Laboratory of Radioactive Isotopes, G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, FRC Pushchino Center for Biological Research of Russian Academy of Sciences, Pushchino, Russia, 142290.
Antonie Van Leeuwenhoek. 2020 Jan;113(1):101-116. doi: 10.1007/s10482-019-01320-5. Epub 2019 Aug 28.
The genome of methylotrophic bacteria Methylorubrum extorquens DM4 contains two homologous groESL operons encoding the 60-kDa and 10-kDa subunits of GroE heat shock chaperones with highly similar amino acid sequences. To test a possible functional redundancy of corresponding GroEL proteins we attempted to disrupt the groEL1 and groEL2 genes. Despite the large number of recombinants analysed and the gentle culture conditions the groEL1-lacking mutant was not constructed suggesting that the loss of GroEL1 was lethal for cells. At the same time the ∆groEL2 strain was viable and varied from the wild-type by increased sensitivity to acid, salt and desiccation stresses as well as by the impaired growth with a toxic halogenated compound-dichloromethane (DCM). The evaluation of activity of putative P and P promoters using the reporter gene of green fluorescent protein (GFP) showed that the expression of groESL1 operon greatly prevails (about two orders of magnitude) over those of groESL2 under all tested conditions. However the above promoters demonstrated differential regulation in response to stresses. The expression from P was heat-inducible, while the activity of P was upregulated upon acid shock and cultivation with DCM. Based on these results we conclude that the highly conservative groESL1 operon (old locus tags METDI5839-5840) encodes the housekeeping chaperone essential for fundamental cellular processes. On the contrary the second pair of paralogues (METDI4129-4130) is dispensable, but corresponding GroE2 chaperone promotes the tolerance to acid and salt stresses, in particular, during the growth with DCM.
甲基营养菌 Methylorubrum extorquens DM4 的基因组包含两个同源的 groESL 操纵子，分别编码 GroE 热休克伴侣的 60kDa 和 10kDa 亚基，它们具有高度相似的氨基酸序列。为了测试相应 GroEL 蛋白的可能功能冗余性，我们试图敲除 groEL1 和 groEL2 基因。尽管分析了大量的重组子，并采用了温和的培养条件，但仍然无法构建 groEL1 缺失突变体，这表明 GroEL1 的缺失对细胞是致命的。与此同时，ΔgroEL2 菌株是存活的，与野生型相比，其对酸、盐和干燥胁迫的敏感性增加，以及对有毒卤代化合物二氯甲烷（DCM）的生长能力受损。使用绿色荧光蛋白（GFP）的报告基因评估假定的 P 和 P 启动子的活性表明，在所有测试条件下，groESL1 操纵子的表达大大超过 groESL2 的表达（约两个数量级）。然而，这些启动子表现出对不同胁迫的差异调节。P 的表达受热诱导，而 P 的活性在酸冲击和用 DCM 培养时上调。基于这些结果，我们得出结论，高度保守的 groESL1 操纵子（旧的基因标签 METDI5839-5840）编码对基本细胞过程至关重要的管家伴侣。相反，第二对同源基因（METDI4129-4130）是可有可无的，但相应的 GroE2 伴侣有助于耐受酸和盐胁迫，特别是在生长过程中使用 DCM。
