Department of Biology, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA.
Weitzman School of Design, University of Pennsylvaniagrid.25879.31, Philadelphia, Pennsylvania, USA.
Appl Environ Microbiol. 2022 Feb 22;88(4):e0228321. doi: 10.1128/aem.02283-21. Epub 2022 Jan 5.
Most microorganisms exist in biofilms, which comprise aggregates of cells surrounded by an extracellular matrix that provides protection from external stresses. Based on the conditions under which they form, biofilm structures vary in significant ways. For instance, biofilms that develop when microbes are incubated under static conditions differ from those formed when microbes encounter the shear forces of a flowing liquid. Moreover, biofilms develop dynamically over time. Here, we describe a cost-effective coverslip holder, printed with a three-dimensional (3D) printer, that facilitates surface adhesion assays under a broad range of standing and shaking culture conditions. This ultianel hesion (mPAD) mount further allows cultures to be sampled at multiple time points, ensuring consistency and comparability between samples and enabling analyses of the dynamics of biofilm formation. As a proof of principle, using the mPAD mount for shaking, oxic cultures, we confirm previous flow chamber experiments showing that the Pseudomonas aeruginosa wild-type strain and a phenazine deletion mutant (Δ) strain form biofilms with similar structure but reduced density in the mutant strain. Extending this analysis to anoxic conditions, we reveal that microcolony formation and biofilm formation can only be observed under shaking conditions and are decreased in the Δ mutant compared to wild-type cultures, indicating that phenazines are crucial for the formation of biofilms if oxygen as an electron acceptor is unavailable. Furthermore, while the model archaeon Haloferax volcanii does not require archaella for surface attachment under static conditions, we demonstrate that an H. volcanii mutant that lacks archaella is impaired in early stages of biofilm formation under shaking conditions. Due to the versatility of the mPAD mount, we anticipate that it will aid the analysis of biofilm formation in a broad range of bacteria and archaea. Thereby, it contributes to answering critical biological questions about the regulatory and structural components of biofilm formation and understanding this process in a wide array of environmental, biotechnological, and medical contexts.
大多数微生物存在于生物膜中,生物膜由细胞聚集物组成,这些细胞聚集物被细胞外基质包围,从而为其提供了免受外部压力的保护。基于它们形成的条件,生物膜结构在很大程度上有所不同。例如,在微生物处于静态条件下培养时形成的生物膜与在微生物遇到流动液体的剪切力时形成的生物膜不同。此外,生物膜会随着时间的推移而动态发展。在这里,我们描述了一种具有成本效益的盖玻片支架,该支架由 3D 打印机打印而成,可在广泛的静置和摇动培养条件下促进表面粘附测定。这种超粘连(mPAD)支架进一步允许在多个时间点对培养物进行采样,确保样品之间的一致性和可比性,并能够分析生物膜形成的动态。作为原理验证,我们使用 mPAD 支架进行摇床、需氧培养,证实了先前的流动室实验结果,即铜绿假单胞菌野生型菌株和吩嗪缺失突变体(Δ)菌株形成的生物膜具有相似的结构,但在突变菌株中密度降低。将这一分析扩展到缺氧条件下,我们发现只有在摇动条件下才能观察到微菌落形成和生物膜形成,并且与野生型培养物相比,在 Δ 突变体中减少了,这表明如果没有作为电子受体的氧气,吩嗪对生物膜的形成至关重要。此外,虽然模式古菌盐沼盐杆菌在静态条件下不需要菌毛进行表面附着,但我们证明,缺乏菌毛的 H. volcanii 突变体在摇动条件下的生物膜形成早期阶段受到损害。由于 mPAD 支架的多功能性,我们预计它将有助于分析广泛的细菌和古菌的生物膜形成。从而有助于回答关于生物膜形成的调节和结构成分的关键生物学问题,并在广泛的环境、生物技术和医学背景下理解这一过程。