Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA
mBio. 2019 Feb 12;10(1):e02273-18. doi: 10.1128/mBio.02273-18.
Due to their intimate physical interactions with the environment, surface polysaccharides are critical determinants of fitness for bacteria. produces a specialized structure at one of its cell poles called the holdfast that enables attachment to surfaces. Previous studies have shown that the holdfast is composed of carbohydrate-based material and identified a number of genes required for holdfast development. However, incomplete information about its chemical structure, biosynthetic genes, and regulatory principles has limited progress in understanding the mechanism of holdfast synthesis. We leveraged the adhesive properties of the holdfast to perform a saturating screen for genes affecting attachment to cheesecloth over a multiday time course. Using similarities in the temporal profiles of mutants in a transposon library, we defined discrete clusters of genes with related effects on cheesecloth colonization. Holdfast synthesis, flagellar motility, type IV pilus assembly, and smooth lipopolysaccharide (SLPS) production represented key classes of adhesion determinants. Examining these clusters in detail allowed us to predict and experimentally define the functions of multiple uncharacterized genes in both the holdfast and SLPS pathways. In addition, we showed that the pilus and the flagellum control holdfast synthesis separately by modulating the holdfast inhibitor This report defines a set of genes contributing to adhesion that includes newly discovered genes required for holdfast biosynthesis and attachment. Our data provide evidence that the holdfast contains a complex polysaccharide with at least four monosaccharides in the repeating unit and underscore the central role of cell polarity in mediating attachment of to surfaces. Bacteria routinely encounter biotic and abiotic materials in their surrounding environments, and they often enlist specific behavioral programs to colonize these materials. Adhesion is an early step in colonizing a surface. produces a structure called the holdfast which allows this organism to attach to and colonize surfaces. To understand how the holdfast is produced, we performed a genome-wide search for genes that contribute to adhesion by selecting for mutants that could not attach to cheesecloth. We discovered complex interactions between genes that mediate surface contact and genes that contribute to holdfast development. Our genetic selection identified what likely represents a comprehensive set of genes required to generate a holdfast, laying the groundwork for a detailed characterization of the enzymes that build this specialized adhesin.
由于其与环境的密切物理相互作用，表面多糖是细菌适应能力的关键决定因素。产生一种特殊的结构，称为固着器，位于其细胞的一个极上，使它能够附着在表面上。以前的研究表明，固着器由基于碳水化合物的物质组成，并确定了一些固着器发育所必需的基因。然而，关于其化学结构、生物合成基因和调节原则的不完全信息限制了对固着合成机制的理解。我们利用固着器的粘附特性，在多天的时间过程中，对影响奶酪布附着的基因进行了饱和筛选。利用转座子文库中突变体的时间曲线相似性，我们定义了具有相关奶酪布定殖效应的离散基因簇。固着合成、鞭毛运动、IV 型菌毛组装和光滑脂多糖 (SLPS) 产生代表了主要的粘附决定因素类别。详细研究这些簇使我们能够预测和实验确定固着器和 SLPS 途径中多个未被表征基因的功能。此外，我们还表明，通过调节固着抑制剂，菌毛和鞭毛分别控制固着器的合成。本报告定义了一组有助于粘附的基因，包括新发现的固着生物合成和附着所需的基因。我们的数据提供了证据表明，固着器含有一种复杂的多糖，在重复单元中至少含有四种单糖，并强调了细胞极性在介导对表面的附着中的核心作用。细菌在其周围环境中经常遇到生物和非生物物质，它们经常利用特定的行为程序来定殖这些物质。粘附是定殖表面的第一步。产生一种称为固着器的结构，使这种生物能够附着和定殖表面。为了了解固着器是如何产生的，我们通过选择不能附着在奶酪布上的突变体来进行了一个全基因组搜索，以寻找有助于粘附的基因。我们发现了介导表面接触的基因和有助于固着器发育的基因之间的复杂相互作用。我们的遗传选择确定了可能代表产生固着器所需的一组全面基因，为详细表征构建这种特殊黏附素的酶奠定了基础。
