Functional Identification of Na <sup>+</sup>/H <sup>+</sup> Antiporter in Novel YdjM Superfamily Members

doi:10.13523/j.cb.20181205

China Biotechnology

2018, Vol. 38

Issue (12): 32-40 DOI: 10.13523/j.cb.20181205

Orginal Article

Functional Identification of Na ⁺/H ⁺ Antiporter in Novel YdjM Superfamily Members

WANG Yan-hong,LIU Yan-shuang,SHI De-xi,ZHU Bao-guo,LV Bao-lei,FU Shi-yu,XU Miao,WANG We,YIN Kui-de(

)

College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, China

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Abstract

In prokaryotes, Na ⁺/H ⁺ antiporters catalyze the efflux of intracellular alkali cations, such as Na ⁺, Li ⁺ or K ⁺ in exchange for external protons, which play vital roles in reducing the cytoplasmic concentration of toxic alkali cations and supporting intracellular pH homeostasis under alkaline conditions. To obtain as many (especially novel) Na ⁺/H ⁺ antiporter genes as possible, genomic DNA from the Halobacillus Y5 was screened, then partially digested with Sau3AI selected in E. coli KNabc, which lacks three major Na ⁺/H ⁺ antiporters. The gene designated Ha_ydjM (ydjM from the Halobacillus Y5) was screened, which showed the noval Na ⁺/H ⁺ antiporter, belong to a member of YdjM superfamilly. It is a membrane protein of unknown function. Phylogenetic analysis based on the neighbour-joining algorithm showed that Ha-YdjM showed the closer phylogenetic relationship with YdjM(accession version No. WP_101846656.1) from Halobacillus sp. Marseille -P 3789 but it is a separate branch. The presence of Ha-YdjM conferred tolerance of E. coli KNabc to NaCl up to 0.2mol/L and to LiCl up to 5mmol/L and to an alkaline pH 8.0. pH-dependent Na ⁺/H ⁺ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc/Ha-YdjM but not those of KNabc/pUC18. The Michaelis-Menten kinetics analysis showed that K_m values for Na ⁺, K ⁺, Li ⁺ were 0.43±0.05mmol/L、0.49±0.06mmol/L、0.64±0.06mmol/L, respectively, indicating that the preference of Ha-YdjM for the transported cations was Na ⁺ > K ⁺ > Li ⁺. Taken together, Ha_ydjM represents a novel Na ⁺/H ⁺ antiport. This study found new members of the YdjM superfamily and provided insights for functions of other unknown membrane members.

Key words： Na ⁺/H ⁺ antiporter Membrane proteins Functional identification YdjM superfamily Halobacillus

Received: 20 July 2018 Published: 10 January 2019

ZTFLH:

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Corresponding Authors: Kui-de YIN E-mail: yinkuide@163.com

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	Yan-hong WANG
	Yan-shuang LIU
	De-xi SHI
	Bao-guo ZHU
	Bao-lei LV
	Shi-yu FU
	Miao XU
	We WANG
	Kui-de YIN

Cite this article:

WANG Yan-hong,LIU Yan-shuang,SHI De-xi,ZHU Bao-guo,LV Bao-lei,FU Shi-yu,XU Miao,WANG We,YIN Kui-de. Functional Identification of Na ⁺/H ⁺ Antiporter in Novel YdjM Superfamily Members. China Biotechnology, 2018, 38(12): 32-40.

URL:

https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20181205 OR https://manu60.magtech.com.cn/biotech/Y2018/V38/I12/32

Fig.1 Subcloning strategy of Ha_YdjM

Fig.2 Multiple alignment of Ha_YdjM along with other proteins from Halobacillus

Fig.3 Neighbour-joining phylogenetic tree of Ha_YdjM based on the amino acid sequence

Fig.4 Prediction of transmembrane helices for Ha_YdjM Red square:Transmembrane; Pink line:Hydrophilic loop near the periplasmic space

Fig.5 Hydrophobic property of deduced proteins for Ha_YdjM Positive: Hydrophobic amino acid; Negative: Hydrophilic amino acid

Table 1 Five putative transmembrane segments of Ha_YdjM

Fig.6 Growth of Ha_YdjM 1: pUC-HaY; 2: Ha_YdjM; 3: ORF2; 4: pUC18 plasmid; 5: uncultured bacteria

Fig.7 Growth test for pH resistence of Ha_YdjM

Fig.8 Assays for Na⁺(Li⁺,K⁺)/H⁺ antiport activity of KNabc/Ha_YdjM

Fig.9 Na⁺(Li⁺,K⁺)/H⁺ antiport activity of KNabc/Ha_YdjM at different pH

Fig.10 Calculation of K_m values of KNabc/Ha_YdjM for Na⁺, Li⁺ and K⁺


[1]	Uzdavinys P, Coincon M, Nji E , et al. Dissecting the proton transport pathway in electrogenic Na +/H + antiporters . Proceedings of the National Academy of Sciences of the United States of America, 2017,114(7):1101-1110. doi: 10.1073/pnas.1614521114 pmid: 28154142

[2]	Padan E, Schuldiner S . Molecular physiology of Na +/H + antiporters, key transporters in circulation of Na + and H + in cells . Biochim Biophys Acta, 1994,1185(2):129-151. doi: 10.1016/0005-2728(94)90204-6

[3]	Ito M, Guffanti A A, Oudega B , et al. mrp, a multigene, multifunctional locus in Bacillus subtilis with roles in resistance to cholate and to Na + and in pH homeostasis . J Bacteriol, 1999,181(8):2394-2402. pmid: 10198001

[4]	Padan E, Bibi E, Ito M , et al. Alkaline pH homeostasis in bacteria: new insights. Biochim Biophys Acta, 2005,1717(2):67-88.

[5]	Saier Jr M H, Reddy V S, Tamang D G , et al. The transporter classification database. Nucleic Acids Research, 2014,42(D1):D251-D258. doi: 10.1093/nar/gkt1097 pmid: 19022853

[6]	Quinn M J, Resch C T, Sun J , et al. NhaP1 is a K +(Na +)/H + antiporter required for growth and internal pH homeostasis of Vibrio cholerae at low extracellular pH . Microbiology, 2012,158(4):1094-1105. doi: 10.1099/mic.0.056119-0 pmid: 3949420

[7]	杨礼富, 赵百锁, 杨苏声 . 细菌钠离子输出系统的类型及其可能机制. 微生物学报, 2007,47(6):1110-1114.

[7]	Yang L F, Zhao B S, Yang S S . Sodium ion transportation system and its possible mechanisms in bacteria. Acta Microbiologica Sinica, 2007,47(6):1110-1114.

[8]	Padan E . Functional and structural dynamics of NhaA, a prototype for Na + and H + antiporters, which are responsible for Na + and H + homeostasis in cells . Biochimica Et Biophysica Acta, 2014,1837(7):1047-1062. doi: 10.1016/j.bbabio.2013.12.007 pmid: 24361841

[9]	Dwivedi M, Sukenik S, Friedler A , et al. The Ec-NhaA antiporter switches from antagonistic to synergistic antiport upon a single point mutation. Scientific Reports, 2016,6:23339. doi: 10.1038/srep23339 pmid: 4810432

[10]	Zhang H, Wang Z, Wang L , et al. Cloning and identification of a novel NhaD-type Na +/H + antiporter from metagenomic DNA of the halophilic bacteria in soil samples around Daban Salt Lake . Extremophiles, 2014,18(1):89-98. doi: 10.1007/s00792-013-0600-2 pmid: 24297704

[11]	Sousa P M, Videira M A, Vorburger T , et al. The novel NhaE-type Na +/H + antiporter of the pathogenic bacterium Neisseria meningitidis . Archives of Microbiology, 2013,195(3):211-217. doi: 10.1007/s00203-012-0856-4

[12]	Pinner E, Carmel O, Bercovier H , et al. Cloning, sequencing and expression of the nhaA and nhaR genes from Salmonella entiritidis. Archives of Microbiology, 1992,157(4):323-328.

[13]	Cui Y, Cheng B, Meng Y , et al. Expression and functional analysis of two NhaD type antiporters from the halotolerant and alkaliphilic Halomonas sp. Y2. Extremophiles, 2016,20(5):1-9. doi: 10.1007/s00792-016-0852-8 pmid: 27315164

[14]	Ohyama T, Igarashi K, Kobayashi H . Physiological role of the chaA gene in sodium and calcium circulations at a high pH in Escherichia coli. Journal of Bacteriology, 1994,176(14):4311-4315.

[15]	徐宁, 程海娇, 刘清岱 , 等. 细菌Na +/H +逆向转运蛋白的研究进展 . 微生物学通报, 2015,42(10):2002-2011.

[15]	Xu N, Cheng H J, Liu Q D , et al. Research progress of the Na +/H + antiporters in bacteria . Microbiology China, 2015,42(10):2002-2011.

[16]	Fakharany E E, Hassan M . A universal method for extraction of genomic DNA from various microorganisms using lysozyme. New Biotechnology, 2016,33:S210. doi: 10.1016/j.nbt.2016.06.1445

[17]	王艳红, 曹宁, 贾桂燕 , 等. 中度嗜盐菌Halobacillus Y5甘氨酸甜菜碱转运蛋白opuD基因的克隆及功能分析. 中国生物制品学杂志, 2017,30(3):258-263.

[17]	Wang Y H, Cao N, Jia G Y , et al. Cloning and functional identification of glycine betaine-cholinecarnitine transporter opuD gene from Halobacillus Y5. Chinese Journal Biologicals, 2017,30(3):258-263.

[18]	Som A, Fuellen G . The effect of heterotachy in multigene analysis using the neighbor joining method. Molecular Phylogenetics & Evolution, 2009,52(3):846-851.

[19]	Meng L, Meng F, Zhang R , et al. Characterization of a novel two-component Na +(Li +, K +)/H + antiporter from Halomonas zhaodongensis . Scientific Reports, 2017,7(1):4221-4234.

[20]	Rosen B P . Ion extrusion system in Escherichia coli. Methods in Enzymology, 1986,125(1):328-336.

[21]	Wang Y, Song N, Yang L , et al. A novel NhaD-type Na +/H + antiporter from the moderate halophile and alkaliphile Halomonas alkaliphila. Canadian Journal of Microbiology, 2017,63(7):596-607.

[22]	Huang L, Khusnutdinova A, Nocek B , et al. A family of metal-dependent phosphatases implicated in metabolite damage-control. Nature Chemical Biology, 2016,12(8):1-8. doi: 10.1038/nchembio.2108 pmid: 27322068

[23]	López J E, Sullivan E D, Fierke C A . Metal-dependent deacetylases: cancer and epigenetic regulators. Acs Chemical Biology, 2016,11(3):706-729. doi: 10.1021/acschembio.5b01067 pmid: 26907466

[24]	Li R, Gong Z, Pan C , et al. Metal-dependent protein phosphatase 1A functions as an extracellular signal-regulated kinase phosphatase. Febs Journal, 2013,280(11):2700-2711. doi: 10.1111/febs.12275 pmid: 23560844

[25]	Mahajan C, Basotra N, Singh S , et al. Malbranchea cinnamomea: a thermophilic fungal source of catalytically efficient lignocellulolytic glycosyl hydrolases and metal dependent enzymes. Bioresource Technology, 2015,200:55-63. doi: 10.1016/j.biortech.2015.09.113 pmid: 26476165

[26]	Dong W, Wang F, Huang F , et al. Metabolic pathway involved in 6-chloro-2-benzoxazolinone degradation by Pigmentiphaga sp. strain DL-8 and identification of the novel metal-dependent hydrolase CbaA. Appl Environ Microbiol, 2016,82(14):4169-4179.

[27]	Marschner A, Klein C D . Metal promiscuity and metal-dependent substrate preferences of Trypanosoma brucei, methionine aminopeptidase 1. Biochimie, 2015,115:35-43. doi: 10.1016/j.biochi.2015.04.012 pmid: 25921435

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