Genetic Engineering of Bee Gut Microbiome Bacteria with a Toolkit for Modular Assembly of Broad-Host-Range Plasmids

Engineering the bacteria
present in animal microbiomes promises
to lead to breakthroughs in medicine and agriculture, but progress
is hampered by a dearth of tools for genetically modifying the diverse
species that comprise these communities. Here we present a toolkit
of genetic parts for the modular construction of broad-host-range
plasmids built around the RSF1010 replicon. Golden Gate assembly of
parts in this toolkit can be used to rapidly test various antibiotic
resistance markers, promoters, fluorescent reporters, and other coding
sequences in newly isolated bacteria. We demonstrate the utility of
this toolkit in multiple species of Proteobacteria that are native
to the gut microbiomes of honey bees (Apis mellifera) and bumble bees (Bombus sp.). Expressing fluorescent
proteins in Snodgrassella alvi, Gilliamella apicola, Bartonella
apis, and Serratia strains enables us to
visualize how these bacteria colonize the bee gut. We also demonstrate
CRISPRi repression in B. apis and use Cas9-facilitated
knockout of an S. alvi adhesion gene to show that
it is important for colonization of the gut. Beyond characterizing
how the gut microbiome influences the health of these prominent pollinators,
this bee microbiome toolkit (BTK) will be useful for engineering bacteria
found in other natural microbial communities.

对动物微生物组内的细菌进行工程化改造，有望在医药与农业领域取得突破性进展，但当前研究进展受限于可用于遗传改造这类群落中多样物种的工具匮乏。本研究开发了一套基于RSF1010复制子（RSF1010 replicon）构建的遗传元件工具包，可用于模块化构建广谱宿主范围质粒。借助该工具包中的元件进行Golden Gate组装，可快速在新分离的细菌中测试多种抗生素抗性标记、启动子、荧光报告基因及其他编码序列。我们在多种源自蜜蜂（Apis mellifera）与熊蜂（Bombus sp.）肠道微生物组的变形菌门（Proteobacteria）物种中验证了该工具包的实用性。在Snodgrassella alvi、Gilliamella apicola、Bartonella apis及Serratia菌株中表达荧光蛋白，可帮助我们直观观察这些细菌如何定殖于蜂类肠道。我们还实现了B. apis中的CRISPRi基因沉默，并借助Cas9介导的S. alvi黏附基因敲除实验，证实该基因对肠道定殖至关重要。除了用于解析肠道微生物组如何影响这类重要传粉昆虫的健康之外，这套蜜蜂微生物组工具包（bee microbiome toolkit, BTK）也可用于对其他自然微生物群落中的细菌进行工程化改造。