Transfer and analysis of Salmonella pdu genes in a range of Gram-negative bacteria demonstrate exogenous microcompartment expression across a variety of species
Transfer and analysis of Salmonella pdu genes in a range of Gram-negative bacteria demonstrate exogenous microcompartment expression across a variety of species
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DOI：
10.1111/1751-7915.12863
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发表时间：
2018-01-01
影响因子：
5.7
通讯作者：
Wilson, James W.
中科院分区：
文献类型：
--
作者：
Graf, Laura;Wu, Kent;Wilson, James W.
细菌微室（MCPs）是蛋白质细胞器，通常容纳参与代谢途径的毒性或挥发性反应中间体。工程细菌表达外源性MCPs将使这些细胞获得包括分子区隔化在内的有用功能。我们从肠炎沙门氏菌血清型鼠伤寒沙门氏菌基因组中克隆了一个38kb的区域，其中包含pdu 1,2丙二醇（1,2 PD）利用和cob/cbi基因，使用FRT-Capture策略克隆和转移大的基因组片段。我们将该克隆转移到一系列革兰氏阴性菌中，发现该克隆在多种物种（包括鼠伤寒沙门氏菌、大肠杆菌、邦戈里沙门氏菌、肺炎克雷伯菌、阪崎克罗诺杆菌、粘质沙雷氏菌和不同的假单胞菌）中具有1,2 PD代谢功能。我们成功地从这些菌株的几个（但不是全部）克隆中分离出表达的MCPs，我们利用一系列不同的培养基和在没有蛋白酶抑制剂的情况下观察到这一点。我们还提出了一种小型准备方案，允许快速，小规模筛选菌株用于MCP生产。迄今为止，这是首次对几种不同革兰氏阴性背景下克隆的外源性微室表达进行分析，并为使用完整的克隆在各种细菌物种中使用MCP提供了基础。
Bacterial microcompartments (MCPs) are protein organelles that typically house toxic or volatile reaction intermediates involved in metabolic pathways. Engineering bacteria to express exogenous MCPs will allow these cells to gain useful functions involving molecule compartmentalization. We cloned a 38kb region from the Salmonella enterica serovar Typhimurium genome containing the pdu 1,2 propanediol (1,2 PD) utilization and cob/cbi genes using the FRT-Capture strategy to clone and transfer large genomic segments. We transferred this clone to a range of Gram-negative bacteria and found the clone to be functional for 1,2 PD metabolism in a variety of species including S. Typhimurium pdu, Escherichia coli, Salmonella bongori, Klebsiella pneumoniae, Cronobacter sakazakii, Serratia marcescens, and different Pseudomonas species. We successfully isolated MCPs expressed from the clone from several, but not all, of these strains, and we observed this utilizing a range of different media and in the absence of protease inhibitor. We also present a mini-prep protocol that allows rapid, small-scale screening of strains for MCP production. To date, this is the first analysis of cloned, exogenous microcompartment expression across several different Gram-negative backgrounds and provides a foundation for MCP use in a variety of bacterial species using a full, intact clone.