Frontier Science Center for Synthetic Biology (Ministry of Education), Key Laboratory of Systems Bioengineering, Tianjin University, Tianjin 300072, China.
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China.
ACS Synth Biol. 2023 Jun 16;12(6):1645-1656. doi: 10.1021/acssynbio.2c00636. Epub 2023 May 4.
Outer membrane vesicles (OMVs) of Gram-negative bacteria play an essential role in cellular physiology. The underlying regulatory mechanism of OMV formation and its impact on extracellular electron transfer (EET) in the model exoelectrogen MR-1 remain unclear and have not been reported. To explore the regulatory mechanism of OMV formation, we used the CRISPR-dCas9 gene repression technology to reduce the crosslink between the peptidoglycan (PG) layer and the outer membrane, thus promoting the OMV formation. We screened the target genes that were potentially beneficial to the outer membrane bulge, which were classified into two modules: PG integrity module (Module 1) and outer membrane component module (Module 2). We found that downregulation of the penicillin-binding protein-encoding gene for peptidoglycan integrity (Module 1) and the -acetyl-d-mannosamine dehydrogenase-encoding gene involved in lipopolysaccharide synthesis (Module 2) exhibited the highest production of OMVs and enabled the highest output power density of 331.3 ± 1.2 and 363.8 ± 9.9 mW m, 6.33- and 6.96-fold higher than that of the wild-type MR-1 (52.3 ± 0.6 mW m), respectively. To elucidate the specific impacts of OMV formation on EET, OMVs were isolated and quantified for UV-visible spectroscopy and heme staining characterization. Our study showed that abundant outer membrane -type cytochromes (-Cyts) including MtrC and OmcA and periplasmic -Cyts were exposed on the surface or inside of OMVs, which were the vital constituents responsible for EET. Meanwhile, we found that the overproduction of OMVs could facilitate biofilm formation and increase biofilm conductivity. To the best of our knowledge, this study is the first to explore the mechanism of OMV formation and its correlation with EET of , which paves the way for further study of OMV-mediated EET.
革兰氏阴性细菌的外膜囊泡（OMVs）在细胞生理学中起着至关重要的作用。然而，OMV 形成的潜在调节机制及其对模型外泌电子供体 MR-1 中外电子转移（EET）的影响尚不清楚，也尚未有相关报道。为了探索 OMV 形成的调节机制，我们使用 CRISPR-dCas9 基因抑制技术减少肽聚糖（PG）层与外膜之间的交联，从而促进 OMV 的形成。我们筛选了可能有利于外膜膨出的目标基因，这些基因被分为两个模块：PG 完整性模块（模块 1）和外膜成分模块（模块 2）。我们发现，下调与肽聚糖完整性相关的青霉素结合蛋白编码基因（模块 1）和参与脂多糖合成的 N-乙酰-d-甘露糖胺脱氢酶编码基因（模块 2），可产生最高产量的 OMVs，并使最大功率密度分别达到 331.3 ± 1.2 和 363.8 ± 9.9 mW m，比野生型 MR-1（52.3 ± 0.6 mW m）分别高出 6.33 倍和 6.96 倍。为了阐明 OMV 形成对 EET 的具体影响，我们对 OMV 进行了分离和定量，并用紫外可见光谱和血红素染色进行了表征。我们的研究表明，丰富的外膜型细胞色素（-Cyts），包括 MtrC 和 OmcA 以及周质 -Cyts，暴露在 OMV 的表面或内部，这是负责 EET 的重要组成部分。同时，我们发现 OMV 的过度产生可以促进生物膜的形成并增加生物膜的导电性。据我们所知，这项研究首次探索了 OMV 形成的机制及其与 的 EET 之间的相关性，为进一步研究 OMV 介导的 EET 铺平了道路。
