Deletion of degQ gene enhances outer membrane vesicle production of Shewanella oneidensis cells
Deletion of degQ gene enhances outer membrane vesicle production of Shewanella oneidensis cells
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DOI：
10.1007/s00203-016-1315-4
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发表时间：
2017-04
影响因子：
2.8
通讯作者：
Yoshihiro Ojima;Thivagaran Mohanadas;K. Kitamura;Shota Nunogami;Reiki Yajima;M. Taya
中科院分区：
文献类型：
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作者：
Yoshihiro Ojima;Thivagaran Mohanadas;K. Kitamura;Shota Nunogami;Reiki Yajima;M. Taya
希瓦氏菌是一种革兰氏阴性兼性厌氧菌，可利用多种末端电子受体进行厌氧呼吸。在本研究中，S.克隆并表达了oneidensis degQ基因，该基因编码一种假定的周质丝氨酸蛋白酶。纯化的DegQ的活性被典型的丝氨酸蛋白酶特异性抑制剂氟磷酸二异丙酯抑制，表明DegQ是丝氨酸蛋白酶。进行了degQ的框内缺失和随后的互补，以检查包膜应力对外膜囊泡（OMV）产生的影响。结果表明，S.其中一个菌株显示degQ的缺失诱导了蛋白质的积累，并导致周质空间内蛋白酶活性的显著降低。从野生型和突变株的OMV进行纯化，并通过透射电子显微镜观察。OMV的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示在约37 kDa处有一条显著条带。纳米液相色谱-串联质谱分析鉴定了3个外膜孔蛋白（SO 3896、SO 1821和SO 3545）作为条带的主要组分，表明这些蛋白可用作比较S.一种密度定量评估显示degQ缺陷细胞与野生型细胞相比，OMV产量增加了5倍。因此，DegQ缺失后OMV产量的增加可能是造成膜胁迫增加的原因。
Shewanella oneidensisis a Gram-negative facultative anaerobe that can use a wide variety of terminal electron acceptors for anaerobic respiration. In this study,S. oneidensis degQgene, encoding a putative periplasmic serine protease, was cloned and expressed. The activity of purified DegQ was inhibited by diisopropyl fluorophosphate, a typical serine protease-specific inhibitor, indicating that DegQ is a serine protease. In-frame deletion and subsequent complementation of thedegQwere carried out to examine the effect of envelope stress on the production of outer membrane vesicles (OMVs). Analysis of periplasmic proteins from the resultingS. oneidensisstrain showed that deletion ofdegQinduced protein accumulation and resulted in a significant decrease in protease activity within the periplasmic space. OMVs from the wild-type and mutant strains were purified and observed by transmission electron microscopy. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the OMVs showed a prominent band at ~37 kDa. Nanoliquid chromatography–tandem mass spectrometry analysis identified three outer membrane porins (SO3896, SO1821, and SO3545) as dominant components of the band, suggesting that these proteins could be used as indices for comparing OMV production byS. oneidensisstrains. Quantitative evaluation showed thatdegQ-deficient cells had a fivefold increase in OMV production compared with wild-type cells. Thus, the increased OMV production following the deletion of DegQ inS. oneidensismay be responsible for the increase in envelope stress.