Abstract
In this study, genomic DNA was screened for novel Na+/H+ antiporter genes from Halomonas zhaodongensis by selection in Escherichia coli KNabc lacking three major Na+/H+ antiporters. Co-expression of two genes designated umpAB, encoding paired homologous unknown membrane proteins belonging to DUF1538 (domain of unknown function with No. 1538) family, were found to confer E. coli KNabc the tolerance to 0.4 M NaCl and 30 mM LiCl, and an alkaline pH resistance at 8.0. Western blot and co-immunoprecipitation establish that UmpAB localize as a hetero-dimer in the cytoplasmic membranes. Functional analysis reveals that UmpAB exhibit pH-dependent Na+(Li+, K+)/H+ antiport activity at a wide pH range of 6.5 to 9.5 with an optimal pH at 9.0. Neither UmpA nor UmpB showed homology with known single-gene or multi-gene Na+/H+ antiporters, or such proteins as ChaA, MdfA, TetA(L), Nap and PsmrAB with Na+/H+ antiport activity. Phylogenetic analysis confirms that UmpAB should belong to DUF1538 family, which are significantly distant with the above-mentioned proteins with Na+/H+ antiport activity. Taken together, we propose that UmpAB represent a novel two-component Na+(Li+, K+)/H+ antiporter. To the best of our knowledge, this is the first report on the functional analysis of unknown membrane proteins belonging to DUF1538 family.
Highlights
Western blot and co-immunoprecipitation establish that UmpAB localize as a hetero-dimer in the cytoplasmic membranes
Combined on the number of encoding genes with the specificity for the substrates, Na+/H+ antiporters are divided into two major categories: one category of Na+/H+ antiporters mainly with the substrates for Na+ and Li+ are encoded by a single gene such as nhaA5, 8, nhaB9, 10, nhaC11, nhaD12–16, nheE17, napA18, nhaP19, nhaG20 or nhaH21, all of which are grouped into the monovalent Cation/Proton Antiporter 1 (CPA-1) family with the exception of NapA sharing the high identity with K+/H+ antiporters that are grouped into the CPA-2 family[22]
For the cloning of Na+/H+ antiporter genes, Sau3AI-digested genomic DNA from strain NEAU-ST10-25T was electroporated into E. coli KNabc and its transformants were screened on the LBK medium plates containing 0.2 M NaCl
Summary
Western blot and co-immunoprecipitation establish that UmpAB localize as a hetero-dimer in the cytoplasmic membranes. Na+/H+ antiporters are ubiquitous secondary transporters that catalyze the efflux of intracellular alkali cations such as Na+, Li+ or K+ in exchange for external protons, which play a vital role in reducing the cytoplasmic concentration of toxic alkali cations and supporting Na+(K+)-dependent intracellular pH homeostasis under alkaline conditions[1,2,3,4] They were designated Na+(Li+)/H+ antiporters, due to Na+/H+ antiport activity together with Li+/H+ antiport activity. Strain NEAU-ST10-25T isolated from Na2CO3-type saline-alkaline soils in Zhaodong City, Heilongjiang Province, China, has been identified to represent a novel species of the genus Halomonas, Halomonas zhaodongensis[40] This novel strain is a moderately halophilic and alkaliphilic bacterium with the growth range of 0–2.5 M NaCl (optimum 0.5 M) and pH 6–12 (optimum pH 9.0), and could have developed sophisticated mechanisms to maintain its intracellular steady osmotic and ionic states. Since almost all halophilic microorganisms have the ability to expel Na+ from the interior of the cells using Na+/H+ antiporters[41, 42], it is very likely that a variety of important Na+/H+ antiporters, or even novel proteins which have not been reported to possess Na+/H+ antiport activity, exist in this novel strain, NEAU-ST10-25T, a moderate halophile and alkaliphile which can tolerate up to 2.5 M NaCl and pH 12
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