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

Abstract

In this study, genomic DNA was screened from Halobacillus andaensis NEAU-ST10-40T 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.

An Uncharacterized Major Facilitator Superfamily Transporter From Planococcus maritimus Exhibits Dual Functions as a Na+(Li+, K+)/H+ Antiporter and a Multidrug Efflux Pump

“…P. maritimus DSM 17275 T was incubated in modified Luria-Bertani (LB) medium with the composition of 1.0% tryptone, 0.5% yeast extract plus 0.34 M NaCl (optimal) at pH 7.2–7.4, 28°C ( Wang et al, 2015 ). E. coli KNabc ( Nozaki et al, 1996 ) with the absence of three major Na + /H + antiporters (NhaA, NhaB, and ChaA) and its transformants were grown overnight to OD 600 of 1.0 at 37°C in KCl-modified LB (LBK) medium with the substitution of NaCl by 87 mM KCl, as described by our recent studies ( Dong et al, 2017 ; Meng et al, 2017 ; Wang et al, 2017 ). Growth tests for salt tolerance or alkaline pH resistance were performed by growing E. coli KNabc transformants in LBK media with the addition of NaCl or LiCl at indicated concentrations or supplemented by 50 NaCl with the adjustment of pH by Tris-HCl buffer at the final concentration of 100 mM, as described by our recent studies ( Dong et al, 2017 ; Meng et al, 2017 ; Wang et al, 2017 ).…”

Characterization of a Functionally Unknown Arginine–Aspartate–Aspartate Family Protein From Halobacillus andaensis and Functional Analysis of Its Conserved Arginine/Aspartate Residues

“…Ampicillin or kanamycin was used for the selection and growth of E. coli KNabc transformants at a final concentration of 50 μg.ml -1 . Preparation and electroporation of electrocompetent E. coli cells were carried out as described by Dong et al (2017) .…”

“…Screening of the gene with Na + /H + antiport activity from strain NEAU-ST10-40 T was performed through a series of routine procedures for DNA manipulation and functional complementation with E. coli KNabc on the LBK medium plates containing 0.2 M NaCl, as described in the study by Dong et al (2017) . A tiny modification is that DNA fragments with both 4–10 kb and 0–10 kb were used for the ligation with pUC18 to increase the possibility for obtaining genes with Na + /H + antiport activity.…”

“…Either ORF included in the 1145-bp DNA fragment inserted in the recombinant plasmid pUC-SL38 was subcloned separately to analyze which one can complement functionally with E. coli KNabc. The subcloning strategy for rdd gene was carried out similarly to that of upf0118 gene, as described by Dong et al (2017) . To reflect the response of rdd gene to the change of saline-alkaline stress, its predicted native promoter and ribosomal site (RBS) was inserted between the corresponding Bgl II and Nco I sites of pET22b (Novagen Ltd., United States) to replace DNA fragment including the T7 promoter and RBS between the two enzymatic sites of the expression vector through PCR amplification, purification, double digestion, and ligation.…”

“…All screened resultant genes have not been reported to possess Na + /H + antiport activity as yet. For example, a UPF0118 family protein with uncharacterized function from this strain has recently been reported to represent a novel class of Na + (Li + )/H + antiporters ( Dong et al, 2017 ). Of other genes, one gene designated rdd showed the highest identity of 64% with an unannotated gene encoding an uncharacterized protein belonging to arginine–aspartate–aspartate (RDD) family from Pontibacillus halophilus , but shares no identity with all known genes with Na + /H + antiport activity.…”