In Vivo Titration of Folate Pathway Enzymes.
In Vivo Titration of Folate Pathway Enzymes.
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叶酸途径酶的体内滴定。
DOI：
10.1128/aem.01139-18
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发表时间：
2018
影响因子：
4.4
通讯作者：
Howell,ElizabethE
中科院分区：
文献类型：
--
作者：
Nambiar,Deepika;Berhane,Timkhite-Kulu;Shew,Robert;Schwarz,Bryan;DuffJr,MichaelR;Howell,ElizabethE
酶在细胞中的行为可能与它们在试管中的行为不同。以前的体外研究发现，渗透剂与叶酸的相互作用很弱。从叶酸的溶剂化壳中除去渗透剂比除去水更困难，这削弱了叶酸与其酶伴侣的结合。为了检查这种现象是否发生在体内，用大肠杆菌进行渗透压应力滴定。采用了两种策略：对抗菌药物的抗性和通过克隆到质粒中的适当基因对敲除菌株的互补，所述质粒允许严格控制表达水平以及通过降解标签进行标记。比较了敲除菌株和补充菌株在渗透胁迫下的生长能力。通常，敲除菌株可以在补充培养基上生长至高渗透压，而补充菌株在基本培养基上在较低渗透压下停止生长。在拯救Δ folA菌株的R67二氢叶酸还原酶克隆、拯救Δ metF菌株的亚甲基四氢叶酸还原酶克隆和拯救Δ glyA菌株的丝氨酸羟甲基转移酶克隆中观察到这种模式。此外，R67二氢叶酸还原酶克隆允许E.大肠杆菌DH 5 α在甲氧苄啶的存在下生长，直到达到0.81的渗透压，而在缺乏抗生素的对照滴定中，细胞可以生长到1.90渗透压。大肠杆菌可以在干旱和洪水条件下存活，并且可以耐受渗透压的大的变化。然而，在高渗透胁迫条件下限制细菌生长的细胞过程尚不清楚。在本研究中，四种不同的酶在E.大肠杆菌中，通过使用由可调质粒中携带的基因互补的缺失菌株来降低。在限制酶浓度的条件下（低于通过染色体基因表达实现的浓度），细胞生长可以被通常耐受的渗透胁迫条件阻断。这些结果表明，E.大肠杆菌已经进化到能够应对其渗透环境的变化，正常的蛋白质水平足以缓冲细胞免受环境变化的影响。渗透压响应中涉及的其他因素可能包括改变的蛋白质浓度/活性水平、与配体的弱溶质相互作用（这可能使蛋白质更难以在体内结合其底物/抑制剂/辅因子）和/或粘度效应。
How enzymes behave in cells is likely different from how they behave in the test tube. Previousin vitrostudies find that osmolytes interact weakly with folate. Removal of the osmolyte from the solvation shell of folate is more difficult than removal of water, which weakens binding of folate to its enzyme partners. To examine if this phenomenon occursin vivo, osmotic stress titrations were performed with Escherichia coli. Two strategies were employed: resistance to an antibacterial drug and complementation of a knockout strain by the appropriate gene cloned into a plasmid that allows tight control of expression levels as well as labeling by a degradation tag. The abilities of the knockout and complemented strains to grow under osmotic stress were compared. Typically, the knockout strain could grow to high osmolalities on supplemented medium, while the complemented strain stopped growing at lower osmolalities on minimal medium. This pattern was observed for an R67 dihydrofolate reductase clone rescuing a ΔfolAstrain, for a methylenetetrahydrofolate reductase clone rescuing a ΔmetFstrain, and for a serine hydroxymethyltransferase clone rescuing a ΔglyAstrain. Additionally, an R67 dihydrofolate reductase clone allowed E. coli DH5α to grow in the presence of trimethoprim until an osmolality of ∼0.81 is reached, while cells in a control titration lacking antibiotic could grow to 1.90 osmol.IMPORTANCEE. coli can survive in drought and flooding conditions and can tolerate large changes in osmolality. However, the cell processes that limit bacterial growth under high osmotic stress conditions are not known. In this study, the dose of four different enzymes in E. coli was decreased by using deletion strains complemented by the gene carried in a tunable plasmid. Under conditions of limiting enzyme concentration (lower than that achieved by chromosomal gene expression), cell growth can be blocked by osmotic stress conditions that are normally tolerated. These observations indicate that E. coli has evolved to deal with variations in its osmotic environment and that normal protein levels are sufficient to buffer the cell from environmental changes. Additional factors involved in the osmotic pressure response may include altered protein concentration/activity levels, weak solute interactions with ligands which can make it more difficult for proteins to bind their substrates/inhibitors/cofactorsin vivo, and/or viscosity effects.