Transcriptome Analysis Of Halomonas Sp.Y2 And Functional Analysis Of Mrp Antiporter

Halomonas sp.Y2 is a halotolerant alkaliphilic strain from Na+-rich pulp mill wastewater with high alkalinity(pH>11).Totally,four putative Na+/H+antiporters were identified from the genome of Halomonas sp.Y2,including three single subunit antiporters NhaD 1,NhaD2,NhaP,and a multisubunits antiporter Mrp.The transcriptome analysis of this strain at different pH(6.21,8.0,10.17)conditions showed that this strain could use various transport systems to maintain the acid-base balance.Compared with the acidic conditions,the genes encoding four Na+/H+showed obvious differentially expression in acidic or alkaline conditions.In our previous study,the heterologous expression of four Na+/H+antiporters preliminarily verified the functional properties of the four Na+/H+antiporters.To further determine the physiological function of these Na+/H+antiporters in Halomonas sp.Y2,we constructed four Na+/H+antiporters deleted strains and further demonstrated that the Na+/H+antiporters from Halomonas sp.Y2 work in a labor division way in dealing with saline and alkaline environments.Ha-NhaD2 showed strong Na+(Li+)transport capacity in E.coli KNabc and a Halomonas sp.Y2/?nhaD2 mutant,andexhibited growth inhibition at high Na+(Li+)concentrations at pH values of 6.2,8.0,and 10.0,suggesting its physiological role in the osmotic homeostasis of Halomonas sp.Y2.However,the similar growth status of a Halomonas sp.Y2/?nhaD1 mutant and Halomonas sp.Y2 suggested that Ha-nhaDl possesses weaker ability in saline alkali tolerance.Ha-Mrp integrated the properties of Mrp transporters from some alkalophilic Bacillus and neutral bacteria,in which not onlyexhibited the prominent Na+(Li+,K+)transport capacity,but also displayed a key role in maintaining acid-base balance of Halomonas sp.Y2,as that of the alkalophilic Bacillus.A Ha-mrp-disrupted mutant,Halomonas sp.Y2/?mrpF,was seriously inhibited by high concentrations of Na+(Li+,K+)but merely under alkaline conditions.The decreased growth of Halomonas sp.Y2/?nhaP mutant upon high concentration K+also demonstrated its strong K+transport capacity,and the Halomonas sp.Y2/?nhaP showed a growth inhibition under high K+concentration at different pH conditions.In order to further confirm the physiological ability of Ha-Mrp in K+transport at high pH conditions and investigate the mechanism of K+transport in Halomonas sp.Y2,four putative potassium efflux transporters in the Halomonas genome(Potassium efflux system KefA protein/Small-conductance mechanosensitive channel:(KE1),Potassium efflux system KefA protein/Small-conductance mechanosensitive channel:(KE2),Glutathione-regulated potassium-efflux system ATP-binding protein:(KE3)and Potassium efflux system KefA protein/Small-conductance mechanosensitive channel:(KE4))were further investigated.Then,the genes of four putative potassium efflux transporters were knocked out respectively.The growth results showed that only the mutant strain Halomonas sp.Y2/?kel exhibited growth inhibition under different pH or K+conditions.In the meanwhhile,the mutant strain Halomonas sp.Y2/?mrpF showed the most significant growth inhibition at high pH with different K+conditions.In addition,in the presence of different NaCl concentraions,the growth of all mutant strains were restored under the pH 8 and 1.6 M KCl,except Halomonas sp.Y2/?ke1.On the contrary,at pH 10,the growth of all mutant strains were restored to the level of wild type except Halomonas sp.Y2/?mrpF.By the addition of NaCl,the growth of the defective strains was restored.This indicated the possibility that the K+efflux was promoted by Na+.In other words,there may be a mechanism of Na+/K+-couple transport in Halomonas sp.Y2.At high pH condition,the strain Halomonas sp.Y2/?mrpF was only restored in a low range of NaCl,while the other mutant strains were all restored after the addition of NaCl.Therefore,we supposed that Mrp also plays an important role in the Na+/K+cotransport mechanism of Halomonas sp.Y2.In a word,Ha-Mrp may possess the function of Na+/K+-coupled transport.At pH 10 and different Na+conditions,the fluorescence quantitative analysis results of the genes of K+efflux proteins:mrpA,mrpG,nhaP,kel,ke2,ke3 and ke4,showed that mrpA and mrpG were obvious up-regulaed in response to high K+stress compared with those of low K+conditons.Conversely,at pH 8 and different Na+conditions,the fluorescence quantitative analysis results of the genes of K+efflux proteins showed that nhaP was up-regulated upon high K+stress.The fluorescence quantitative analysis results further proved that the function and characteristics of Mrp on potassium transport at pH 10.Results described above confirmed that Ha-Mrp plays an important role in pH homeostasis and K+transport under high pH conditions.To further reveal the mechanism of Ha-Mrp on K+transport,12 sites were selected for site directed mutagenesis on Ha-MrpA.As a result,MrpA-A233E retained the Na+/Li+transport capacity while lost the K+efflux ability.On the contrast,most acidic amino acid sites mutations resulted in a significant decreased in Na+/Li+/K+efflux capacity.The mutantion of MrpA-A233 resulted in a substrate variation,with similar spectrum to those of alkaliphilic strains.This indicates that the 233 site may be an important site for the difference of Mrp transport substrates.In addition,MrpA-R666H retained the Na+transport capacity and obvious Li+transport capacity while lost the K+efflux ability.Therefore,666 may also be an important site for the difference of Mrp transport substrates.