微生物学报

Halophilic and halotolerant related antiporters	K_m [Na⁺] (mmol/L)	V_max (μmol/(min·g))	Substrates	Main characteristics	References
−: No relevant information found in literature.
CPA-1 family
NhaP	6.0 (pH 7.5)	42	Na⁺, Li⁺, K⁺, Rb⁺, Ca²⁺, NH₄⁺	The transport process depends on the potential energy. Most of them are single-subunit secondary sodium pumps, mainly involved in regulating intracellular pH homeostasis and resisting salt stress	[12-13]
NhaK	88.0 (pH 8.0) 24.0 (pH 8.5)	−	Na⁺, Li⁺, K⁺, Rb⁺	[14]
YjcE	−	−	Na⁺	[15]
CvrA	−	−	Na⁺, K⁺	[15]
NhaG	−	−	Na⁺, Li⁺	[16]
CPA-2 family
NhaA	1.98	253	Na⁺, Li⁺	Mainly found in prokaryotes such asEnterococcus haideri andBacillus cereus, with functional features similar to the CPA-1 family	[17]
NapA	1.0 (pH 7.5)	−	Na⁺, Li⁺	[18]
KefA	−	−	K⁺	[19]
KefB	−	−	Na⁺, K⁺	[20]
GerN	1.5 (pH 8.0) 25.0 (pH 7.0)	−	Na⁺, Li⁺	[21]
KefC	−	−	Na⁺, Li⁺, K⁺, Rb⁺	[20]
CPA-3 family
Mrp	−	−	Na⁺, Li⁺, K⁺	Mrp antiporter, the only member of this family, is a polysubunit secondary sodium pump, containing 6−7 subunits that function in the form of a heterologous complex	[22]
Antiporter superfamily
MFS	−	−	Na⁺, Li⁺, K⁺	The homology with other families is low, and some members have the function of transporting Na⁺, which is still to be explored	[23]
Ha-ydjM	0.43±0.05 (pH 8.0)	−	Na⁺, Li⁺, K⁺	[24]
Other antiporters
NhaD	0.89 (NhaD1，pH 8.5) 0.47 (NhaD2，pH 9.5) 0.42 (Ha-NhaD, pH 9.0)	−	Na⁺, Li⁺	It not only makes the strain tolerant to hypersaline environments, but also to highly alkaline environments, where it is generally most active	[25-26]
NhaH	0.83 (pH 8.5)	−	Na⁺, Li⁺	It’s the first Na⁺/H⁺ antiporter cloned from a moderately halophilic bacterium	[27]
NhaB	1.3 (pH 8.0)	404	Na⁺, Li⁺	It functions primarily at low Na⁺ concentrations and low pH	[12]
NhaC	−	−	Na⁺	It has a limited role in maintaining Na⁺-dependent pH homeostasis and does not participate in high salt-induced adaptive responses	[28]
RDD	1.29±0.14 (pH 9.0)	−	Na⁺, Li⁺, K⁺	It has Na⁺(Li⁺, K⁺)/H⁺ reverse transport activity which can be affected by amino acid residues, and has no homology with other ion transporters	[4]
UPF0118	1.13±0.09 (pH 9.0)	23.08±0.48	Na⁺, Li⁺	It has Na⁺ (Li⁺)/H⁺ reverse transport activity, and has no homology with other ion transporters	[5]
DUF	0.25±0.06 (pH 9.0)	−	Na⁺, Li⁺, K⁺	DUF1 and DUF2 form DUF1-2 complex, which has Na⁺ (Li⁺, K⁺)/H⁺ reverse transport activity	[29]

Halophilic and halotolerant related antiporters	K_m [Na⁺] (mmol/L)	V_max (μmol/(min·g))	Substrates	Main characteristics	References
−: No relevant information found in literature.
CPA-1 family
NhaP	6.0 (pH 7.5)	42	Na⁺, Li⁺, K⁺, Rb⁺, Ca²⁺, NH₄⁺	The transport process depends on the potential energy. Most of them are single-subunit secondary sodium pumps, mainly involved in regulating intracellular pH homeostasis and resisting salt stress	[12-13]
NhaK	88.0 (pH 8.0) 24.0 (pH 8.5)	−	Na⁺, Li⁺, K⁺, Rb⁺	[14]
YjcE	−	−	Na⁺	[15]
CvrA	−	−	Na⁺, K⁺	[15]
NhaG	−	−	Na⁺, Li⁺	[16]
CPA-2 family
NhaA	1.98	253	Na⁺, Li⁺	Mainly found in prokaryotes such asEnterococcus haideri andBacillus cereus, with functional features similar to the CPA-1 family	[17]
NapA	1.0 (pH 7.5)	−	Na⁺, Li⁺	[18]
KefA	−	−	K⁺	[19]
KefB	−	−	Na⁺, K⁺	[20]
GerN	1.5 (pH 8.0) 25.0 (pH 7.0)	−	Na⁺, Li⁺	[21]
KefC	−	−	Na⁺, Li⁺, K⁺, Rb⁺	[20]
CPA-3 family
Mrp	−	−	Na⁺, Li⁺, K⁺	Mrp antiporter, the only member of this family, is a polysubunit secondary sodium pump, containing 6−7 subunits that function in the form of a heterologous complex	[22]
Antiporter superfamily
MFS	−	−	Na⁺, Li⁺, K⁺	The homology with other families is low, and some members have the function of transporting Na⁺, which is still to be explored	[23]
Ha-ydjM	0.43±0.05 (pH 8.0)	−	Na⁺, Li⁺, K⁺	[24]
Other antiporters
NhaD	0.89 (NhaD1，pH 8.5) 0.47 (NhaD2，pH 9.5) 0.42 (Ha-NhaD, pH 9.0)	−	Na⁺, Li⁺	It not only makes the strain tolerant to hypersaline environments, but also to highly alkaline environments, where it is generally most active	[25-26]
NhaH	0.83 (pH 8.5)	−	Na⁺, Li⁺	It’s the first Na⁺/H⁺ antiporter cloned from a moderately halophilic bacterium	[27]
NhaB	1.3 (pH 8.0)	404	Na⁺, Li⁺	It functions primarily at low Na⁺ concentrations and low pH	[12]
NhaC	−	−	Na⁺	It has a limited role in maintaining Na⁺-dependent pH homeostasis and does not participate in high salt-induced adaptive responses	[28]
RDD	1.29±0.14 (pH 9.0)	−	Na⁺, Li⁺, K⁺	It has Na⁺(Li⁺, K⁺)/H⁺ reverse transport activity which can be affected by amino acid residues, and has no homology with other ion transporters	[4]
UPF0118	1.13±0.09 (pH 9.0)	23.08±0.48	Na⁺, Li⁺	It has Na⁺ (Li⁺)/H⁺ reverse transport activity, and has no homology with other ion transporters	[5]
DUF	0.25±0.06 (pH 9.0)	−	Na⁺, Li⁺, K⁺	DUF1 and DUF2 form DUF1-2 complex, which has Na⁺ (Li⁺, K⁺)/H⁺ reverse transport activity	[29]

Halophilic and halotolerant related transporters	K_m [K⁺] (mmol/L)	V_max (μmol/(min·g))	Substrates	Main characteristics	References
−: No relevant information found in literature.
Trk family
TrkA	−	−	−	TrkA is a binding protein which needs to cooperate with other transporters and has the highest transport activity at pH 7.5−8.5	[69]
TrkH	6.0±1.0	800±180	K⁺, Rb⁺	TrkH transporter has a low affinity for K⁺, and is dependent on other transporters	[70]
TrkG	0.8	200	K⁺, Rb⁺, Na⁺	TrkG is an integral membrane protein consisting of twelve transmembrane helices and requires Na⁺ for its K⁺ transport function	[70-72]
TrkI	1.12	176	K⁺	Similar to TrkH, TrkI is also dependent on other transporters	[69]
Ktr family
KtrA	−	−	−	It usually binds to the transmembrane transporter KtrB to form the K⁺ transporter complex	[73-74]
KtrB	0.015 (KtrB) 1 (KtrAB)	16 (KtrB) 40−100 (KtrAB)	K⁺, Na⁺	There was a strong Na⁺ dependence when KtrA and KtrB work together for K⁺ transport. However, KtrB still retained low K⁺ transport capacity when present alone	[73-75]
KtrC	−	−	−	It usually binds to KtrD to form the K⁺ transporter complex	[73]
KtrD	10	40−100	K⁺	It binds to KtrC to form the K⁺ transporter complex, which has a low affinity for K⁺	[73]
KtrE	−	−	−	KtrE was a regulatory protein to supplement the K⁺ transport system	[76]
Kdp family
KdpFABC	−	−	K⁺	KdpA is responsible for the transport of K⁺ in the complex, and all components are essential in this process	[77]
KdpD/E	−	−	−	It is mainly present in bacteria, and KdpD is the most important transporter component of the Kdp family, receiving stimulus and transmitting the signal to KdpE, thus activating the Kdp transporter system	[78]
KdpQ	−	−	−	It is present in archaea and responsible for initiating the Kdp transporter system	[79]
Kup family
Kup	0.37±0.13	27±5	K⁺, Rb⁺, Cs⁺	It is mainly involved in K⁺ transport in low potassium environments, and has low homology with other transporter families	[80-81]
KimA	0.215±0.024	245±7	K⁺	It has strong activity in acidic environments, and has low homology with other K⁺ transporter families	[6]

Halophilic and halotolerant related transporters	K_m [K⁺] (mmol/L)	V_max (μmol/(min·g))	Substrates	Main characteristics	References
−: No relevant information found in literature.
Trk family
TrkA	−	−	−	TrkA is a binding protein which needs to cooperate with other transporters and has the highest transport activity at pH 7.5−8.5	[69]
TrkH	6.0±1.0	800±180	K⁺, Rb⁺	TrkH transporter has a low affinity for K⁺, and is dependent on other transporters	[70]
TrkG	0.8	200	K⁺, Rb⁺, Na⁺	TrkG is an integral membrane protein consisting of twelve transmembrane helices and requires Na⁺ for its K⁺ transport function	[70-72]
TrkI	1.12	176	K⁺	Similar to TrkH, TrkI is also dependent on other transporters	[69]
Ktr family
KtrA	−	−	−	It usually binds to the transmembrane transporter KtrB to form the K⁺ transporter complex	[73-74]
KtrB	0.015 (KtrB) 1 (KtrAB)	16 (KtrB) 40−100 (KtrAB)	K⁺, Na⁺	There was a strong Na⁺ dependence when KtrA and KtrB work together for K⁺ transport. However, KtrB still retained low K⁺ transport capacity when present alone	[73-75]
KtrC	−	−	−	It usually binds to KtrD to form the K⁺ transporter complex	[73]
KtrD	10	40−100	K⁺	It binds to KtrC to form the K⁺ transporter complex, which has a low affinity for K⁺	[73]
KtrE	−	−	−	KtrE was a regulatory protein to supplement the K⁺ transport system	[76]
Kdp family
KdpFABC	−	−	K⁺	KdpA is responsible for the transport of K⁺ in the complex, and all components are essential in this process	[77]
KdpD/E	−	−	−	It is mainly present in bacteria, and KdpD is the most important transporter component of the Kdp family, receiving stimulus and transmitting the signal to KdpE, thus activating the Kdp transporter system	[78]
KdpQ	−	−	−	It is present in archaea and responsible for initiating the Kdp transporter system	[79]
Kup family
Kup	0.37±0.13	27±5	K⁺, Rb⁺, Cs⁺	It is mainly involved in K⁺ transport in low potassium environments, and has low homology with other transporter families	[80-81]
KimA	0.215±0.024	245±7	K⁺	It has strong activity in acidic environments, and has low homology with other K⁺ transporter families	[6]

嗜盐耐盐微生物抗盐胁迫相关离子转运蛋白研究进展

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马欣 , 马想蓉 , 朱德锐 , 李永臻 , 邢江娃 ^*

微生物学报 | 综述 2024,64(3): 651-671

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微生物学报 | 综述 2024, 64(3): 651-671

嗜盐耐盐微生物抗盐胁迫相关离子转运蛋白研究进展

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马欣, 马想蓉, 朱德锐, 李永臻, 邢江娃^*

作者信息

青海大学医学部基础医学研究中心, 青海西宁 810016

Advances in ion transporters associated with tolerance of halophilic and halotolerant microorganisms to salt stress

Xin MA, Xiangrong MA, Derui ZHU, Yongzhen LI, Jiangwa XING^*

Affiliations

Research Center of Basic Medical Sciences, Medical College, Qinghai University, Xining 810016, Qinghai, China

出版时间: 2024-03-04 doi: 10.13343/j.cnki.wsxb.20230534

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摘要

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离子转运蛋白在维持细胞内pH稳态、离子动态平衡等方面发挥着重要作用。钠离子转运体和钾离子转运体在嗜盐耐盐微生物中广泛存在，其“保钾排钠”机制是微生物抗盐胁迫的两大策略之一。近年来，嗜盐耐盐微生物中许多新型钠、钾离子转运体被陆续发现，如RDD蛋白、UPF0118蛋白、DUF蛋白和KimA蛋白等；Fe³⁺、Mg²⁺等其他金属离子的转运蛋白也被证实可通过影响微生物胞内相容性溶质的合成起到渗透调节的作用。本文综述了嗜盐耐盐微生物中抗盐胁迫相关的各类离子转运蛋白，分析其分子结构和工作机理，并对这些蛋白在农业方面的应用进行了展望。继续发现新的离子转运蛋白，探究抗盐胁迫相关离子转运蛋白的结构和机理，解析各转运系统的协同作用及分子调控机制，将进一步加深对嗜盐耐盐微生物抗盐胁迫调控的认识，并为盐碱地农作物的改良等提供新的思路。

关键词

盐胁迫 / 微生物 / 离子转运蛋白 / 钠离子转运体 / 钾离子转运体

Abstract

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Ion transporters play an important role in maintaining intracellular pH homeostasis and ionic equilibrium. Sodium ion transporters and potassium ion transporters exist widely in halophilic and halotolerant microorganisms, and their function of retaining potassium and excreting sodium is one of the two major strategies for microbial tolerance to salt stress. In recent years, new sodium and potassium ion transporters, such as RDD, UPF0118, DUF, and KimA, have been discovered in halophilic and halotolerant microorganisms. The transporters of other metal ions, such as Fe³⁺ and Mg²⁺, have been proved to play a role in microbial osmoregulation by participating in the synthesis of intracellular compatible solutes. This paper reviews the ion transporters associated with salt stress tolerance in halophilic and halotolerant microorganisms, analyzes their molecular structures and working mechanisms, and prospects for their applications in agriculture. Discovering new ion transporters, revealing the structures and mechanisms of ion transporters associated with salt stress tolerance, and analyzing the synergistic effect of coexisting transporter systems and their regulation mechanisms will deepen the understanding of the regulatory mechanisms of salt stress tolerance of halophilic and halotolerant microorganisms and provide new ideas for the improvement of crops in saline-alkali land.

Key words

salt stress / microorganism / ion transporter / sodium ion transporter / potassium ion transporter

引用本文

马欣, 马想蓉, 朱德锐, 李永臻, 邢江娃. 嗜盐耐盐微生物抗盐胁迫相关离子转运蛋白研究进展. 微生物学报, 2024 , 64 (3) : 651 -671 . DOI: 10.13343/j.cnki.wsxb.20230534

Xin MA, Xiangrong MA, Derui ZHU, Yongzhen LI, Jiangwa XING. Advances in ion transporters associated with tolerance of halophilic and halotolerant microorganisms to salt stress[J]. Acta Microbiologica Sinica, 2024 , 64 (3) : 651 -671 . DOI: 10.13343/j.cnki.wsxb.20230534

正文

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钠、钾等是微生物生存所必需的微量元素，通常以离子形态存在，在维持微生物的生理状态和正常生长代谢等方面发挥着不可或缺的作用。然而，当外界环境中Na⁺浓度过高时，会破坏微生物细胞内的渗透压平衡，使其生长繁殖受阻甚至死亡。嗜盐耐盐微生物是一类能在一定盐度范围内进行专性或兼性生长的微生物^[1]，在长期进化过程中，主要形成了两种对抗盐胁迫的生理机制。一种是在胞内合成和累积糖、多元醇、甜菜碱、氨基酸及其衍生物等一类被称为相容性溶质的小分子极性物质，以调节细胞内外渗透压平衡，起到渗透保护作用^[2]；另一种是通过离子转运调节Na⁺、K⁺等离子的排出和输入，以维持胞内各离子浓度的动态平衡^[3]。由于依赖相容性溶质的抗盐胁迫机制需要微生物进行一系列的反应以生成所需的相容性溶质，离子转运相较于相容性溶质合成来说耗能较少，是更直接快速的微生物应对高盐环境的调节工具^[1]。

近年来，盐境适应机制相关的离子转运蛋白研究日益热门。研究者们在NhaA蛋白、TrkA蛋白等常见离子转运蛋白的基础上，新发现了RDD蛋白^[4]、UPF0118蛋白^[5]、KimA蛋白^[6]等新型的钠、钾离子转运蛋白。同时，越来越多的研究表明，离子转运在嗜盐耐盐的过程中可能发挥着比传统认识上更为重要的作用，在依赖相容性溶质合成的嗜盐耐盐微生物中也是不可或缺的^[7]。通过离子转运对抗盐胁迫的生理机制主要由位于细胞膜上的离子转运蛋白介导。离子转运蛋白广泛存在于细菌、植物和动物体中，在维持细胞内离子动态平衡、pH稳态等方面发挥着重要作用^[8]。本文主要关注嗜盐耐盐微生物的离子转运蛋白，以嗜碱盐单胞菌(Halomonas alkalicola)、盐沼盐杆菌(Halobacterium salinarum)、大肠杆菌(Escherichia coli)和蓝藻(Cyanobacteria)等为例，从离子转运蛋白的种类、结构和应用等方面进行综述。

1 钠氢离子转运蛋白

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1.1 Na⁺/H⁺转运蛋白的类型和特征

在高盐环境中，嗜盐耐盐微生物依赖的Na⁺外排系统主要是各种Na⁺/H⁺转运蛋白，其转运过程以质子电化学梯度为动力，大多可独立发挥转运功能^[9]。目前已发现的Na⁺/H⁺转运蛋白包括CPA-1家族、CPA-2家族、CPA-3家族、Na⁺/H⁺转运蛋白超家族以及其他一些尚未明确结构和机制的离子转运蛋白等^[10-11] (表1)。值得一提的是，虽然这些Na⁺/H⁺转运蛋白的英文名称各不相同，但却都表示同样的意思，如数量最多的Nhaxx蛋白，是从“Na⁺/H⁺ antiporter”中得到^[30]，说明其是一个同时转运钠离子和氢离子的逆向转运蛋白，还有CPA-2家族的NapA蛋白，意为“NaH-antiporter”^[31]，与前者几乎同义。

1.1.1 CPA-1家族

阳离子/质子逆向转运蛋白-1家族(the cation/proton antiporter-1 family, CPA-1 family)广泛分布于革兰氏阳性菌、革兰氏阴性菌、真菌、植物和动物等膜系统上，具有Na⁺/H⁺逆向转运蛋白活性，主要参与调控胞内pH稳态和对抗盐胁迫。该家族大多是单亚基型次级钠泵，到目前为止，主要包括YjcE、CvrA^[15]、NhaG^[16]、NhaP和NhaK等，其中得到广泛关注的是NhaP蛋白和NhaK蛋白。

(1) NhaP蛋白

NhaP是CPA-1家族的主要成员之一^[32]，在结构和功能方面具有一定的多样性。系统发育分析表明，NhaP转运蛋白经长期进化后，主要分为NhaP1和NhaP2两种类型^[33]，且NhaP2除转运Na⁺外，对K⁺也有一定的亲和力^[13]。NhaP蛋白广泛存在于极端嗜热古生菌(Pyrococcus abyssi)和詹氏甲烷球菌(Methanocaldococcus jannaschii)等嗜盐古菌^[34-35]，以及铜绿假单胞菌(Pseudomonas aeruginosa)、霍乱弧菌(Vibrio cholerae)、蓝藻等嗜盐耐盐微生物中^[13,32-33]。王姝杰等将蓝藻中NhaP的编码基因转移到烟草中^[36]，发现转基因烟草的耐盐性从0.1 mol/L NaCl提高到了0.2 mol/L NaCl，且该基因可在烟草中稳定遗传。

(2) NhaK蛋白

NhaK蛋白也称为YvgP蛋白，最早发现于枯草芽孢杆菌(Bacillus subtilis)中，后又在金黄色葡萄球菌(Staphylococcus aureus)中发现了它的存在^[14,37]。研究表明，枯草芽孢杆菌中的NhaK蛋白具有Na⁺ (K⁺, Li⁺, Rb⁺)/H⁺逆向转运活性，且Mg²⁺、Ca²⁺和Mn²⁺会抑制其转运活性^[14]。截至目前，未见其他嗜盐耐盐微生物中发现NhaK蛋白的报道，其转运机制和特性也有待进一步研究。

1.1.2 CPA-2家族

阳离子/质子逆向转运蛋白-2家族(the cation/proton antiporter-2 family, CPA-2 family)主要存在于细菌和古菌等原核生物中，较少出现在真核生物膜系统中，其家族成员主要有NhaA、NapA、KefA^[19]、KefB、KefC^[20]和GerN^[21]等。目前重点研究的主要是以下3种。

(1) NhaA蛋白

NhaA蛋白是从细菌中分离出来的第一种Na⁺/H⁺转运蛋白，也是目前研究最丰富的转运蛋白^[38]。NhaA最早从大肠杆菌中克隆得到^[30]，在维持其胞内渗透压和pH稳态方面发挥着至关重要的作用，缺失会导致菌株在高盐环境中出现生长缺陷。NhaA蛋白活性具有pH依赖性，对周围环境的pH值十分敏感，只有当pH值达到6.5时才会被激活^[39]。将一株嗜盐假单胞菌的NhaA蛋白编码基因转移到大肠杆菌表达载体中，发现转化子的耐盐极限由对照组的1 mol/L NaCl提升到1.1 mol/L NaCl，且相同盐度条件下生长速度和最终菌浓度远高于对照组^[40]。

(2) NapA蛋白

NapA蛋白是从海氏肠球菌(Enterococcus hirae)中鉴定出的一种CPA-2家族蛋白。研究者们使用基因片段切除与插入技术，证实了NapA蛋白在高盐环境中的离子转运能力。破坏NapA编码基因导致大肠杆菌克隆子在0.15 mol/L NaCl的情况下无法生长，而恢复该蛋白的表达之后，菌株又对高盐环境表现出耐受能力^[31]。在一项针对假坚强芽孢杆菌(Bacillus pseudofirmus)的研究中，研究者通过基因序列分析发现其可能有NapA蛋白的表达，但具体的功能分析尚不明确^[41]。同时，基因序列分析也表明，钠氢离子转运蛋白GerN与NapA来源于同一个祖先^[9]，说明该蛋白也是CPA-2家族的一员。

(3) KefB蛋白

KefB是从大肠杆菌中分离出的CPA-2家族蛋白^[20]，是一种谷胱甘肽门控的K⁺离子输出蛋白，早期还被称为TrkB。在盐胁迫下，嗜盐古菌科库里盐红菌(Halorubrum kocurii) 2020YC7中的kefB转录水平可随NaCl浓度的升高显著上调^[42]。研究者将结核分枝杆菌(Mycobacterium tuberculosis)的KefB蛋白基因剔除，发现突变子仅在高K⁺环境下生存受损，而在低K⁺环境下的生存不受影响，说明KefB蛋白主要在高K⁺环境中发挥K⁺的外排功能^[43]。

1.1.3 CPA-3家族

阳离子/质子逆向转运蛋白-3家族(the cation/proton antiporter-3 family, CPA-3 family)广泛存在于原核生物中。钠离子/质子逆向转运蛋白Mrp (multiple resistance and pH-related antiporter)是目前该家族的唯一分支，也是最重要成员，属于多亚基型次级钠泵^[11]。由于早期命名系统的混乱，Mrp蛋白曾被赋予多种名称，例如Mnh、Pha、Sha和Sno等^[8]。

Mrp蛋白最早发现于耐盐芽孢杆菌(Bacillus halotolerans)中^[44]，其蛋白结构通常包含6−7个亚基(MrpA−MrpG)，以复合体的形式发挥功能，在维持胞内外渗透压平衡与酸碱平衡方面具有重要作用。根据亚基数量的不同，可以将Mrp蛋白分为Mrp-Ⅰ和Mrp-Ⅱ两种类型，分别主要存在于革兰氏阳性菌和革兰氏阴性菌中^[45]。Mrp-Ⅰ由完整的7个亚基组成；Mrp-Ⅱ由6个亚基组成，缺少MrpB，但其MrpA亚基中包含了MrpB的结构域，所以功能并未受到影响^[9]。2019年，Mormile等从华盛顿州皂湖中分离出一株皂湖7号盐单胞菌(Halomonas sp. Soap Lake #7)，是已知的首个体内同时存在Mrp-Ⅰ和Mrp-Ⅱ两种转运复合体的嗜盐嗜碱微生物^[22]。在嗜碱盐单胞菌(Halomonas alkalicola) CICC 11012s中，研究者发现Mrp蛋白在中性和碱性环境中发挥作用，在pH 11.0时表达量最高，说明Mrp蛋白可能主要在碱性条件下参与菌株的pH调节^[46]。嗜碱性拟坚强芽孢杆菌(Bacillus pseudofirmus)中的Mrp蛋白在维持细胞Na⁺平衡和酸碱平衡中都发挥着重要作用^[47]。此外，Mrp蛋白还存在于许多其他的嗜盐耐盐微生物中，如超嗜热古菌(Thermococcus eurythermalis) A501^[48]、嗜中性枯草芽孢杆菌^[49]、中华根瘤菌(Sinorhizobium)^[50]以及金黄色葡萄球菌^[51]等。

1.1.4 转运蛋白超家族

除已经明确家族分类的离子转运蛋白之外，还存在一些具有Na⁺转运功能，但与已知家族序列同源性较低的蛋白质，分属于不同的质子转运体超家族(proton antiporter-superfamily)，其中较具有代表性的是MFS超家族和Ha-ydjM蛋白。

(1) MFS超家族

主要促进者超家族(major facilitator superfamily, MFS)是目前已知最大的膜转运蛋白超家族之一^[52]，家族成员众多，可促进糖、药物分子、肽、Na⁺和H⁺等溶质在电化学梯度下进行跨膜运输。研究者在海洋扁球菌(Planococcus maritimus) DS 17275^T中发现了一种功能未知的MFS转运体，命名为Mdrp蛋白^[23]，发现其具有Na⁺ (Li⁺, K⁺)/H⁺逆转运活性，对3种离子表观亲和力的大小为Na⁺ > K⁺ > Li⁺。在pH 9.0时，Mdrp蛋白对Na⁺和Li⁺的转运活性最高；在pH 8.5时，对K⁺的转运活性最高。王艳红等在盐单胞菌(Halomonas) YH-I中发现MFS转运蛋白基因，但经过转基因筛选实验之后，发现菌株中起耐盐作用的并不是MFS超家族成员，而可能是开放阅读框3 (open reading frame 3, ORF3)和ORF4两个结构未知的膜蛋白^[53]。截至目前，MFS超家族依然有十几个功能未知的亚家族蛋白^[54]，这些蛋白中具有Na⁺转运功能的究竟有多少种，仍待研究者们后续探索。

(2) Ha-ydjM蛋白

Ha-ydjM蛋白由殷奎德课题组于2018年发现于中度嗜盐菌喜盐芽孢杆菌(Halobacillus) Y5中，研究者通过基因功能互补的方法，证实它是YdjM超家族的一个新成员，对细菌胞内外的Na⁺/H⁺转运具有重要影响^[24]。Ha-ydjM蛋白能够恢复大肠杆菌突变株KNabc在0.2 mol/L NaCl条件下的生长能力，并呈现出pH依赖的Na⁺/H⁺转运蛋白活性，在pH 8.0时，可以更好地发挥转运作用。YdjM超家族的其他成员大多不具有Na⁺转运功能，所以Ha-ydjM蛋白的发现不仅丰富了离子转运蛋白的研究，也为更多新型Na⁺/H⁺转运蛋白的发现提供了一个新思路。

1.1.5 其他蛋白

随着全基因组测序技术和分子生物学技术的发展，除了上文描述的Na⁺/H⁺转运蛋白家族外，嗜盐耐盐微生物中相继发现了许多其他类型的Na⁺/H⁺逆向转运蛋白，如NhaH^[27]、NhaC^[28]、NhaB^[55]和NhaD^[56]，以及新发现的RDD蛋白和UPF0118蛋白等。这些蛋白已被证实参与了细菌在高盐环境中的适应作用，但其具体的蛋白分类还不明确，本文主要介绍以下4种蛋白。

(1) NhaD蛋白

NhaD蛋白最早发现于副溶血弧菌(Vibrio parahemolyticus)中，能够使菌株耐受高盐、高碱环境。插入了NhaD蛋白编码基因的大肠杆菌转化子KNabc可在高盐环境中生长，且表达的NhaD蛋白仅在碱性条件下具有转运活性，在pH 8.5−9.0时活性最高^[56]。通过改变细菌生长的盐度和pH值，发现霍乱弧菌的NhaD蛋白同样在碱性环境(pH 7.25−8.50)中发挥作用^[57]。除转运Na⁺以外，拥有NhaD蛋白的大肠杆菌缺陷菌株还表现出了Li⁺耐受性，甚至在0.5 mol/L Na⁺浓度、10 mmol/L Li⁺浓度的情况下，仍然保留了较强的生长能力^[58]。NhaD蛋白可分为不同的亚型，其活性特点略有不同。Cui等从盐单胞菌(Halomonas sp.) Y2的基因组DNA中克隆出NhaD1和NhaD2两种NhaD蛋白，可在中性及弱酸性条件下检测到转运活性，但在碱性条件下活性最高^[25]。其中NhaD1的最高活性值在pH 8.0−8.5，NhaD2的最高活性值在pH 9.5左右。宋娜在嗜碱盐单胞菌(Halomonas alkaliphila) DSM 16354^T中发现了NhaD蛋白的另一亚型，命名为Ha-NhaD蛋白，插入了这个蛋白编码基因的大肠杆菌转化菌株在pH 9.0时拥有最大的耐盐生长活性^[26]。这些研究结果表明，同一离子转运蛋白的不同亚型保持活性的最佳pH值可能不同，进一步加深了关于离子转运蛋白的研究。

(2) NhaH蛋白

Yang等通过功能互补的方法，首次从中度嗜盐菌达坂盐杆菌(Halobacillus dabanensis) D-8^T中克隆得到一个编码Na⁺转运蛋白的基因，并将其编码的蛋白命名为NhaH^[27]。NhaH蛋白对Na⁺具有高亲和性，主要参与碱性环境下Na⁺的转运，可提高大肠杆菌转化子的耐盐能力^[59]。进一步的蛋白质亲疏水性图谱分析显示，NhaH蛋白的C端区域仅存在9个亲水性氨基酸残基，Na⁺转运活性较低。

(3) UPF0118、RDD蛋白

UPF0118和RDD蛋白均由姜巨全课题组从安达喜盐芽孢杆菌(Halobacillus andaensis) NEAU-ST10-40^T中先后筛选发现，与已知的Na⁺/H⁺逆向转运蛋白无相似性^[4-5]。其中，UPF0118具有Na⁺ (Li⁺)/H⁺逆向转运活性，RDD蛋白具有Na⁺ (Li⁺, K⁺)/H⁺逆向转运活性，且氨基酸残基R124、R129和D158在后者的逆转运活性中发挥重要作用^[4]。将upf0118和rdd基因分别转移到大肠杆菌突变株KNabc中，均可恢复突变株的抗盐能力(最高耐受盐度为0.2 mol/L)，并提高突变株对碱性环境的耐受性，使其可以在pH 8.0−8.5的条件下生长^[5]。

(4) DUF蛋白

贾桂燕等从盐单胞菌DSM 16354^T中筛选出了2个具有耐盐功能的蛋白编码基因duf1和duf2，将其分别导入大肠杆菌缺陷菌株中发现，只有DUF1蛋白和DUF2蛋白共同表达时，菌株才具有Na⁺ (Li⁺、K⁺)/H⁺逆向转运活性，最高可在0.5 mol/L NaCl条件下生长，表现出显著的耐盐能力^[29]。进一步的结构分析发现，DUF1蛋白和DUF2蛋白均含有7个跨膜区和10个螺旋，螺旋的两侧末端连续排布亲水性氨基酸，是构成离子通道的重要组成部分。

1.2 Na⁺/H⁺转运蛋白的结构和机理

Na⁺转运系统可分为初级钠泵和次级钠泵，Na⁺/H⁺转运蛋白多属于后者，是一类高度保守的膜蛋白。根据Na⁺/H⁺转运蛋白所包含亚基的数量，可进一步划分为单亚基型钠泵和多亚基型钠泵^[8]。本文以单亚基型钠泵NhaA蛋白和多亚基型钠泵Mrp蛋白为例，初步揭示抗盐胁迫相关Na⁺/H⁺转运蛋白的分子作用机制。

1.2.1 单亚基型钠泵NhaA

NhaA蛋白是从细菌中分离出来的第一种Na⁺/H⁺转运蛋白，在每转运2个H⁺到胞内的同时，可转运1个Na⁺到胞外^[60]。其空间构型中存在2个漏斗状结构，两者朝向相反。其中，由TMSⅡ、Ⅸ、Ⅳc和Ⅴ形成的带负电荷的离子漏斗开口于细胞基质，终止于细胞质膜内推测的离子结合位点；另一个带负电荷的漏斗由TMSⅡ、Ⅷ和Ⅺp形成，开口于细胞质周质^[61]。NhaA蛋白的pH感应器位于TMS Ⅸ跨膜区，在胞质漏斗结构的开口部位；Na⁺结合位点则位于TMS IV-XI结构的延伸链上，非常靠近胞质漏斗结构的底部区域。pH感应器能够感应环境信号，引起TMS Ⅸ跨膜螺旋区构型的改变，并最终完全释放Na⁺结合位点(图1)^[62]。当NhaA蛋白与Na⁺结合后，引起电荷失衡，打开周质离子屏障，将Na⁺离子结合位点暴露于周质漏斗结构的底部区域，最终完成Na⁺的释放。

1.2.2 多亚基型钠泵Mrp

Mrp蛋白属于多亚基型次级钠泵，每个亚基都参与复合体的激活过程^[8]，但各亚基之间是如何发挥协同作用的，目前尚没有明确定论。Lee等研究发现，Mrp蛋白可分为MrpA–MrpD和MrpE–MrpG两个模块，两者之间相互影响，任何一个模块的亚基发生改变，都会影响另外一个模块的功能^[63]。MrpA和MrpD是Mrp系统中最大的2个亚基，其中MrpA是Na⁺转运通道，MrpD是H⁺转运通道，两者共同形成Na⁺/H⁺逆向转运体^[64]，其高表达可能与菌株的强耐碱性有关^[65]。Li等通过构建Mrp复合体的分子模型，提出MrpE、MrpF、MrpG这3种膜蛋白会在作用时维持复合体的稳定性^[66]。Mrp系统的具体结构和功能尚未研究透彻，以上发现为后续的进一步研究奠定了基础。

2 钾离子转运蛋白

收起

2.1 K⁺转运蛋白的类型和特征

钾是生物体必需的微量元素，通常以K⁺的形式存在。盐胁迫下，许多嗜盐耐盐微生物可通过升高胞内K⁺浓度来维持渗透压的平衡^[67]。这些微生物拥有一系列负责K⁺摄入的蛋白转运系统，其转运过程需要消耗ATP，且大多需要多个蛋白相互配合，共同发挥转运作用^[68]。目前已发现的K⁺蛋白转运系统主要包括Trk家族、Ktr家族、Kdp家族和Kup家族等(表2)。K⁺转运蛋白的命名规律与Na⁺/H⁺转运蛋白非常相似，如Trk家族蛋白和Ktr家族蛋白，均是指“K⁺ transporter”，虽字母顺序不一样，但都表达了同样的意思^[69]。

2.1.1 Trk家族

Trk系统是嗜盐耐盐微生物转运K⁺的主要系统之一，本质上是一个质子转运体，由跨膜蛋白和结合核苷酸的外周膜蛋白组成，在转运过程中需要质子动力和ATP^[82]。主要家族成员包括TrkI^[69]、TrkA、TrkG^[72]和TrkH等，其中研究较多的是TrkA和TrkH。

(1) TrkA蛋白

细胞膜表面蛋白TrkA主要存在于细菌和古菌中，是一种NAD⁺结合蛋白，主要负责Trk系统的激活，与Ktr家族的KtrA蛋白具有一定的同源性^[83]。一项针对嗜盐放线菌(Haloactinomyces) AFM 10258^T的研究表明，在高盐条件下，TrkA蛋白的表达在转录和蛋白水平均上调^[84]，说明该菌株可能通过加强K⁺转运来提高胞内渗透压，以对抗高盐环境。Kraegeloh等发现，在大肠杆菌突变体中，只保留TrkA蛋白和只剔除TrkA蛋白的菌株转运K⁺的能力都受到限制^[69]，说明TrkA蛋白是一个结合蛋白，需要与其他的转运蛋白协同发挥转运功能。在嗜盐解淀粉碱单胞菌(Alkalimonas amylolytica) N10中发现，TrkA可与K⁺通道蛋白TrkH结合，发挥K⁺转运功能，且在pH 7.5−8.5时，转运活性最高^[85]。进一步的研究表明，TrkA是TrkH的转运调节蛋白，可通过与ATP的结合打开TrkH离子转运通道^[86]。

(2) TrkH蛋白

跨膜蛋白TrkH属于典型的Trk家族K⁺转运蛋白，在同样的条件下，缺少TrkH蛋白的大肠杆菌菌株表现出的生长抑制不明显，说明其对K⁺的亲和力不高^[72]，但仍可表现出较高的离子转运速率^[87]。从嗜盐解淀粉碱单胞菌N10中分离的TrkH蛋白被证明在高盐培养基中对菌株的生长起关键作用^[85]。同时，TrkH蛋白需要借用TrkA蛋白作为ATP结合亚单位完成对K⁺的转运，表现出对其他转运蛋白的“依赖”现象^[88]。

2.1.2 Ktr家族

Ktr系统是一种Na⁺依赖的K⁺转运系统，主要存在于细菌中，可以在高盐高渗环境中通过调节K⁺的转运来对抗盐胁迫。Ktr家族成员主要分为膜嵌入型蛋白(如KtrB和KtrD)和膜连接型蛋白(如KtrA、KtrC和KtrE)两种^[73,76]，通过复合体的形式来发挥作用，目前得到深入研究的是KtrA和KtrB。

(1) KtrA蛋白

KtrA是Ktr家族研究最深入的转运蛋白，最早发现于溶藻弧菌(Vibrio lysolyticus)中，通常与跨膜蛋白KtrB结合形成复合物，发挥K⁺转运功能^[74]。同时，Ktr家族的KtrC和KtrD蛋白也是通过复合体的形式发挥作用，其中KtrC与KtrA同源，KtrD与KtrB同源，但整体对K⁺的亲和力较低，只有KtrAB蛋白的十分之一^[73]。鲜先毅等用RT-PCR和基因敲除的方法，发现缺少KtrA编码基因的耐辐射异常球菌(Deinococcus radiodurans) R1菌株在面对高盐环境时抗性降低，说明KtrA蛋白在菌株耐受高盐和高渗透胁迫方面有一定的保护作用^[89]。

(2) KtrB蛋白

KtrB蛋白是Ktr转运系统复合体的核心组件，在微生物转运K⁺、维持胞内外渗透压平衡方面起着至关重要的作用^[90]。虽然KtrB经常和KtrA共同发挥作用，但Zulkifli等研究发现，在较高的K⁺浓度(10 mmol/L KCl)下，单独表达KtrB蛋白也能够维持蓝藻菌株PCC 6803的生长。当KtrA与KtrB共表达时，其转运效应表现出强烈的Na⁺依赖性；而当KtrB蛋白单独存在时，即使不施加额外的Na⁺刺激，KtrB依旧保留了较低的K⁺转运能力^[76]。对KtrB蛋白的深入研究将进一步加深对离子转运蛋白家族的认识。

2.1.3 Kdp家族

Kdp系统是一种可诱导、高亲和性的K⁺转运系统^[84]，主要存在于细菌和古菌中，与前面两种K⁺转运家族相比，转运效率较低^[91]。Kdp家族成员包括KdpA、KdpB、KdpC、KdpD、KdpE、KdpF和KdpQ等。其中，KdpA、KdpB、KdpC和KdpF形成钾离子泵KdpFABC，负责Kdp系统中K⁺的转运；KdpD和KdpE主要存在于嗜盐耐盐细菌中，形成双组分系统KdpD/E，负责Kdp转运系统的激活；KdpQ仅存在于嗜盐古菌中，取代KdpD/E、激活钾离子泵KdpFABC。

(1) KdpFABC复合体

KdpFABC复合体是一种ATP驱动的多亚基钾泵^[77]。其中，KdpA蛋白负责K⁺的转运；KdpB是一种ATP水解酶，提供离子转运过程中所需要的能量；KdpC充当催化伴侣，以增加KdpB亚单位与ATP结合的亲和力；KdpF负责维持KdpFABC复合体的稳定性。研究者在一项针对沙门氏菌的研究中发现，低钾时kdpF的过表达会降低kdpD和kdpA的转录水平，说明KdpF蛋白在复合物中主要发挥调节作用^[92]。

(2) KdpD/E系统

KdpD可以响应胞内外Na⁺浓度的变化^[78]。被激活后，磷酸化的KdpD可将磷酸基团传送给KdpE蛋白，启动Kdp转运系统，维持细胞内外的渗透压平衡。张燕飞等曾敲除溶藻弧菌HY9901中的KdpD编码基因，发现KdpD编码基因的缺失影响了菌株对K⁺的吸收能力，降低了对高渗环境的抗性^[93]。

(3) KdpQ

KdpQ是在嗜盐古菌中发现的Kdp家族成员。Strahl等在嗜盐古菌盐沼盐杆菌R1和极端嗜盐古菌(Halobacterium sp.) NRC-1中检测到了一个功能未知的基因cat3^[79,94]。将盐沼盐杆菌R1中的kdpFABC和cat3一起敲除，只恢复kdpFABC基因的表达，菌株没有恢复对K⁺的摄取能力；同时恢复kdpFABC和cat3的表达，菌株重新拥有了K⁺摄取功能。这说明cat3对Kdp系统的转运有重要的调节作用，研究者将其命名为kdpQ。

2.1.4 Kup家族

Kup家族对K⁺的转运能力十分微弱，且与其他的K⁺转运蛋白家族同源性极低，其本质是K⁺/H⁺同向转运蛋白。Sato等用火焰光度法测量了大肠杆菌中由Kup蛋白介导的K⁺摄取转运活性，发现Kup蛋白主要参与低钾环境中的离子转运^[95]。Trchounian课题组用两种大肠杆菌的突变体对比Kup蛋白、TrkA蛋白和Kdp蛋白在pH为5.5的酸性环境中对K⁺的摄取情况，发现Kup蛋白是酸胁迫下大肠杆菌主要的K⁺吸收系统^[96]。2020年，研究者对枯草芽孢杆菌中发现的KimA蛋白做了功能表征^[6]，验证了其是Kup家族的一个新成员。

2.2 K⁺转运蛋白的结构和机理

抗盐胁迫相关K⁺转运蛋白广泛存在于各种嗜盐耐盐微生物中，因家族分类的不同，其结构和机理存在较大差异。Trk家族和Ktr家族同源性较高，转运K⁺的机制相似；Kdp家族的K⁺转运机制则与前两者不同。本文以这两类不同的K⁺转运蛋白为例，简要阐述其转运K⁺的分子机制。

TrkA蛋白与KtrA蛋白同源，TrkH蛋白与KtrB蛋白同源，研究者常把其放在一起，进行对比研究。TrkH和KtrB分别与TrkA和KtrA蛋白结合形成TrkHA和KtrBA复合物，通过RCK门控环介导和调节K⁺的转运^[88]。研究发现，ATP在Trk和Ktr转运家族的K⁺转运过程中发挥着重要作用。ATP对于Trk家族的K⁺转运是必需的^[85]。当与ADP结合时，TrkA呈四聚体，使TrkH呈现闭合构象，K⁺通道关闭；当与ATP结合时，TrkA环分裂成2个TrkA二聚体，释放对TrkH的限制，K⁺通道开放(图2)^[97]。对于Ktr家族来说，当ATP结合在KtrA等转运蛋白上时，会增强Ktr家族的K⁺转运能力^[67]。KtrA蛋白的RCK结构域可与ATP或NADH结合，为K⁺的转运提供能量^[98]。

Kdp家族同样是通过复合体的形式发挥作用，其KdpFABC复合体属于多亚基钾泵，在嗜盐古菌和嗜盐耐盐细菌中均广泛存在，负责K⁺的转运。然而，KdpFABC复合体的激活在细菌和古菌中存在明显差异。嗜盐耐盐细菌中，由双组分系统KdpD/E负责盐浓度的感知和Kdp转运系统的激活^[78]；嗜盐古菌中，并不能检测到kdpD和kdpE的基因同源物，而是由KdpQ取代KdpD和KdpE，发挥着启动Kdp转运系统的作用^[79]。

此外，在K⁺稳态调节过程中，环状二腺苷单磷酸(cyclic dianosine monophosphate, c-di-AMP)是非常重要的调节因子，可在细胞内高钾的情况下抑制各K⁺蛋白转运系统，以降低环境中钾离子的摄取。C-di-AMP可以降低KtrCD、Kim A等K⁺转运蛋白的转录表达水平^[99]，并可与TrkH、KtrA、Kim A和Kup^[68,100-102]等K⁺转运蛋白结合，降低其K⁺转运效率。

3 其他金属离子转运蛋白

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除了Na⁺、H⁺、K⁺等直接参与微生物盐境调节的离子之外，其他金属离子的转运如Fe³⁺、Mg²⁺等，也在微生物的盐境适应过程中发挥了一定调节作用。与Na⁺和K⁺转运蛋白对胞内渗透压大小的直接调节作用不同，Fe³⁺和Mg²⁺的转运蛋白主要通过影响胞内相容性溶质的合成起到渗透调节的作用。

3.1 Fur蛋白

Argandoña等首次证明了铁离子转运蛋白Fur是需盐色盐杆菌(Chromohalobacter salexigens) DSM 3043应对高盐胁迫复杂通路的一部分^[103]。研究者构建了缺失Fur蛋白的突变菌株，通过RT-PCR的方法，将其与拥有Fur蛋白的野生株进行对比观察，结果发现在高盐环境中，突变菌株的ectABC基因表达水平下调了10倍左右，说明Fur蛋白是相容性溶质合成基因的正调节器。在需盐色盐杆菌CHR61的培养基中额外添加FeCl₃，可显著增加胞内的铁含量，并使高盐条件(2.5 mol/L NaCl)下菌株羟基四氢嘧啶的产量增加20%，而四氢嘧啶的产量却有所下降，菌株的总体生长趋势强于对照组^[104]。这些结果表明，铁离子转运可调节微生物在应对高盐环境时的相容性溶质合成产量，提高微生物的盐适应能力。

3.2 CorA蛋白

Mg²⁺是所有生物最重要的金属离子之一，可参与各种生理生化反应。CorA蛋白是第一个被鉴别出来的Mg²⁺转运体，也是细菌中最主要的Mg²⁺转运体^[105]。Mg²⁺在微生物应对盐境时发挥的作用与Fe³⁺较为相似，同样可以促进微生物胞内相容性溶质的生成。笔者课题组在盐单胞菌(Halomonas sp.) QHL5的培养基中添加了不同浓度的MgCl₂，结果显示四氢嘧啶的积聚量可随Mg²⁺的浓度增加而逐步升高^[106]。类似地，顾頔测定了不同MgSO₄浓度下一株中度嗜盐杆菌(Brachybacterium muris)的生长曲线，发现菌株在高MgSO₄浓度(10 g/L)的条件下生长状态更好，同时，菌株内谷氨酸的产量也随着MgSO₄浓度的升高而升高^[107]。四氢嘧啶和谷氨酸都属于微生物在胞内积聚的相容性溶质，这二者产量的升高说明了微生物在面对盐胁迫时，胞内一定浓度的Mg²⁺会增强微生物的耐盐能力，而介导这一作用的转运蛋白，就是CorA蛋白。

4 展望

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Na⁺、K⁺等离子的转运调控对于嗜盐耐盐微生物在盐胁迫下的生存极为重要，有助于维持胞内外的渗透压平衡和pH稳态。在具体的转运过程中，同一微生物中一种离子的转运可能依靠多种转运蛋白来共同发挥作用，且不同菌株中存在的转运蛋白不同。如盐单胞菌Y2中同时存在Mrp转运蛋白、NhaP转运蛋白和KefA转运蛋白以共同完成Na⁺转运功能^[108]，而笔者课题组从青藏高原盐湖中分离出的盐单胞菌新种柴达木盐单胞菌(Halomonas qaidamensis) XH36中参与Na⁺转运的主要为Mrp蛋白和NhaC蛋白^[109]。目前，现有的研究多集中在各种新型离子转运蛋白的发现、转运蛋白的分子结构和工作机理，以及具体的分子调控机制等方面，但对这些冗余的多种转运系统如何协同发挥离子转运功能、以帮助菌株适应各种生存环境方面探索不足。最新的研究表明，冗余的K⁺转运系统可保证粪肠球菌(Enterococcus faecalis)在不同应激条件下得以存活^[110]，但具体的分子调控网络和调控机制尚不明确。

与此同时，Fe³⁺、Mg²⁺等离子的转运虽被证明可通过提高相容性溶质的合成和累积过程来促进嗜盐耐盐微生物适应盐胁迫，但其具体的作用机制仍待阐明。笔者课题组最新的研究表明，除Fe³⁺和Mg²⁺外，Ca²⁺的跨膜转运可能也在嗜盐耐盐微生物的盐境适应过程中发挥了一定调节作用^[111]。运用转录组学技术分析盐激条件下坎帕尼亚盐单胞菌(Halomonas campaniensis) XH26的上调表达基因，筛选出一个随盐度增加而显著上调的新型基因orf03282。通过克隆该新型基因并将其异源表达到大肠杆菌中发现，重组菌株胞内的Ca²⁺浓度和盐耐受性均明显提高。结合蛋白序列比对和结构功能预测结果，推测Orf03282蛋白可能与Ca²⁺离子转运相关，可通过增加胞内Ca²⁺浓度提高细胞的耐盐能力。这些金属离子转运蛋白参与渗透压调节的分子作用机制究竟是什么，以及Cu²⁺、Mn²⁺等其他金属离子通道是否也与嗜盐耐盐微生物的渗透压调节有关，是后续亟待解答的问题。

近年来，嗜盐耐盐微生物中抗盐胁迫相关Na⁺、K⁺等离子转运蛋白已陆续被应用于盐碱地农作物的改良。如在大豆植株中异源表达施氏假单胞菌(Pseudomonas stutzeri)中的NhaA蛋白后，能够显著提高大豆对盐胁迫的耐受性，促进大豆耐盐新品种的选育和广泛应用^[112]；在棉花中异源表达中度嗜盐菌菌株的NhaD蛋白可以提高棉花的抗旱耐盐碱能力^[113-114]；在烟草和玉米中异源表达细菌TrkH蛋白，可明显改善植物在缺钾且盐渍化的土壤上的生长状态等^[115-116]。然而，由于菌株的嗜盐耐盐特性更多是多基因协同作用的结果，现有的单一基因过表达仍具有一定的局限性。采用高通量筛选和其他分子生物学等研究手段，发现新的抗盐胁迫相关离子转运蛋白，探索各离子转运蛋白的应答和协同作用机制，将为转基因工程技术在培育抗盐碱农作物方面的进一步应用提供更多的理论和实践支撑。

基金

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国家自然科学基金(31860030)
人社部2021年度高层次留学人才回国资助项目
青海大学青年科研基金(2022-QYY-15)

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2024年第64卷第3期

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doi: 10.13343/j.cnki.wsxb.20230534

接收时间：2023-08-17
首发时间：2026-03-19
出版时间：2024-03-04

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收稿日期：2023-08-17
录用日期：2023-11-30

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National Natural Science Foundation of China(31860030)

国家自然科学基金(31860030)

2021 Return of Overseas High-level Talents Program by the Ministry of Human Resources and Social Security

人社部2021年度高层次留学人才回国资助项目

Youth Science and Technology Project of Qinghai University(2022-QYY-15)

青海大学青年科研基金(2022-QYY-15)

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青海大学医学部基础医学研究中心, 青海西宁 810016

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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Halophilic and halotolerant related antiporters	K_m [Na⁺] (mmol/L)	V_max (μmol/(min·g))	Substrates	Main characteristics	References
−: No relevant information found in literature.
CPA-1 family
NhaP	6.0 (pH 7.5)	42	Na⁺, Li⁺, K⁺, Rb⁺, Ca²⁺, NH₄⁺	The transport process depends on the potential energy. Most of them are single-subunit secondary sodium pumps, mainly involved in regulating intracellular pH homeostasis and resisting salt stress	[12-13]
NhaK	88.0 (pH 8.0) 24.0 (pH 8.5)	−	Na⁺, Li⁺, K⁺, Rb⁺	[14]
YjcE	−	−	Na⁺	[15]
CvrA	−	−	Na⁺, K⁺	[15]
NhaG	−	−	Na⁺, Li⁺	[16]
CPA-2 family
NhaA	1.98	253	Na⁺, Li⁺	Mainly found in prokaryotes such asEnterococcus haideri andBacillus cereus, with functional features similar to the CPA-1 family	[17]
NapA	1.0 (pH 7.5)	−	Na⁺, Li⁺	[18]
KefA	−	−	K⁺	[19]
KefB	−	−	Na⁺, K⁺	[20]
GerN	1.5 (pH 8.0) 25.0 (pH 7.0)	−	Na⁺, Li⁺	[21]
KefC	−	−	Na⁺, Li⁺, K⁺, Rb⁺	[20]
CPA-3 family
Mrp	−	−	Na⁺, Li⁺, K⁺	Mrp antiporter, the only member of this family, is a polysubunit secondary sodium pump, containing 6−7 subunits that function in the form of a heterologous complex	[22]
Antiporter superfamily
MFS	−	−	Na⁺, Li⁺, K⁺	The homology with other families is low, and some members have the function of transporting Na⁺, which is still to be explored	[23]
Ha-ydjM	0.43±0.05 (pH 8.0)	−	Na⁺, Li⁺, K⁺	[24]
Other antiporters
NhaD	0.89 (NhaD1，pH 8.5) 0.47 (NhaD2，pH 9.5) 0.42 (Ha-NhaD, pH 9.0)	−	Na⁺, Li⁺	It not only makes the strain tolerant to hypersaline environments, but also to highly alkaline environments, where it is generally most active	[25-26]
NhaH	0.83 (pH 8.5)	−	Na⁺, Li⁺	It’s the first Na⁺/H⁺ antiporter cloned from a moderately halophilic bacterium	[27]
NhaB	1.3 (pH 8.0)	404	Na⁺, Li⁺	It functions primarily at low Na⁺ concentrations and low pH	[12]
NhaC	−	−	Na⁺	It has a limited role in maintaining Na⁺-dependent pH homeostasis and does not participate in high salt-induced adaptive responses	[28]
RDD	1.29±0.14 (pH 9.0)	−	Na⁺, Li⁺, K⁺	It has Na⁺(Li⁺, K⁺)/H⁺ reverse transport activity which can be affected by amino acid residues, and has no homology with other ion transporters	[4]
UPF0118	1.13±0.09 (pH 9.0)	23.08±0.48	Na⁺, Li⁺	It has Na⁺ (Li⁺)/H⁺ reverse transport activity, and has no homology with other ion transporters	[5]
DUF	0.25±0.06 (pH 9.0)	−	Na⁺, Li⁺, K⁺	DUF1 and DUF2 form DUF1-2 complex, which has Na⁺ (Li⁺, K⁺)/H⁺ reverse transport activity	[29]

Halophilic and halotolerant related antiporters

K_m [Na⁺] (mmol/L)

V_max
(μmol/(min·g))

Substrates

Main characteristics

References

−: No relevant information found in literature.

CPA-1 family

NhaP

6.0 (pH 7.5)

Na⁺, Li⁺, K⁺, Rb⁺, Ca²⁺, NH₄⁺

The transport process depends on the potential energy. Most of them are single-subunit secondary sodium pumps, mainly involved in regulating intracellular pH homeostasis and resisting salt stress

[12-13]

NhaK

88.0 (pH 8.0)
24.0 (pH 8.5)

−

Na⁺, Li⁺, K⁺, Rb⁺

[14]

YjcE

−

Na⁺

[15]

CvrA

−

Na⁺, K⁺

[15]

NhaG

−

Na⁺, Li⁺

[16]

CPA-2 family

NhaA

1.98

253

Na⁺, Li⁺

Mainly found in prokaryotes such asEnterococcus haideri andBacillus cereus, with functional features similar to the CPA-1 family

[17]

NapA

1.0 (pH 7.5)

−

Na⁺, Li⁺

[18]

KefA

−

K⁺

[19]

KefB

−

Na⁺, K⁺

[20]

GerN

1.5 (pH 8.0)
25.0 (pH 7.0)

−

Na⁺, Li⁺

[21]

KefC

−

Na⁺, Li⁺, K⁺, Rb⁺

[20]

CPA-3 family

Mrp

−

Na⁺, Li⁺, K⁺

Mrp antiporter, the only member of this family, is a polysubunit secondary sodium pump, containing 6−7 subunits that function in the form of a heterologous complex

[22]

Antiporter superfamily

MFS

−

Na⁺, Li⁺, K⁺

The homology with other families is low, and some members have the function of transporting Na⁺, which is still to be explored

[23]

Ha-ydjM

0.43±0.05 (pH 8.0)

−

Na⁺, Li⁺, K⁺

[24]

Other antiporters

NhaD

0.89 (NhaD1，pH 8.5)
0.47 (NhaD2，pH 9.5)
0.42 (Ha-NhaD, pH 9.0)

−

Na⁺, Li⁺

It not only makes the strain tolerant to hypersaline environments, but also to highly alkaline environments, where it is generally most active

[25-26]

NhaH

0.83 (pH 8.5)

−

Na⁺, Li⁺

It’s the first Na⁺/H⁺ antiporter cloned from a moderately halophilic bacterium

[27]

NhaB

1.3 (pH 8.0)

404

Na⁺, Li⁺

It functions primarily at low Na⁺ concentrations and low pH

[12]

NhaC

−

Na⁺

It has a limited role in maintaining Na⁺-dependent pH homeostasis and does not participate in high salt-induced adaptive responses

[28]

RDD

1.29±0.14 (pH 9.0)

−

Na⁺, Li⁺, K⁺

It has Na⁺(Li⁺, K⁺)/H⁺ reverse transport activity which can be affected by amino acid residues, and has no homology with other ion transporters

[4]

UPF0118

1.13±0.09 (pH 9.0)

23.08±0.48

Na⁺, Li⁺

It has Na⁺ (Li⁺)/H⁺ reverse transport activity, and has no homology with other ion transporters

[5]

DUF

0.25±0.06 (pH 9.0)

−

Na⁺, Li⁺, K⁺

DUF1 and DUF2 form DUF1-2 complex, which has Na⁺ (Li⁺, K⁺)/H⁺ reverse transport activity

[29]

Halophilic and halotolerant related transporters	K_m [K⁺] (mmol/L)	V_max (μmol/(min·g))	Substrates	Main characteristics	References
−: No relevant information found in literature.
Trk family
TrkA	−	−	−	TrkA is a binding protein which needs to cooperate with other transporters and has the highest transport activity at pH 7.5−8.5	[69]
TrkH	6.0±1.0	800±180	K⁺, Rb⁺	TrkH transporter has a low affinity for K⁺, and is dependent on other transporters	[70]
TrkG	0.8	200	K⁺, Rb⁺, Na⁺	TrkG is an integral membrane protein consisting of twelve transmembrane helices and requires Na⁺ for its K⁺ transport function	[70-72]
TrkI	1.12	176	K⁺	Similar to TrkH, TrkI is also dependent on other transporters	[69]
Ktr family
KtrA	−	−	−	It usually binds to the transmembrane transporter KtrB to form the K⁺ transporter complex	[73-74]
KtrB	0.015 (KtrB) 1 (KtrAB)	16 (KtrB) 40−100 (KtrAB)	K⁺, Na⁺	There was a strong Na⁺ dependence when KtrA and KtrB work together for K⁺ transport. However, KtrB still retained low K⁺ transport capacity when present alone	[73-75]
KtrC	−	−	−	It usually binds to KtrD to form the K⁺ transporter complex	[73]
KtrD	10	40−100	K⁺	It binds to KtrC to form the K⁺ transporter complex, which has a low affinity for K⁺	[73]
KtrE	−	−	−	KtrE was a regulatory protein to supplement the K⁺ transport system	[76]
Kdp family
KdpFABC	−	−	K⁺	KdpA is responsible for the transport of K⁺ in the complex, and all components are essential in this process	[77]
KdpD/E	−	−	−	It is mainly present in bacteria, and KdpD is the most important transporter component of the Kdp family, receiving stimulus and transmitting the signal to KdpE, thus activating the Kdp transporter system	[78]
KdpQ	−	−	−	It is present in archaea and responsible for initiating the Kdp transporter system	[79]
Kup family
Kup	0.37±0.13	27±5	K⁺, Rb⁺, Cs⁺	It is mainly involved in K⁺ transport in low potassium environments, and has low homology with other transporter families	[80-81]
KimA	0.215±0.024	245±7	K⁺	It has strong activity in acidic environments, and has low homology with other K⁺ transporter families	[6]

Halophilic and halotolerant related transporters

K_m [K⁺]
(mmol/L)

V_max
(μmol/(min·g))

Substrates

Main characteristics

References

−: No relevant information found in literature.

Trk family

TrkA

−

TrkA is a binding protein which needs to cooperate with other transporters and has the highest transport activity at pH 7.5−8.5

[69]

TrkH

6.0±1.0

800±180

K⁺, Rb⁺

TrkH transporter has a low affinity for K⁺, and is dependent on other transporters

[70]

TrkG

0.8

200

K⁺, Rb⁺, Na⁺

TrkG is an integral membrane protein consisting of twelve transmembrane helices and requires Na⁺ for its K⁺ transport function

[70-72]

TrkI

1.12

176

K⁺

Similar to TrkH, TrkI is also dependent on other transporters

[69]

Ktr family

KtrA

−

It usually binds to the transmembrane transporter KtrB to form the K⁺ transporter complex

[73-74]

KtrB

0.015 (KtrB)
1 (KtrAB)

16 (KtrB)
40−100 (KtrAB)

K⁺, Na⁺

There was a strong Na⁺ dependence when KtrA and KtrB work together for K⁺ transport. However, KtrB still retained low K⁺ transport capacity when present alone

[73-75]

KtrC

−

It usually binds to KtrD to form the K⁺ transporter complex

[73]

KtrD

40−100

K⁺

It binds to KtrC to form the K⁺ transporter complex, which has a low affinity for K⁺

[73]

KtrE

−

KtrE was a regulatory protein to supplement the K⁺ transport system

[76]

Kdp family

KdpFABC

−

K⁺

KdpA is responsible for the transport of K⁺ in the complex, and all components are essential in this process

[77]

KdpD/E

−

It is mainly present in bacteria, and KdpD is the most important transporter component of the Kdp family, receiving stimulus and transmitting the signal to KdpE, thus activating the Kdp transporter system

[78]

KdpQ

−

It is present in archaea and responsible for initiating the Kdp transporter system

[79]

Kup family

Kup

0.37±0.13

27±5

K⁺, Rb⁺, Cs⁺

It is mainly involved in K⁺ transport in low potassium environments, and has low homology with other transporter families

[80-81]

KimA

0.215±0.024

245±7

K⁺

It has strong activity in acidic environments, and has low homology with other K⁺ transporter families

[6]