Enhancement of membrane vesicle production by disrupting the degP gene in Meiothermus ruber H328.
Enhancement of membrane vesicle production by disrupting the degP gene in Meiothermus ruber H328.
复制标题
通过破坏Meiothermus ruber H328中的degP基因来增强膜囊泡的产生。
DOI：
10.1186/s13568-021-01328-z
复制
发表时间：
2021-12-15
期刊：
影响因子：
3.7
通讯作者：
Watanabe K
中科院分区：
文献类型：
--
作者：
Asano Y;Onishi M;Nishi K;Kawasaki K;Watanabe K
众所周知，膜泡（MV）的产生是所有细菌细胞共同的现象。虽然MV有望在各种应用中使用，但提高MV的生产率对应用来说至关重要。由于在大肠杆菌中删除degP基因（一种周质双功能蛋白酶和伴侣蛋白）成功地提高了MV的生产能力，我们试图通过删除degP基因来提高嗜热的M. ruber H328的MV产量。从H328基因组中选择1个基因（mrH_0331）作为degP基因，通过同源重组将H328菌株的degP基因删除，代之以嗜热卡那霉素耐药的htk基因，构建突变株∆degP。突变菌株∆degP在60°C时生长平稳，但浑浊度较低，而野生菌株和突变菌株在55°C时的生长没有差异。最后，我们用DiI和FM4-64两种荧光染料证实，在60°C的孵育下，突变株∆degP的MV增加了约5倍，随后进行了TEM分析。degP基因的缺失可能会导致60°C下变性蛋白的增加，从而导致MV的产生增加。同时，突变体细胞在60℃孵育下制备的MV片段中，H328菌株外膜中包含的s层蛋白增加。这表明该方法对病毒MV的产生是有效的，并且degP的缺失增强了菌株H328的MV的产生。在线版本包含补充材料，可在10.1186/s13568-021-01328-z获得。
The phenomenon of membrane vesicle (MV) production is known to be common to all bacterial cells. Although MVs are expected to be employed in a variety of applications, improving MV productivity is essential for applications. Since the deletion of the degP gene, a periplasmic dual-function protease and chaperone, in Escherichia coli has successfully improved MV production capacity, we tried to enhance MV productivity in the thermophilic M. ruber H328 by deleting the degP gene. One gene (mrH_0331) was selected for degP gene from the H328 genome and we constructed the mutant strain ∆degP by deleting the degP gene of the H328 strain that was replaced with the htk gene showing thermophilic kanamaycin resistance by homologous recombination. The mutant strain ∆degP exhibited smooth growth but a lower level of turbidity at 60 °C although there was no difference in growth at 55 °C between the wild strain and the mutant strain. Finally, we have confirmed that incubation at 60 °C increases MV in the mutant strain ∆degP strain about fivefold by using two fluorescent dyes, DiI and FM4-64, which is followed by TEM analysis. The deletion of the degP gene presumably causes an increase in denatured proteins at 60 °C, leading to enhanced MV production. Meanwhile, the S-layer protein included in the outer membrane of the H328 strain increased in the MV fraction prepared from the mutant cells incubated at 60 °C. This indicates that this method is effective for MV production and that degP deletion enhances it in strain H328. The online version contains supplementary material available at 10.1186/s13568-021-01328-z.
登录
查看更多内容
影响因子：
4.4
作者：
Daleke-Schermerhorn, Maria H.;Felix, Tristan;Luirink, Joen
通讯作者：
Luirink, Joen
DOI：
10.1128/genomea.00176-13
发表时间：
2013-05-02
期刊：
Genome announcements
影响因子：
--
作者：
Inada S;Watanabe K
通讯作者：
Watanabe K
影响因子：
4.4
作者：
Turner, Lorinda;Praszkier, Judyta;Ferrero, Richard L.
通讯作者：
Ferrero, Richard L.
影响因子：
3.5
作者：
Hashimoto, Y;Yano, T;Kagamiyama, H
通讯作者：
Kagamiyama, H
影响因子：
3.2
作者：
MacDonald, Ian A.;Kuehn, Meta J.
通讯作者：
Kuehn, Meta J.