<i>P</i>. <i>aeruginosa</i> wild type in monoculture.

Duration 3.5 hours. Three hours post-inoculation. Acquisition interval 1 second. Output interval every 40th frame at 50 ms/frame. (MOV)</p

<i>P</i>. <i>aeruginosa</i> Δ<i>pilK</i> has bimodal pilus-mediated motility.

Separate ΔpilK cultures plated onto 4 individual experimental dishes and 4 fields of view in each culture dish were simultaneously imaged at 5 hours postinoculation. Agarose pads were made from the same media and dried under the same conditions at the same time. A range of motility phenotypes are seen between all microcolonies imaged. (TIFF)</p

Strains and key resources used in this study.

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.</div

Methylation sites of PilJ.

Amino acid sequence of P. aeruginosa PA14 PilJ (PA14_05360) with conserved MCP methylation motifs highlighted in pale orange and predicted methyl modification glutamate/glutamine residue pairs in bold. (TIFF)</p

Random walk analysis functions.

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.</div

Methyl modification of PilJ is necessary for TFP-mediated chemotaxis response to <i>S</i>. <i>aureus</i>.

(A) Schematic of PilJ with cytoplasmic methylation sites represented as black circles. The methylation site Q412, E413, whose mutant retains wild type motility and is further studied for chemotaxis response, is highlighted in pale orange. (B) Twitching motility diameters of P. aeruginosa wild type and methylation mutants. Motility diameters were analyzed by a one-way ANOVA followed by Dunnett’s multiple comparisons test. **** indicates p p ns indicates no statistically significant difference. (C) Migration towards S. aureus secreted factors of P. aeruginosa wild type and methylation mutants pilJQ412A, E413A, pilJQ623A, Q624A, and pilJQ639A, E640A. Statistical significance of directional motility was determined with a one-way ANOVA followed by Dunnett’s multiple comparisons test. **** indicates p D) Rose graph of the principal angle of motility for each cell trajectory relative to starting position for P. aeruginosa pilJQ412A, E413A in coculture with S. aureus. Position of S. aureus relative to the center of the pilJQ412A, E413A cells is represented by the gray cocci on the perimeter. Trajectory angles are shown by colored vectors with the average angle of all trajectories represented by the dotted black line. Larger vectors indicate more cells for the given principal angle. One rose graph representative of at least 3 replicates is shown. The underlying data can be found in S1 and S5 Data. IM, inner membrane; LBD, ligand binding domain; OM, outer membrane; PG, peptidoglycan; TFP, type IV pilus; WT, wild type.</p

<i>P</i>. <i>aeruginosa pilJ</i>_ΔLBD1-2 in coculture with <i>S</i>. <i>aureus</i> wild type.

Duration 2 hours. Three hours postinoculation. Acquisition interval 1 second. Output interval every 40th frame at 50 ms/frame. (MOV)</p

Trajectories for <i>P</i>. <i>aeruginosa pilJ</i>_{Q412A, E413A}.

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.</div

<i>P</i>. <i>aeruginosa</i> Δ<i>pilK</i> in monoculture.

Hypermotile cells. Duration 3 hours. Three hours postinoculation. Acquisition interval 1 second. Output interval every 40th frame at 50 ms/frame. (MOV)</p

van Hove distributions for all mutants.

Step size distributions for each P. aeruginosa strain are displayed by distance of displacement (Δr) of cells. Step size PDFs are shown for movers (solid lines) and resters (dotted lines) in the parallel (darker lines, ||) and perpendicular (lighter lines, ⊥) directions. Step sizes for each P. aeruginosa strain were calculated from cell trajectories with a 1-second (left), 10-second (middle), and 100-second (right) time step (Δt). Right- and left-facing triangles (movers, ||+ and ||−, respectively) and up- and down-facing triangles (movers, ⊥+ and ⊥−, respectively) highlight the nonzero sharp-shoulder peak step size, when present. The underlying data can be found in S2–S6 Data. (TIFF)</p