Specificity profile of NAT/NCS2 purine transporters in Sinorhizobium (Ensifer) meliloti

Sinorhizobium (Ensifer) meliloti is a model example of a soil alpha‐proteobacterium which induces the formation of nitrogen‐fixing symbiotic nodules on the legume roots. In contrast to all other rhizobacterial species, S. meliloti contains multiple homologs of nucleobase transporter genes that belong to NAT/NCS2 family (Nucleobase‐Ascorbate Transporter/Nucleobase‐Cation Symporter‐2). We analyzed functionally all (six) relevant homologs of S. meliloti 1,021 using Escherichia coli K‐12 as a host and found that five of them are high‐affinity transporters for xanthine (SmLL9), uric acid (SmLL8, SmLL9, SmX28), adenine (SmVC3, SmYE1), guanine (SmVC3), or hypoxanthine (SmVC3). Detailed analysis of substrate profiles showed that two of these transporters display enlarged specificity (SmLL9, SmVC3). SmLL9 is closely related in sequence with the xanthine‐specific XanQ of E. coli. We subjected SmLL9 to rationally designed site‐directed mutagenesis and found that the role of key binding‐site residues of XanQ is conserved in SmLL9, whereas a single amino‐acid change (S93N) converts the xanthine/uric‐acid transporter SmLL9 to a xanthine‐preferring variant, due to disruption of an essential hydrogen bond with the C8 oxygen of uric acid. The results highlight the presence of several different purine nucleobase transporters in S. meliloti and imply that the purine transport might be important in the nodule symbiosis involving S. meliloti. Sinorhizobium meliloti contains a set of purine nucleobase transporters which account for the transport of a range of purines and are absent from other rhizobia. The ability of one of these transporters to utilize the uric acid in addition to xanthine was proved to be related to a binding‐site Ser residue. The identified purine transporters might be relevant to the symbiotic relationship of S. meliloti with their associated legume hosts.