in vitro system will be necessary to define the energetics of DauA transport activity.
A previous study describing the structure of the soluble DauA (YchM) STAS domain led the authors to propose that DauA is a bicarbonate transporter involved in fatty acid metabolism (Babu et al., 2010). However, direct proof of this activity is so far lacking and phylogenetic analysis by ourselves and others (Fig. 1) (Felce and Saier, 2004; Price and Howitt, 2011) shows that DauA does not belong to the subfamily of bicarbonate/carbonate transporters from the SLC26A/SulP family, consistent with the likelihood that the primary function of DauA is not bicarbonate transport. In support of this we have repeatedly failed to detect any DauA-dependent bicarbonate accumulation under conditions where the protein is clearly active for succinate and fumarate transport (data not shown), making it unlikely that bicarbonate is a major substrate for DauA.
The results we report here indicate that DauA is not only acting as a transporter, but also plays a role in the regulation of C4-dicarboxylic acid metabolism, as illustrated by the reduced expression and/or activity of DctA in the absence of DauA. Under aerobic conditions and in response to external C4-dicarboxylic acids, expression of dctA is stimulated by the DcuSR two-component system (Zientz et al., 1998; Davies et al., 1999; Golby et al., 1999). Moreover we have shown that under the experimental conditions we have used in this study, the expression of DctA is completely dependent on DcuSR. It is, therefore, plausible that DauA modifies the expression of dctA via DcuSR. Such a dual role as a transporter and a sensor has already been described for two other E. coli C4-dicarboxylic acid transporters, DctA and DcuB, where DcuS interacts with DctA or DcuB to form a functional sensor unit under aerobic and anaerobic conditions respectively (Davies et al., 1999; Kleefeld et al., 2009; Witan et al., 2012). It should also be noted that other secondary carriers such as the lysine-specific permease, LysP (Tetsch et al., 2008), the hexose phosphate transporter, UhpC (Schwoppe et al., 2003) and the ABC transporters PstSCAB2 (the high-affinity phosphate transporter) (Wanner, 1996) and MalEFGK (the maltose translocation system) (Antoine et al., 2005) also act as co-sensors of membrane-bound transcriptional activators and histidine kinases, reflecting the fact that co-sensing by membrane-bound transporters is an increasingly common phenomenon. It is therefore tempting to speculate that DauA acts as a co-sensor of DcuS.
In addition to the DcuSR two-component system that controls exogenous induction of genes required for general C4-dicarboxylic acid metabolism by extracellular substrates (Kneuper et al., 2005; Scheu et al., 2010), E. coli contains two other regulators that control specific pathways of C4-dicarboxylate metabolism. The cytoplasmic regulatory systems TtdR and DmlR control endogenous induction of the genes for l-tartrate fermentation and d-malate degradation respectively (Oshima and Biville, 2006; Kim et al., 2009; Lukas et al., 2010). Therefore, C4-dicarboxylate metabolism in E. coli is regulated by a combination of exogenous and endogenous signals which regulate the expression of the pathways of general and specific C4-dicarboxylate metabolism for metabolic integration (Scheu et al., 2010). In light of the dual exogenous/endogenous sensing of C4-dicarboxylate, a role for DauA and specifically the cytoplasmic STAS domain in a potential sensing mechanism related to C4-dicarboxylic acid metabolism is very attractive. The function of the STAS domain in bacterial SLC26/SulP proteins is currently unknown; however, studies on other proteins containing STAS domains have shown that they function as sensors, interaction/transduction modules and ligand-activated transcription factors (Sharma et al., 2011). Therefore, the STAS domain of DauA appears to be a prime candidate for a sensing function. Clearly further studies are required to clarify the role of DauA and its STAS domain in C4-dicarboxylate-mediated regulation.
Taken together, the results presented in this study allow us to develop a model for DauA function (Fig. 10):
At pH 7 in the presence of succinate, DctA is the main transporter and DauA is inactive under these conditions. However, in the ΔdauA strain we observe a reduced growth phenotype with succinate, a significant decrease of dctA expression, DctA protein in the membrane and DctA-dependent transport activity. This shows that under these conditions, DauA is essential for optimal expression and activity of DctA. It is not yet clear why DauA is inactive as a transporter under these conditions, further studies are needed to clarify this point.
At pH 5 in the presence of succinate, DauA is the main transporter under these growth conditions. The kinetic analysis shows that DauA has a weaker affinity and lower apparent transport rate for succinate than DctA, in agreement with previous conclusions (Davies et al., 1999; Janausch et al., 2001). DctA is not produced, most likely because the DcuS component of the DcuSR system seems to only bind di-carboxylates and at pH 5 succinate is mainly present as a mono-carboxylate (Kneuper et al., 2005; Cheung and Hendrickson, 2008). Since a ΔdcuS mutant is able to grow under these conditions (our unpublished data and Golby et al., 1999), and the fact that we detect DauA independently of the presence of DcuS (Fig. 9), it is unlikely that dauA expression depends strongly on DcuSR.
Bacterial strains and growth conditions
The E. coli K12 strains and plasmids used in this study are listed in Table 2. Luria–Bertani broth (LB) and LB agar were used for routine bacterial growth and genetic studies (Sambrook et al., 1989). For growth experiments E. coli was cultivated in M9 minimal medium (Yanisch-Perron et al., 1985), enriched M9 (eM9) medium (Kramer et al., 2007) or MOPS minimal medium (Neidhardt et al., 1974). All media were supplemented with 50 mM of the specified carbon source. When required, antibiotics where added at the following final concentrations: Ampicillin 100 μg ml−1, Kanamycin 50 μg ml−1 and Chloramphenicol 30 μg ml−1.
Table 2. Strains and plasmids used in this study
a and b denotes proteins with in-frame C-terminal His tag or FLAG tag fusions respectively.
EK1 and EK2 originate from strains JW5189-1 and JW3469-1 respectively (Table 2) in which the Kan-resistance cassette has been removed using the temperature-sensitive vector pCP20 that encodes the FLP recombinase (Datsenko and Wanner, 2000). Strain EK3 (ΔdauA/ΔdctA) was constructed by P1 transduction. A P1 lysate was prepared from JW3469-1 and transduced into EK1 according to standard methods (Miller, 1992). The KanR cassette was then subsequently removed using the pCP20-encoded FLP recombinase (Datsenko and Wanner, 2000).
Strains EK4 and EK5 were constructed as follows: a fragment (A) covering the last 501 base pairs (bp) of dauA and introducing six histidines before the termination codon was amplified by PCR using primers EKO1 Fw and EKO2 Rv (Table S2) with BW25113 chromosomal DNA as template. The product was digested with XbaI and BamHI. A second fragment (B) covering 500 bp sequence downstream of dauA was amplified by PCR using primers EKO3 Fw and EKO4 Rv (Table S2) with BW25113 chromosomal DNA as template. The product was digested with BamHI and PstI. Both fragments were cloned simultaneously into pBluescript (Stratagene). The dauA (His)6-tagged allele was excised by digestion with XbaI and PstI and subcloned into pMAK705. The mutant allele of dauA was transferred into the chromosome of BW25113 and EK2 as described (Hamilton et al., 1989) to give strains EK4 and EK5 respectively.
Strains EK5 and EK6 were constructed similarly using the primers EKO5 Fw – EKO6 Rv for fragment A and EKO7 Fw – EKO8 Rv for fragment B (Table S2). The mutant allele of dctA was transferred to the chromosome of BW25113 and EK1 to give strains EK5 and EK6 respectively.
Strain EK9 was constructed as follows: primers EKO9 Fw and EKO10 Rv (Table S2) were used to replace by PCR the His-tag with a FLAG tag on the pMAK705 vector carrying the mutated allele of dctA. The FLAG tagged mutate allele was transferred into the chromosome of EK4 as described (Hamilton et al., 1989) to give strain EK9.
Strain EK10 was constructed by P1 transduction. A P1 lysate was prepared from JW4086-1 and transduced into EK9 according to standard methods (Miller, 1992). The KanR cassette was then subsequently removed using the pCP20-encoded FLP recombinase (Datsenko and Wanner, 2000).
The technology of Phenotypic Microarrays (Biolog) was used (Bochner, 2009). Briefly, the strains were grown overnight on LB agar. A cell suspension at a standardized cell density was prepared in MOPS-based minimal medium supplemented with the appropriate nutrient source and a redox dye, tetrazolium violet. One hundred microlitres of this cell suspension was pipetted in a 96-well microtitre plate and measured for 24 h at 37°C in a microtitre plate reader (OMNILOG instrument). Seven hundred non-redundant trophic conditions were screened (Carbon, Nitrogen, Phosphorus and Sulphur metabolism, nutrient supplements, osmotic and pH sensitivity).
Escherichia coli strains were cultured overnight in eM9 medium supplemented with the appropriate carbon source at pH 7 or 5. The next day the cells were washed twice in eM9 (carbon source free) at pH 5 or pH 7 and diluted to an optical density at 600 nm of 0.05 in 150 μl of eM9 medium containing the appropriate carbon source at the appropriate pH into a 96-well microtitre plate. Growth was monitored for 24 h using a Synergy 2 platereader (Biotek).
Escherichia coli strains were cultured overnight in M9 minimal medium containing glucose as sole carbon source. The next day the cells were washed twice in M9 (carbon source free) at pH 5 or pH 7 and diluted to an optical density at 600 nm of 0.6 in M9 medium containing succinate (50 mM) at pH 5 or pH 7. After 6 h, cells were harvested for measurement of β-galactosidase activity. For measurement of β-galactosidase activity, cells were permeabilized with toluene and added to Z buffer (52 g of Na2HPO4, 6.24 g of NaH2PO4·2H2O, 0.75 g of KCl, 0.25 g of MgSO4·7H2O and 0.7 ml of β-mercaptoethanol per litre) in a total volume of 180 μl. The reaction was started by the addition of 30 μl of 4 mg ml−1o-nitrophenyl β-galactoside and the rate of increase of A405 (ΔA405 min−1) at 37°C was measured immediately in an ELx808 absorbance microplate reader (BioTek). β-Galactosidase activity is reported as ΔA405 min−1 ml−1 OD600−1.
Cells were grown as for the β-galactosidase assays using the carbon source and pH indicated and fractionated according to procedures described previously (Coutts et al., 2002). The total protein concentration in each fraction was measured using the DC protein assay kit (Bio-Rad Laboratories) using bovine serum albumin as the standard.
Western blotting was performed as described previously (Coutts et al., 2002). Proteins were detected using one of the following antibodies: anti-His antibody (Qiagen); anti-FLAG antibody (Sigma) and rabbit polyclonal anti-TatC antibody raised against purified E. coli TatC protein (kind gift of T. Palmer).
In vivo [14C]-succinate, [14C]-fumarate and [14C]-aspartate transport assays
In vivo transport assays were adapted from Jack et al. (1999). Briefly, cells were cultured in the same way as for the β-galactosidase assays using the carbon source and pH indicated. After 6 h, cells were harvested, washed twice in M9 minimal medium without a carbon source, at the appropriate pH and resuspended in the same medium to give a final OD600 of 0.6. At zero time, [14C]-aspartate, -fumarate or -succinate (55 mCi mmol−1) was added to give the final concentration specified for each experiment. Samples of 300 μl were taken at 15 s, 30 s, 1, 2, 3, 4, 5 and 6 min and uptake was terminated by filtration through nitrocellulose filters (Millipore type HA; 0.45 μm pore size) under a constant vacuum. The radioactivity present on the filter was determined using a scintillation counter. Data were calibrated by using internal standards spotted on filters and counted in the same experiment.
For the competition experiments, unlabelled competitor (50-fold excess) was added at the same time as the [14C]-labelled substrate (40 μM). For the uptake experiments at pH 7 and pH 5, the cells were cultured in the same way as for the other transport assays, at pH 7 or pH 5, after which they were split in two equal batches, washed quickly twice in M9 minimal medium without a carbon source at pH 7 or 5 and assayed immediately for uptake activity (10 μM substrate). The pH dependence of the concentrations (expressed as %) of the different protonation states of succinate and fumarate were calculated as follows: [COOH – COOH] = ([H+]2)/([H+]2 + Ka1[H+] + Ka1Ka2), [COOH – COO–] = (Ka1[H+])/([H+]2 + Ka1[H+] + Ka1Ka2), [COO– – COO–] = (Ka1 Ka2)/([H+]2 + Ka1[H+] + Ka1Ka2) with pKa = –Log10(Ka)
Sequences were obtained from NCBI databases (http://www.ncbi.nlm.nih.gov/) using blastp and the DauA (YchM) sequence from E. coli as the input sequence. Amino acid sequence alignments were performed with Muscle in MEGA version 5 (Tamura et al., 2011). The evolutionary history was inferred using the Neighbour-Joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 111.73872566 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the main branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method (Zuckerkandl and Pauling, 1965) and are in the units of the number of amino acid substitutions per site. The analysis involved 766 amino acid sequences. All ambiguous positions were removed for each sequence pair. There were a total of 1868 positions in the final data set. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011).
We would like to thank Prof. Tracy Palmer for her constructive comments on the manuscript and for providing us with the anti-TatC antibody, Prof. Gottfried Unden for providing strain IMW385 and Lucia Licandro-Lado for technical help. E.K. was supported by a studentship from the Biotechnology and Biological Sciences Research Council doctoral training grant (BB/F017022/1), E.C. by a postdoctoral fellowship from the Medical Research Council (G1000054) and A.J. by a Scottish government/Royal Society of Edinburgh personal research fellowship.