微生物学报  2020, Vol. 60 Issue (7): 1335-1344   DOI: 10.13343/j.cnki.wsxb.20190460.
http://dx.doi.org/10.13343/j.cnki.wsxb.20190460
中国科学院微生物研究所,中国微生物学会,中国菌物学会
0

文章信息

唐雅倩, 许玉荣, 蔡新露, 吴杭, 张部昌. 2020
Tang Yaqian, Xu Yurong, Cai Xinlu, Wu Hang, Zhang Buchang. 2020
放线菌中亮氨酸应答调控蛋白的生物学功能及其调控机理
Biological function and regulation mechanism of Leucine-responsive regulatory proteins in actinomycetes
微生物学报, 60(7): 1335-1344
Acta Microbiologica Sinica, 60(7): 1335-1344

文章历史

收稿日期:2019-10-09
修回日期:2019-11-18
网络出版日期:2019-11-29
放线菌中亮氨酸应答调控蛋白的生物学功能及其调控机理
唐雅倩1 , 许玉荣2 , 蔡新露1 , 吴杭1 , 张部昌1     
1. 安徽大学, 物质科学与信息技术研究院, 生命科学学院, 安徽 合肥 230601;
2. 合肥师范学院化学与化学工程学院, 安徽 合肥 230601
摘要:放线菌是一类革兰氏阳性细菌,可产生氨基酸等初级代谢产物和抗生素等次级代谢产物,其广泛用于食品、医药、添加剂及化妆品行业。此外,还有少数放线菌,如分枝杆菌等,是可以引起人和动植物病害的病原菌。亮氨酸应答调控蛋白(Leucine-responsive regulatory protein,Lrp)是一类在氨基酸代谢及其相关代谢过程中的重要转录调控子,能够应答各种氨基酸,参与调控微生物细胞的多个生理过程,例如氨基酸代谢和转运、中心代谢、细菌的持久性和毒力等。本文总结了放线菌Lrp的生物学功能,并综述了放线菌中不同种属Lrp以及天蓝色链霉菌和红色糖多孢菌Lrp调控机理的研究进展。
关键词放线菌    初级代谢    次级代谢    亮氨酸应答调控蛋白    
Biological function and regulation mechanism of Leucine-responsive regulatory proteins in actinomycetes
Tang Yaqian1 , Xu Yurong2 , Cai Xinlu1 , Wu Hang1 , Zhang Buchang1     
1. Institution of Physical Science and Information Technology, School of Life Sciences, Anhui University, Hefei 230601, Anhui Province, China;
2. Hefei Normal University, School of Chemistry and Chemical Engineering, Hefei 230601, Anhui Province, China
Abstract: Actinomycetes are a class of Gram-positive bacteria that can produce primary metabolites such as amino acids and secondary metabolites such as antibiotics. Actinomycetes are widely used in food, pharmaceutical, additive and cosmetic industries. In addition, a few actinomycetes, such as Mycobacterium, are pathogen that can cause human, animal and plant diseases. Leucine-responsive regulatory protein (Lrp) is a category of global transcriptional regulator involved in amino acid metabolism and its relevant metabolic processes. They are capable of responding to a variety of amino acids and participating in the regulation of multiple physiological processes in microbial cells, such as amino acid metabolism and transport, central metabolism, bacterial persistence and virulence, etc. This paper summarizes the biological functions of Lrp in actinomycetes, and reviews the research advance of regulatory mechanism of Lrp from different actinomycetes, especially Lrp from Streptomyces coelicolor and Saccharopolyspora erythraea studied.
Keywords: Actinomycetes    primary metabolism    secondary metabolism    Leucine-responsive regulatory protein    

放线菌是一类主要以孢子繁殖和菌丝状生长的革兰氏阳性细菌。放线菌目包括放线菌亚目、链霉菌亚目(含链霉菌属)、链孢囊菌亚目、小单孢菌亚目、糖霉菌亚目、细链孢菌亚目、放线多孢菌亚目、动球菌亚目、假诺卡氏亚目(含糖多孢菌属)、弗兰克氏菌属亚目、棒杆菌亚目(含棒杆菌属和分枝杆菌属)、微球菌亚目和丙酸杆菌亚目。放线菌与人类的生活密切相关,对人类的健康作出了突出的贡献。放线菌产生的次级代谢产物占到目前临床使用抗生素的三分之二[1],它们还是许多酶、维生素、氨基酸等的产生菌,例如谷氨酸棒杆菌可产生多种氨基酸[2-3]。弗兰克氏菌属对非豆科植物的共生固氮具有重大作用[4]。此外,还有少数放线菌能引起人和动植物病害,如结核分枝杆菌[5]

Lrp最早被认为是影响分支氨基酸转运的基因座(livR)[6]。亮氨酸(Leu)通过IHB调控蛋白影响ilvIH(乙酰羟酸合成酶,参与分支氨基酸的生物合成)和oppABCDF(参与多肽转运)操纵子的转录,因此IHB被称为亮氨酸应答调控蛋白,它参与氨基酸代谢和多肽的转运[7]。Lrp广泛存在于细菌和古细菌中,可调控微生物细胞的代谢过程[8]。AsnC是第一个在大肠杆菌中发现的与Lrp相关的蛋白质,是天冬酰胺合成酶(AsnA)的一种特异性的转录激活因子[9],因此,Lrp也被称为Lrp/AsnC家族蛋白[10],它们也因在饥饿环境中的适应性潜能而被称为贫/富调控蛋白[11]

放线菌中最早被发现的Lrp/AsnC家族成员是结核分枝杆菌中的LrpA(Rv3291c)[12],与大肠杆菌中的Lrp具有高度同源性[11]。与大多数Lrp蛋白相似,LrpA的大小约18 kDa,包括两个功能域:一个是N端的螺旋-转角-螺旋DNA结合域,包含HTH结构,能够识别靶基因DNA;另一个是C端的氨基酸响应结构域(RAM),包含着αβ三明治结构[11]。近年来研究发现,Lrp可控制放线菌中多样的生理代谢过程,包括抗生素生物合成、菌丝体形态分化、氨基酸代谢、细菌持久性和毒力等。基因组测序表明放线菌中(如链霉菌、红色糖多孢菌、分枝杆菌)普遍分布Lrp/AsnC家族蛋白。例如,天蓝色链霉菌基因组中有9个Lrp/AsnC家族蛋白编码基因,阿维链霉菌基因组中有14个Lrp/AsnC家族蛋白编码基因,红色糖多孢菌基因组中有15个Lrp/AsnC家族蛋白编码基因。但目前已被文献报道的放线菌中Lrp/AsnC家族蛋白仅有10个,包括分枝杆菌中3个,棒杆菌中1个,链霉菌中4个和红色糖多孢菌中2个(表 1)。

表 1. 放线菌中Lrp的功能和配体 Table 1. Functions and ligands of Lrp in Actinomycetes
Lrp Strains Function of Lrp Ligand Reference
LrpA Mycobacterium tuberculosis Virulence and persistence Amino acids, vitamins [11, 13-16]
AldR Mycobacterium tuberculosis Feast/famine regulator [17-18]
AldR Mycobacterium smegmatis Regulate alanine dehydrogenase [19-20]
Lrp Corynebacterium glutamicum Amino acid production [2, 21-22]
SCO2140 Streptomyces coelicolor Antibiotic biosynthesis and morphological differentiation [23]
BkdR Streptomyces coelicolor Antibiotic production and morphogenesis [24]
SCO3361 Streptomyces coelicolor Actinorhodin biosynthesis and morphogenesis Phenylalanine and cysteine [25]
SSP_Lrp Streptomyces spiramyceticus Spiramycin and bitespiramycin biosynthesis [26]
SACE_Lrp Saccharopolyspora erythraea Erythromycin biosynthesis Lysine, arginine and histidine [27]
SACE_5717 Saccharopolyspora erythraea Erythromycin biosynthesis Arginine, tyrosine and tryptophan [28]

1 Lrp的研究进展 1.1 分枝杆菌中的亮氨酸应答调控子

结核分枝杆菌(Mycobacterium tuberculosis)是一种引起结核病的病原菌,研究发现结核分枝杆菌中存在亮氨酸应答调控子LrpA(Rv3291c),它位于sigF上游,可参与细菌在饥饿环境中的应激反应[15],调控多个基因的表达,Lat是一种赖氨酸氨基转移酶,能够参与抗生素的合成,LrpA与lat启动子结合形成DNA-蛋白质复合体,促使lat的表达量提高42倍[11]。偶发分枝杆菌(Mycobacterium fortuitum)是一种不需要形成结核而可以快速生长的分枝杆菌病原菌,将偶发分枝杆菌的MT13分离,细菌的毒力和持久性明显下降,而在突变株中插入LrpA,细菌的毒力和持久性得到恢复[13, 16]。表明LrpA在体内的持久性有可能被用作抗结核病的新型药物靶点[14]

结核分枝杆菌中还发现另一个亮氨酸应答调控子AldR(Rv2779c),其是一种贫/富调控蛋白,也被认为在病原体的潜伏/持续阶段中起重要作用[29]。丙氨酸脱氢酶(Ald)是利用丙氨酸作为氮源的生长所需的酶,它可以使丙氨酸通过氧化脱氨反应为细胞生长提供氮源,研究发现Rv2779c还可以调控丙氨酸脱氢酶的表达[19],并且在耻垢分枝杆菌(Mycobacterium smegmatis)中也有相同的调控方式[20],推测在分枝杆菌中普遍存在AldR调控方式。本文就已报道的结核分枝杆菌中Lrp,总结了其调控模式图(图 1)。

图 1 结核分枝杆菌中亮氨酸应答调控子调控模式 Figure 1 Regulatory mode of Lrp in Mycobacterium tuberculosis. Rv3414c: ECF subfamily sigma subunit; Rv3290c: lysine aminotransferase; Rv3289c: a possible transmembrane protein; Rv3288c: hypothetical protein; Rv3287c: antisigma B factor; Rv3286c: ECF subfamily sigma subunit; Rv2780c: alanine dehydrogenase.

1.2 谷氨酸棒状杆菌中的亮氨酸应答调控子

革兰氏阳性土壤细菌谷氨酸棒杆菌(Corynebacterium glutamicum)被用于工业生产各种氨基酸,每年产生超过2160000 t谷氨酸和1330000 t赖氨酸[2]。它也可被改造用于生产其他氨基酸,例如丝氨酸[30]、异亮氨酸[31]和缬氨酸[22, 32-33]。有报道显示谷氨酸棒杆菌中brnEbrnF组成一个双组份的转运系统BrnFE,属于LIV-E转运蛋白家族,能够特异性地转运支链氨基酸和甲硫氨酸[2, 34]lrpbrnFE符合排布在相邻位置且转录方向相反的特点。当lrp基因缺失后,brnFE也不能正常转录,进一步通过凝胶迁移实验(electrophoretic mobility shift assays,EMSAs)和DNA酶I足迹法(DNase I footprinting)分析,确定Lrp能够与lrp-brnF的间隔区域结合,表明brnFE启动子的表达受Lrp调控,并且同时过表达lrpbrnFE可大幅度提高异亮氨酸的产量[2-3, 21]。除此之外,利用Lrp调控brnFE改变胞内的分支氨基酸和甲硫氨酸浓度的特征,谷氨酸棒杆菌的Lrp还被开发为生物传感器,用于监测胞内效应氨基酸的浓度,有效地将胞内氨基酸浓度的变化转化为荧光信号输出[34-36]

1.3 链霉菌中的亮氨酸应答调控子

在链霉菌的模式菌株——天蓝色链霉菌(Streptomyces coelicolor)中已发现3个Lrp家族成员,它们分别是BkdR、SCO2140和SCO3361。其中BkdR具有调控相邻bkd操纵子表达的作用,当敲除bkdR后,其突变株基本失去了产孢子和放线紫红素能力,暗示BkdR具有全局性的调控作用,但是该研究中并没有BkdR蛋白体外功能的验证[24]。而在阿维链霉菌(S. avermitilis)中也仅仅对bkd操纵子进行了体内缺失分析,但遗憾的是没有对bkd操纵子调控方面的研究[37]。Yu等还发现了天蓝色链霉菌中另一个Lrp蛋白SCO2140,它不仅能够调控菌丝体的生长,而且还会导致放线紫红素以及钙依赖抗生素产量的下降,但是SCO2140缺少典型的DNA结合域[23]

本实验室研究发现另一个天蓝色链霉菌中的Lrp蛋白SCO3361,它通过直接激活放线紫红素的正调控子ActII-ORF4[38],来提高放线紫红素的产量;并同时通过直接调控amfC[39]以及间接调控whiB[40]ssgB[41],影响菌株的形态分化。SCO3361在抑制自身的同时还可以激活其临近编码赖氨酸外排蛋白的基因SCO3362,并且特异性应答苯丙氨酸(Phe)和半胱氨酸(Cys)[25]。SCO3361与已发现红色糖多孢菌中的SACE_Lrp具有高度的同源性,且它们可以相互结合到彼此的启动子区,表明它们之间存在相互调控[25, 27]。链霉菌和红色糖多孢菌中广泛分布着SCO3361的同源蛋白(图 2),这暗示放线菌Lrp对抗生素生物合成具有普适性的调控作用。该研究成果一经发表就获得英国皇家科学院院士Keith F. Chater教授推荐,作为F1000Prime推荐论文(https://f1000.com/prime/727702556)。

图 2 链霉菌和红色糖多孢菌中SCO3361的同源蛋白 Figure 2 Homologous proteins of SCO3361 in Streptomyces and Saccharopolyspora erythraea. A: phylogenetic tree of SCO3361 homologous proteins in Streptomyces and Saccharopolyspora erythraea; B: Amino acid sequence alignment of SCO3361 homologous proteins in Streptomyces and Saccharopolyspora erythraea (Saccharopolyspora erythraea SACE_Lrp, Streptomyces venezuelae SVEN_0224, Streptomyces avermitilis SAV_3764, Streptomyceshygroscopicus SHJG_4727, Streptomyces clavuligerus SCLAV_5669, Streptomyces bingchenggens SBI_09408, Streptomyces coelicolor SCO3361, Saccharopolyspora erythraea SACE_5717, Nocardia farcinica NFA_44740, Streptomyces roseum Sros_7861).

最近,Lu等在螺旋链霉菌(Streptomyces spiramyceticus)中也发现了Lrp家族蛋白SSP_Lrp,它负调控螺旋霉素和可利霉素的生物合成,通过实验发现敲除SSP_Lrp的L-Leu结合结构域,即可提高这两种抗生素的产量[26]

1.4 红色糖多孢菌中的亮氨酸应答调控子

近年来,本实验室在研究红色糖多孢菌(Saccharopolyspora erythraea)的Lrp方面取得了一定进展,已经对红色糖多孢菌中SACE_Lrp和SACE_5717进行深入研究。SACE_Lrp对红霉素生物合成具有负调控作用,而且SACE_Lrp对分支氨基酸转运等代谢过程具有调控作用,对精氨酸(Arg)、赖氨酸(Lys)和组氨酸(His)三种氨基酸有特异性的应答作用。通过在红霉素工业高产菌株中缺失SACE_Lrp并过表达靶基因SACE_5387SACE_5386,添加调控底物缬氨酸(Val),在摇瓶发酵中可将红霉素产量提高48%,在发酵罐水平下可将产量提高到41%[27]。这是第一例对放线菌Lrp调控抗生素生物合成的分子机理进行深入研究的报道,为工业上提高其他放线菌次级代谢产物的产量提供了新的方法。近期研究发现,SACE_5717对红霉素生物合成也具有负调控作用,并且它可以特异性应答精氨酸(Arg)、色氨酸(Trp)和酪氨酸(Tyr),通过基因转录和EMSA分析,发现SACE_5717可以直接抑制自身和其临近的编码赖氨酸外排蛋白基因SACE_5716的转录,并证实SACE_5716可以外排赖氨酸(Lys)、组氨酸(His)、苏氨酸(Thr)和甘氨酸(Gly),另外这四种氨基酸可以影响红霉素产量,但是其作用机理并不清楚[28]。在红霉素工业高产菌株中缺失SACE_5717,并过表达其靶基因SACE_5716,在摇瓶发酵情况下,可将红霉素产量提高36%,在发酵罐水平上,能将产量提高到41%[28]。这与本实验室之前发现的Lrp的功能有所不同,虽然它们都可以调控抗生素的生物合成,但SCO3361还可以调控菌丝体的形态分化,SACE_Lrp是通过调控其临近基因对分支氨基酸内运来调控红霉素的生物合成,而SACE_5717却是通过调控其临近基因对氨基酸的外排来调控红霉素的生物合成,这也暗示着在其他产抗生素的放线菌中Lrp同源蛋白行使着不同的功能。基于以上研究,我们绘制了红色糖多孢菌中Lrp的调控网络(图 3)。

图 3 红色糖多孢菌中亮氨酸应答调控子的调控网络 Figure 3 Regulatory network of Lrp in Saccharopolyspora erythraea.

2 展望

在放线菌复杂的基因调控网络中,Lrp是非常重要的成员,在氨基酸代谢、中心代谢、物质转运等方面发挥着非常重要的作用。根据我们构建的放线菌Lrp系统发育树(图 4),可以看出同样是产抗生素放线菌中的Lrp,却分布在不同的分支上,暗示这些Lrp的功能存在差异性,为以后研究放线菌中Lrp提供一个新的视角。因此,研究放线菌中Lrp的功能,不仅对深入研究放线菌基因调控具有理论指导意义,而且在氨基酸或抗生素的工业生产方面也有实际应用价值。

图 4 放线菌中亮氨酸应答调控子的进化树分析 Figure 4 Phylogenetic tree analysis of Lrp in actinomycetes. Streptomyces spiramyceticus SSP_Lrp (GenBank accession number: MH460452); Streptomyces coelicolor SCO2140 (GenBank accession number: NP_626396); Streptomyces coelicolor BkdR (GenBank accession number: NP_628020); Streptomyces coelicolor SCO3361 (GenBank accession number: NP_627569); Mycobacterium tuberculosis LrpA (GenBank accession number: NP_217808); Mycobacterium tuberculosis Rv2779c (GenBank accession number: NP_217295); Corynebacterium glutamicum Lrp (GenBank accession number: AAM46687); Mycobacterium smegmatis AldR (GenBank accession number: YP_886997); Saccharopolyspora erythraea SACE_5717 (GenBank accession number: CAM04902); Saccharopolyspora erythraea SACE_Lrp (GenBank accession number: CAM04626).

现在对Lrp的研究已经取得了一些进展,人们已经了解到Lrp不仅是微生物代谢的重要调控子,而且还可以应答各种氨基酸,在产抗生素放线菌(链霉菌和红色糖多孢菌)中有报道Lrp均可应答不同的氨基酸[25, 27-28],且应答的氨基酸具有多样性,但其具体原因并不清楚。目前关于放线菌Lrp的研究,还有诸多问题尚未得到解答。如氨基酸作为配体或前体,甚至是信号分子,是如何参与抗生素的生物合成?Lrp直接调控的下游靶点是否存在共性,且下游靶点如何调控抗生素的生物合成?Lrp是单独发挥调控功能还是与其他调控子进行协同调控?这些都有待进一步去探究。随着放线菌中Lrp研究的进一步深入,将使人们更加清晰地认识Lrp作用的分子调控机制,也有助于揭示Lrp更多的生物学功能。

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