Cloning and Analysis of Novel Functional Genes in <i>Halomonas alkaliphila</i> DSM 16354 <sup>T</sup>

doi:10.13523/j.cb.2107020

China Biotechnology

2022, Vol. 42

Issue (3): 27-37 DOI: 10.13523/j.cb.2107020

Cloning and Analysis of Novel Functional Genes in Halomonas alkaliphila DSM 16354 ^T

JIA Gui-yan¹,WANG Yong-jie¹,CHEN Zhi-kang¹,CHEN Xing¹,YIN Kui-de¹,LI Wen²,WANG Yan-hong^1,^3,^**(

)

1 College of Life Science and Biotechnology, Heilongjiang Bayi Agricultural University, Daqing 163319, China
2 Heilongjiang Zhenbao Island Wetland National Nature Reserve Administration, Hulin 158419, China
3 Heilongjiang Provincial Key Laboratory of Environmental Microbiology and Recycling of Argo-weast in Cold Region, Daqing 163319, China

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Abstract

Objective: Halophilic bacteria are widely distributed and have strong adaptability, but the research on their mechanism is not deep enough. In order to strengthen the exploration and research on the mechanism of salt tolerance of halophilic bacteria, potential genes related to salt tolerance were screened from H. alkaliphila DSM 16354 ^T, informatics analysis was carried out, and the physiological functions of the related proteins were verified. Methods: Using the combination of gene library screening and functional complementarity, through the salt tolerance functional complementarity experiment with E.coli salt sensitive defective strain KNabc (ΔnhaA、ΔnhaB、ΔchaA), the protein coding gene with salt tolerance function was screened, and then the reverse transport activity and substrate affinity of the protein were determined by fluorescence quenching recovery experiment. Results: Two protein coding genes with salt tolerance function were screened. Bioinformatics analysis showed that the gene encoded membrane proteins with unknown function from DUF1538(domain of unknown function with No.1538 family), named DUF1 and DUF2, respectively. Phylogenetic tree analysis showed that DUF1 and DUF2 from H. alkaliphila DSM 16354 ^T belonged to an independent branch. It was predicted that these two proteins may be new members of DUF1538 family transporters. The physiological functions of DUF1 and DUF2 were analyzed which did not have salt tolerance when expressed alone, but showed significant salt tolerance when expressed together, indicating that duf1 and duf2 subunits jointly supported the salt tolerance function of the protein. The activity of protein DUF1-2 reverse transport assay showed that the two-component protein DUF1-2 had Na ⁺ (Li⁺、K⁺)/H⁺ antiporter activity. Conclusion: The selected duf1 and duf2 transporters had salt-alkali tolerance function and antiporter activity when they were co-expressed. This laid a foundation for screening new DUF1538 family transporter genes and further exploring the function of DUF1538 family transporters.

Key words： H. alkaliphila DUF1538 family transporter Na⁺/H⁺ antiporter Clone Bioinformatics

Received: 06 July 2021 Published: 07 April 2022

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Corresponding Authors: Yan-hong WANG E-mail: wyh_03@126.com

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	Gui-yan JIA
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	Xing CHEN
	Kui-de YIN
	Wen LI
	Yan-hong WANG

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JIA Gui-yan,WANG Yong-jie,CHEN Zhi-kang,CHEN Xing,YIN Kui-de,LI Wen,WANG Yan-hong. Cloning and Analysis of Novel Functional Genes in Halomonas alkaliphila DSM 16354 ^T. China Biotechnology, 2022, 42(3): 27-37.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.2107020 OR https://manu60.magtech.com.cn/biotech/Y2022/V42/I3/27

Table 1 Primer sequences

Fig.1 Sequence analysis result in recombinant plasmid The mapping of the inserted DNA fragment in the recombinant plasmid pUC-wyh1 and subcloning strategy of duf1 gene, duf2 gene or both. Subcloning of duf1 gene, duf2 gene or both were carried out by PCR amplification, purification and re-ligation into a cloning vector pUC18. The line arrows stand for the primers for subcloning of the different ORFs by PCR

Fig.2 Construction of phylogenetic development tree

Fig.3 Topological structure of protein (a) Topological structure of DUF1 protein (b) Topological structure of DUF2 protein

Fig.4 Salt tolerance and alkaline pH resistance of recombinant strain (a) Growth status under different NaCl concentrations (b) Growth status under different LiCl concentrations (c) Growth status under different pH conditions without salt (d) Growth status under different pH conditions with salt ions

Fig.5 Assays for Na⁺(Li⁺, K⁺)/H⁺ antiport activity in the everted membrane vesicles (a), (c) and(e)are the fluorescence values of Na⁺, Li⁺ and K⁺ transported by KNabc/pET19(b) with empty carrier (b),(d)and(f)are the fluorescence values of KNabc with protein DUF1-2 for Na⁺, Li⁺ and K⁺

Fig.6 pH-dependent activity profile of protein Na⁺/H⁺ antiport activity (filled circle), Li⁺/H⁺ antiport activity (filled square) and K⁺/ H⁺ antiport activity (filled triangle) were measured at the indicated pH values. The wavelength of excitation light was 492 nm and fluorescence was monitored at 526 nm

Fig.7 Calculation of K_0.5 values of DUF1-2 for Na⁺, Li⁺ and K⁺ (a) Calculation of K_0.5 values of DUF1-2 for Na⁺ (b) Calculation of K_0.5 values of DUF1-2 for Li⁺ (c) Calculation of K_0.5 values of DUF1-2 for K⁺


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