摘要
微生物在酸胁迫下的耐受能力对于菌株的生长和工业化生产均具有重要意义。当细菌细胞面临外界酸性环境压力时,周质空间蛋白质相对胞内蛋白受到更大酸性压力,所受酸性伤害也比胞内蛋白质更为严重。在革兰阴性菌耐酸过程中,除了胞内的脱羧酶系统外,分子伴侣作为一种重要的“纠错”机制可参与识别并保护蛋白质的空间结构。本文介绍了HdeA、HdeB、DnaK和GroEL等分子伴侣在功能、结构、耐酸机制以及应用前景等方面的研究现状,阐述了分子伴侣介导的耐酸机制的调控方式。深入探究和解析分子伴侣对酸胁迫环境的生理适应策略,以期利用这些研究对目的菌株进行生理性能改造,提高菌株在酸性胁迫环境下的存活能力和耐受性,对工业化发酵、临床抗菌治疗靶点的发现等具有重要意义。
自然界中微生物生长或工业生产过程中会遇到各种外界环境压力,如高
革兰阴性菌为双层膜结构,外层膜上具有非专一性通道蛋白,允许小分子物质进出,其中氢离子可在相关通道蛋白进入周质空间时,对菌体造成酸胁迫,导致周质空间重要代谢相关蛋白质错误折叠,影响其表
周质空间(periplasmic space),又称周质(periplasm)或壁膜间隙,是革兰阴性菌细胞膜与外膜之间的间隔区域。周质空间蛋白主要分为4类:第一类为水解酶类,例如蛋白酶、核酸酶等;第二类为合成酶类,例如肽聚糖合成酶;第三类为结合蛋白,具有运输营养物质的作用;最后一类为底物蛋白,主要与细胞的趋化性有
多数蛋白质是边翻译边折叠过程,需要相关酶以及分子伴侣的参与。细胞中某些蛋白质分子可以识别正在合成的多肽链或部分折叠的多肽并将这些蛋白质转运至相应功能部位,从而帮助多肽转运、折叠和装配,这类分子本身并不参与最终产物的形成,被称为分子伴
Hsp70是一类从古细菌、植物到人类几乎所有生物体中均有发现且进化最为保守的热休克蛋白质。Hsp70由45 kD N-末端核苷酸结合结构域 (nucleotide binding domain, NBD)和25 kD 的C-末端底物结合结构域 (substrate binding domain,SBD)组
研究表明,HdeA和HdeB是调节细菌细胞在酸性环境下生理活性重要的分子伴侣蛋白。HdeA和HdeB序列相似度较低,但它们的单体结构具有较高的相似性,核心结构均由4个α-螺旋包裹形成疏水
尽管蛋白质的空间结构与其氨基酸序列相关,但细胞在面对外界压力如高温、强离子浓度以及强酸碱度等极端环境时,胞内新生肽链经常不能正确折叠,倾向于形成非天然状态下的空间结构,这些异常空间结构会暴露出内部疏水区,发生蛋白质的聚集,影响细胞正常功能,甚至造成细胞死亡。因此,细菌细胞需要利用胞内相关“纠错”系统,应对外界环境压力的影响,而针对于酸性环境的压力的过程中,位于周质空间区域的蛋白质最易受到pH变化的影响,故其空间中分子伴侣这套“纠错”系统产生了重要的作用,不同的分子伴侣耐酸机制有异同(

图1 分子伴侣耐酸机制
在大肠埃希菌及布氏杆菌Brucella abortus中敲除HdeA/B基因可明显降低其在酸性环境下存活率,回补HdeA或HdeB基因后,则可增加菌株存活
在酸性环境下,周质空间蛋白DegP和SurA是HdeA的主要底物蛋白;在中性环境下,DegP和SurA可帮助受HdeA蛋白保护的底物蛋白重新折叠并恢复其活性,故称DegP和SurA为保护伴侣蛋白的伴侣蛋白(chaperone-protecting-chaperone),是革兰阴性菌重要的耐酸机制之
GroEL是一种由14个亚基组成的同型寡聚复合体,呈双环圆桶状结构。3个主要功能域:含待折叠蛋白结合位点以及GroES结合位点的顶部结构域、中间结构域以及ATP结合位点的赤道结构域。GroES充当桶顶部的圆盖,可以和GroEL分开。待折叠肽链进入圆筒内部以后,顶盖合上,肽链在内部与桶壁发生多次可逆结合,每一次结合由ATP水解所驱动,在最终折叠成特定的三维结构之前可能会消耗大量的AT
研究显示,革兰阴性菌可通过不同热休克基因表达,使其在动态环境中耐受应激环境,尤其是酸性环
DnaK是一种ATP依赖的分子伴侣蛋白,可参与酸性环境刺激下大肠埃希菌中蛋白质的保护过程。在细菌中DnaK属于热休克蛋白70家族,具有NBD以及SBD两结构域。ATP存在情况下,DnaK与ATP紧密结合形成单聚体,无ATP时DnaK以无序低聚物存在。DnaK是大肠埃希菌中重要的应激型分子伴侣,其转录和蛋白表达水平在酸胁迫的细菌细胞中均会提高。在外界酸性环境刺激下,它可与GroEL分子伴侣相互协作,帮助蛋白质正确折叠以及修复损伤蛋
微生物在适应外界酸性环境过程中,进化出多种应对机制来回应酸性环境信号。近年来提高微生物酸胁迫的耐受性成为人们研究热点,主要途径包括全局调控工程、过表达微生物耐酸相关热休克蛋白、人工诱变或基因组改组等以提高微生物对酸的耐受
全局转录调控工程(global transcription machinery engineering,gTME)是通过改造和进化全局转录因子、转录机器等关键蛋白,构建高度多样、复杂的转录调控突变文库,在转录水平上产生新型的丰富多样性,实现对基因表达网络和细胞代谢重程,并以定向进化方式加以迭代,从而使细胞获得所期望的表型。针对细胞酸胁迫的复杂性,可利用gTME从转录全局角度来调控周质空间伴侣分子的活性而抵耐酸胁
ABC转运体(ATP-binding cassette transporter)是所有转运体家族中最大的一类转运体, 广泛分布于从原核生物到人类几乎所有的物种
在革兰阴性菌中还存在调控其他压力反应的基因,通过调节这些基因的活性也可以改变菌体对酸的耐受。RpoS是一般酸胁迫反应的主要调控因子,可调控特异性耐酸基因的表达,感知氧化应激、高温、高压以及酸性环境压
中科院天津工业生物技术研究所刘君课题组通过适应性进化策略结合转录组测序,在谷氨酸棒杆菌C.glutamicum ATCC 13032中,通过连续70 d的酸性环境刺激进化,筛选得到耐酸菌株。通过比较分析进化菌株与野生型菌株细胞结构、生长曲线、活性氧水平以及转录组表达水平,结果显示适应性进化策略可使得菌株更好保持酸胁迫环境下胞内完整性,维持了更高的胞内pH及较低的活性氧水平(较野生型降低61%),酸胁迫下较野生型菌株存活率提高39%,此外,研究中还发现和鉴定出多个候选抗酸元件,如铜伴侣蛋白Cg1328、细胞分裂蛋白FtsE/X、脂肪酸合成酶Fas、分子伴侣HscA等。进一步研究揭示,过氧化氢酶-饥饿诱导DNA保护蛋白(KatA-Dps)介导的胞内活性氧清除过程和硫代谢调控因子(McbR)介导的硫元素同化抑制效应能够协同参与谷氨酸棒杆菌的低酸胁迫耐受应答,是影响谷氨酸棒杆菌抗酸生理性能的重要因素,有助于更好理解菌株酸胁迫下生理适应策略,为耐酸菌株的开发提供了一种全新的思
细菌细胞在各种环境压力下,通过长期进化,形成多种应对机制来应对各种压力环境。其中酸性环境下细菌细胞的耐酸机制是令人关注的研究方向之一。目前已知多种氨基酸脱羧酶系统(AR2-AR5)、细胞间质空间分子伴侣的作用(HdeA、HdeB),此外还有多种关键基因的表达可影响细菌细胞内耐酸机制(H-NS、ABC transporter、RcsF、RcsC等),这些机制的发现对于无论是工业化发酵过程,还是临床抗菌治疗靶点的发现均具有重要作用,也对蛋白质间相互作用的检测提供了理论基础。因此,深入探究和解析分子伴侣对低酸胁迫环境的生理适应策略,以期利用这些知识对目的菌株进行生理性能改造,提高菌株在酸性胁迫环境下的存活能力和耐受性,从而充分发挥其应用价值,具有重要理论和现实指导意义。
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