The type IV pilus chemoreceptor PilJ controls chemotaxis of one bacterial species towards another.

Bacteria live in social communities, where the ability to sense and respond to interspecies and environmental signals is critical for survival. We previously showed the pathogen Pseudomonas aeruginosa detects secreted peptides from bacterial competitors and navigates through interspecies signal gradients using pilus-based motility. Yet, it was unknown whether P. aeruginosa utilizes a designated chemosensory system for this behavior. Here, we performed a systematic genetic analysis of a putative pilus chemosensory system, followed by high-speed live-imaging and single-cell tracking, to reveal behaviors of mutants that retain motility but are blind to interspecies signals. The enzymes predicted to methylate (PilK) and demethylate (ChpB) the putative pilus chemoreceptor, PilJ, are necessary for cells to control the direction of migration. While these findings implicate PilJ as a bona fide chemoreceptor, such function had yet to be experimentally defined, as full-length PilJ is essential for motility. Thus, we constructed systematic genetic modifications of PilJ and found that without the predicted ligand binding domains or predicted methylation sites, cells lose the ability to detect competitor gradients, despite retaining pilus-mediated motility. Chemotaxis trajectory analysis revealed that increased probability and size of P. aeruginosa pilus-mediated steps towards S. aureus peptides, versus steps away, determines motility bias in wild type cells. However, PilJ mutants blind to interspecies signals take less frequent steps towards S. aureus or steps of equal size towards and away. Collectively, this work uncovers the chemosensory nature of PilJ, provides insight into how cell movements are biased during pilus-based chemotaxis, and identifies chemotactic interactions necessary for bacterial survival in polymicrobial communities, revealing putative pathways where therapeutic intervention might disrupt bacterial communication.

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time. Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

Interspecies interactions induce exploratory motility in Pseudomonas aeruginosa.

Microbes often live in multispecies communities where interactions among community members impact both the individual constituents and the surrounding environment. Here, we developed a system to visualize interspecies behaviors at initial encounters. By imaging two prevalent pathogens known to be coisolated from chronic illnesses, Pseudomonas aeruginosa and Staphylococcus aureus, we observed P. aeruginosa can modify surface motility in response to secreted factors from S. aureus. Upon sensing S. aureus, P. aeruginosa transitioned from collective to single-cell motility with an associated increase in speed and directedness - a behavior we refer to as 'exploratory motility'. Explorer cells moved preferentially towards S. aureus and invaded S. aureus colonies through the action of the type IV pili. These studies reveal previously undescribed motility behaviors and lend insight into how P. aeruginosa senses and responds to other species. Identifying strategies to harness these interactions may open avenues for new antimicrobial strategies.