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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.
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- Experimental procedures
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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.
Figure 1. Phylogenetic tree of DauA (YchM) homologues in bacteria.
Neighbour-joining tree of 766 DauA-related sequences retrieved from the NCBI database using blastp. The blue and yellow arrows refer to DauA from E. coli and BicA from Synechococcus sp. respectively. Sequences from Actinobacteria, Bacteroidetes, Chlorobi, Chlamydiae, Cyanobacteria, Firmicutes, Proteobacteria and Spirochaetes are reported. Cluster A is a separate group without any functional annotation, composed of sequences from Actinobacteria, Bacteroidetes, Cyanobacteria and Proteobacteria. Bootstrap values are reported only for main clusters. The scale marker represents 0.1 substitutions per residue.
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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.