Hook Andrew L, Flewellen James L, Dubern Jean-Frédéric, Carabelli Alessandro M, Zaid Irwin M, Berry Richard M, Wildman Ricky D, Russell Noah, Williams Paul, Alexander Morgan R
Advanced Materials and Healthcare Technologies Division, School of Pharmacy, University of Nottingham, Nottingham, United Kingdom.
Immune Receptor Activation Laboratory, The Francis Crick Institute, London, United Kingdom.
mSystems. 2019 Sep 24;4(5):e00390-19. doi: 10.1128/mSystems.00390-19.
Bacteria sense chemicals, surfaces, and other cells and move toward some and away from others. Studying how single bacterial cells in a population move requires sophisticated tracking and imaging techniques. We have established quantitative methodology for label-free imaging and tracking of individual bacterial cells simultaneously within the bulk liquid and at solid-liquid interfaces by utilizing the imaging modes of digital holographic microscopy (DHM) in three dimensions (3D), differential interference contrast (DIC), and total internal reflectance microscopy (TIRM) in two dimensions (2D) combined with analysis protocols employing bespoke software. To exemplify and validate this methodology, we investigated the swimming behavior of a wild-type strain and isogenic flagellar stator mutants ( and ) within the bulk liquid and at the surface at the single-cell and population levels. Multiple motile behaviors were observed that could be differentiated by speed and directionality. Both stator mutants swam slower and were unable to adjust to the near-surface environment as effectively as the wild type, highlighting differential roles for the stators in adapting to near-surface environments. A significant reduction in run speed was observed for the mutants, which decreased further on entering the near-surface environment. These results are consistent with the stators playing key roles in responding to the near-surface environment. We have established a methodology to enable the movement of individual bacterial cells to be followed within a 3D space without requiring any labeling. Such an approach is important to observe and understand how bacteria interact with surfaces and form biofilm. We investigated the swimming behavior of , which has two flagellar stators that drive its swimming motion. Mutants that had only either one of the two stators swam slower and were unable to adjust to the near-surface environment as effectively as the wild type. These results are consistent with the stators playing key roles in responding to the near-surface environment and could be used by bacteria to sense via their flagella when they are near a surface.
细菌能够感知化学物质、表面和其他细胞，并朝着某些物质移动而远离其他物质。研究群体中单个细菌细胞的移动需要复杂的追踪和成像技术。我们通过利用三维（3D）数字全息显微镜（DHM）、二维（2D）微分干涉对比（DIC）和全内反射显微镜（TIRM）的成像模式，并结合使用定制软件的分析协议，建立了在体液体中和固液界面同时对单个细菌细胞进行无标记成像和追踪的定量方法。为了举例说明并验证该方法，我们在单细胞和群体水平上研究了野生型菌株以及同基因鞭毛定子突变体（和）在体液体中和表面的游动行为。观察到多种运动行为，这些行为可以通过速度和方向性来区分。两个定子突变体游动速度都较慢，并且不能像野生型那样有效地适应近表面环境，这突出了定子在适应近表面环境中的不同作用。观察到突变体的游动速度显著降低，在进入近表面环境时进一步下降。这些结果与定子在响应近表面环境中发挥关键作用一致。我们建立了一种方法，能够在三维空间中追踪单个细菌细胞的移动而无需任何标记。这样的方法对于观察和理解细菌如何与表面相互作用并形成生物膜很重要。我们研究了具有两个驱动其游动运动的鞭毛定子的的游动行为。只有两个定子之一的突变体游动速度较慢，并且不能像野生型那样有效地适应近表面环境。这些结果与定子在响应近表面环境中发挥关键作用一致，并且细菌在靠近表面时可以通过其鞭毛来感知。
