How do bacteria sense and navigate chemical gradients within biofilms?

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Most bacteria live attached to surfaces where they form dense communities called biofilms. While some of these assemblages play beneficial roles in our lives, infections within the human body are often very difficult to treat because biofilms protect cells from both antibiotics and the immune system. Understanding the fundamental processes that contribute to biofilm formation is essential to developing new ways to disrupt or manipulate these important bacterial communities.The bacterial species Pseudomonas aeruginosa, which causes dangerous infections in burn victims and cystic fibrosis patients, use tiny grappling hook-like appendages called pili move within biofilms. Our group recently demonstrated that single P. aeruginosa cells can use pili-based motility to navigate to more favourable nutrient environments within a developing biofilm. We found that this process, called chemotaxis, arises because cells pull themselves in the opposite direction when they detect they are moving away from a nutrient source. This remarkable ability likely gives chemotactic cells an advantage in biofilms and opens a new way to control biofilm formation. While other forms of microbial chemotaxis have been extensively studied, relatively little is known about pili-based chemotaxis. This study will address two major gaps in our knowledge:First, we do not know how bacteria actually sense whether they are going towards or away from the source of chemoattractant. In general, there are two different possibilities: cells could either move from one location to another and measure the change in concentration over time (temporal sensing) or they could directly sense changes in concentration over the length of their bodies (spatial sensing). The proposed work will use a combination of novel microfluidic experiments, computer based cell tracking, and bacterial genetics to directly test these two different possibilities. Our preliminary experiments indicate that cells do not increase their probability of reversing when they experience a decrease in the concentration of chemoattractants over time, suggesting that surface attached cells do not use temporal sensing to guide chemotaxis. Future experiments will use novel bacterial strains that relocalise fluorescently labelled proteins to their opposite pole when they reverse direction, which will allow us to test if cells can sense changes in concentration over their length. Second, our experiments indicate that cells tend to travel directly up chemical gradients, yet this observation cannot be explained by known forms of bacterial motility. Surface attached bacteria are thought to have only one behaviour in their repertoire to facilitate chemotaxis: reverse direction. However, reversals alone only allow cells to explore a one-dimensional line, so if a cell happened to land on a surface perpendicular to the gradient, it would be incapable of steering towards the nutrient source. In our preliminary work, we have discovered a new way in which surface attached cells can reorient their motility. Our computer-based image analysis software reveals that cells frequently perform somersault-like manoeuvres we call "twiddles". These reorientations occur when cells steer in either a clockwise or counter-clockwise direction over a period of minutes to hours. This study will use novel bacterial strains and cutting edge super-resolution microscopy to understand how cells generate twiddles. In addition, we will combine data obtained from tracking the movement of tens of thousands of cells with mathematical models to quantify how reversals and twiddles work together to generate chemotaxis.Taken together, this study will provide fundamentally new understanding of how cells regulate their movement within biofilms, potentially giving us new tools to inhibit biofilm development or control the motility of cells in industrial applications.