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In this study, genomic DNA was screened from  Halobacillus andaensis  NEAU-ST10-40 T  by selection in  Escherichia coli  KNabc lacking three major Na + /H +  antiporters. One gene designated  upf0118  exhibiting Na + (Li + )/H +  antiport activity was finally cloned. Protein alignment showed that UPF0118 shares the highest identity of 81.5% with an unannotated gene encoding a protein with uncharacterized protein function belonging to UPF0118 family from  H. kuroshimensis , but shares no identity with all known specific Na + (Li + )/H +  antiporter genes or genes with Na + (Li + )/H +  antiport activity. Growth test, western blot and Na + (Li + )/H +  antiport assay revealed that UPF0118 as a transmembrane protein exhibits pH-dependent Na + (Li + )/H +  antiport activity. Phylogenetic analysis indicated that UPF0118 clustered with all its homologs belonging to UPF0118 family at a wide range of 22–82% identities with the bootstrap value of 92%, which was significantly distant with all known specific single-gene Na + (Li + )/H +  antiporters and single-gene proteins with the Na + (Li + )/H +  antiport activity. Taken together, we propose that UPF0118 should represent a novel class of Na + (Li + )/H +  antiporter. To the best of our knowledge, this is the first report on the functional analysis of a protein with uncharacterized protein function as a representative of UPF0118 family containing the domain of unknown function, DUF20.

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A UPF0118 family protein with uncharacterized function from the moderate halophile Halobacillus andaensis represents a novel class of Na+(Li+)/H+ antiporter