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

Abstract

Arginine–aspartate–aspartate (RDD) family, representing a category of transmembrane proteins containing one highly conserved arginine and two highly conserved aspartates, has been functionally uncharacterized as yet. Here we present the characterization of a member of this family designated RDD from the moderate halophile Halobacillus andaensis NEAU-ST10-40T and report for the first time that RDD should function as a novel Na+(Li+, K+)/H+ antiporter. It’s more interesting whether the highly conserved arginine/aspartate residues among the whole family or between RDD and its selected homologs are related to the protein function. Therefore, we analyzed their roles in the cation-transporting activity through site-directed mutagenesis and found that D154, R124, R129, and D158 are indispensable for Na+(Li+, K+)/H+ antiport activity whereas neither R35 nor D42 is involved in Na+(Li+, K+)/H+ antiport activity. As a dual representative of Na+(Li+, K+)/H+ antiporters and RDD family proteins, the characterization of RDD and the analysis of its important residues will positively contribute to the knowledge of the cation-transporting mechanisms of this novel antiporter and the roles of highly conserved arginine/aspartate residues in the functions of RDD family proteins.

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

“…Most bacteria were predicted to contain 5–9 distinct single-gene or multiple-gene Na + /H + antiporters ( Krulwich et al, 2009 ; Mesbah et al, 2009 ). However, we speculate that halophiles may have evolved significantly more Na + /H + antiporters including even unreported ones to exhibit high capability of halo-alkaline tolerance, which has been demonstrated by our recent reports on several novel Na + /H + antiporters from the different moderate halophiles ( Dong et al, 2017 ; Meng et al, 2017 ; Shao et al, 2018 ). In these studies, we found that the members of three functionally uncharacterized families such as UPF0118, DUF1538 and RDD display Na + (Li + )/H + or Na + (Li + , K + )/H + antiport activity ( Dong et al, 2017 ; Meng et al, 2017 ; Shao et al, 2018 ).…”

“…However, we speculate that halophiles may have evolved significantly more Na + /H + antiporters including even unreported ones to exhibit high capability of halo-alkaline tolerance, which has been demonstrated by our recent reports on several novel Na + /H + antiporters from the different moderate halophiles ( Dong et al, 2017 ; Meng et al, 2017 ; Shao et al, 2018 ). In these studies, we found that the members of three functionally uncharacterized families such as UPF0118, DUF1538 and RDD display Na + (Li + )/H + or Na + (Li + , K + )/H + antiport activity ( Dong et al, 2017 ; Meng et al, 2017 ; Shao et al, 2018 ). Although a random screening method by functional complementation with E. coli KNabc helped us find out their functions as Na + /H + antiporters, we suspect that these ever uncharacterized proteins may possess the additional functions, whose function predictions are, to great extent, restricted by limited bioinformatic knowledge about these uncharacterized proteins with no or significantly low sequence identity with their homologs.…”

“…Known Na + /H + antiporters can be classified on the basis of the number of encoding genes into three major groups: (i) single-gene Na + /H + antiporters including NhaA and NhaB ( Herz et al, 2003 ), NhaC ( Ito et al, 1997 ), NhaD ( Zhang et al, 2014 ; Wang et al, 2017 ; Yang et al, 2018 ), NheE ( Sousa et al, 2013 ), NhaG ( Gouda et al, 2001 ), NhaH ( Jiang et al, 2013b ), NhaP ( Utsugi et al, 1998 ), NapA ( Waser et al, 1992 ), and GerN ( Southworth et al, 2001 ); (ii) double-gene Na + /H + antiporters including PsmrAB ( Jiang et al, 2013a ) and UmpAB ( Meng et al, 2017 ); and (iii) multiple-gene Na + /H + antiporters such as Mrp ( Cheng et al, 2016 ; Xu et al, 2018 ), Mnh ( Hiramatsu et al, 1998 ), Pha ( Jiang et al, 2004 ; Yang et al, 2006a ) or Sha ( Kosono et al, 1999 ). In addition, three MFS multi-drug efflux pumps such as MdfA ( Edgar and Bibi, 1997 ), MdtM ( Holdsworth and Law, 2013 ) and Tet(L) ( Cheng et al, 1994 ), and a HCT (2-hydroxy-carboxylate transporter) family transporter MleN ( Wei et al, 2000 ), and a primary Na + pump Nap of NDH (NADH dehydrogenase) family ( Yang et al, 2006b ); and an UPF0118 family protein ( Dong et al, 2017 ), and a RDD family protein ( Shao et al, 2018 ) have also been increasingly reported to be able to function as Na + /H + antiporters. In this study, MdrP was predicted to be a transmembrane protein with 12 putative TMSs (Supplementary Figure S2 ), which was established by the localization of MdrP by western blot in the cytoplasmic membranes of E. coli KNabc ( Figure 2 ).…”

Discovery of in vivo Virulence Genes of Obligatory Intracellular Bacteria by Random Mutagenesis

“…Himar1 intragenic insertion mutants were also identified in genes for vitamin synthesis [thiamine biosynthesis protein ThiC (Lawhorn et al, 2004) and dethiobiotin synthase (Otsuka et al, 1988)], mutation repair [RmuC (Slupska et al, 2000), DNA mismatch repair proteins MutS and MutL (Mansour et al, 2001)], transcriptional regulation [DNA-3-methyladenine glycosylase (Wyatt et al, 1999), transcriptional regulator NrdR (Torrents et al, 2007)], nutrient transport [sodium:alanine symporter family protein (Khani et al, 2018), and two MFS transporters (Quistgaard et al, 2016)], bacterial division and chromosomal segregation [chromosome partitioning ATPase ParA (Lutkenhaus, 2012), cell division ZapA family protein (Small et al, 2007)], and others [amidophosphoribosyltransferase ComF (Bhagavan and Ha, 2015), tellurium resistance protein TerC (Turkovicova et al, 2016), RDD family protein (Shao et al, 2018), glutathione S-transferase (Nebert and Vasiliou, 2004), aspartate kinase (Min et al, 2015), gamma carbonic anhydrase family protein (Hewett-Emmett and Tashian, 1996), octaprenyldiphosphate synthase (Ashby and Edwards, 1990)] ( Table 1). These genes are apparently not required for HF strain infection of macrophages.…”

A Novel MFS-MDR Transporter, MdrP, Employs D223 as a Key Determinant in the Na+ Translocation Coupled to Norfloxacin Efflux

“…E. coli CM2 or KNabc transformants were used to prepare the everted membrane vesicles by the French press method as described in our recent studies (Dong et al, 2017;Meng et al, 2017;Shao et al, 2018;Xu et al, 2019). Briefly, cultures were harvested and crushed by one passage under high pressure.…”