Ojima Yoshihiro, Mohanadas Thivagaran, Kitamura Kosei, Nunogami Shota, Yajima Reiki, Taya Masahito
Division of Chemical Engineering, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama-cho, Toyonaka, Osaka, 560-8531, Japan.
Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka, 558-8585, Japan.
Arch Microbiol. 2017 Apr;199(3):415-423. doi: 10.1007/s00203-016-1315-4. Epub 2016 Oct 31.
Shewanella oneidensis is a Gram-negative facultative anaerobe that can use a wide variety of terminal electron acceptors for anaerobic respiration. In this study, S. oneidensis degQ gene, 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 the degQ were carried out to examine the effect of envelope stress on the production of outer membrane vesicles (OMVs). Analysis of periplasmic proteins from the resulting S. oneidensis strain showed that deletion of degQ induced 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 by S. oneidensis strains. Quantitative evaluation showed that degQ-deficient cells had a fivefold increase in OMV production compared with wild-type cells. Thus, the increased OMV production following the deletion of DegQ in S. oneidensis may be responsible for the increase in envelope stress.
希瓦氏菌是一种革兰氏阴性兼性厌氧菌，能够利用多种末端电子受体进行厌氧呼吸。在本研究中，编码假定周质丝氨酸蛋白酶的希瓦氏菌degQ基因被克隆并表达。纯化后的DegQ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，这表明DegQ是一种丝氨酸蛋白酶。通过对degQ进行框内缺失及后续互补操作，以研究包膜应激对外膜囊泡（OMV）产生的影响。对所得希瓦氏菌菌株的周质蛋白进行分析，结果显示degQ缺失会诱导蛋白积累，并导致周质空间内的蛋白酶活性显著降低。对野生型和突变型菌株的OMV进行纯化，并通过透射电子显微镜观察。对OMV进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析，结果显示在约37 kDa处有一条明显的条带。纳升液相色谱-串联质谱分析确定三种外膜孔蛋白（SO3896、SO1821和SO3545）为该条带的主要成分，这表明这些蛋白可作为比较希瓦氏菌菌株OMV产生情况的指标。定量评估表明，与野生型细胞相比，degQ缺陷型细胞的OMV产量增加了五倍。因此，希瓦氏菌中DegQ缺失后OMV产量的增加可能是包膜应激增加的原因。
