Figures and data
A. Schematic diagram of the chemotaxis signal transduction network and flagellar motor of P. aeruginosa. Fusion protein CheY-EYFP and fluorescently labeled flagellar filaments were used as markers to indicate the position of the chemotaxis complex and motor, respectively. B. The labeling mechanism of flagellar filaments and chemotaxis regulatory protein CheY. Filaments (with cysteine point mutation FliCT394C) were labeled through sulfhydryl-maleimide conjugation, and cheY-eyfp fusion with a 3× glycine linker was used to visualize chemotaxis complex positions. C. Localization of CheY-EYFP in the wild-type strain of P. aeruginosa. CheY-EYFP is mainly located at the single cell pole, and the white arrow points to individuals with obvious chemotaxis complex at both cell poles, which generally have a large aspect ratio of the cell body. The yellow dashed box marks the cell outline. D. The merged imaging of flagellar filaments and CheY-EYFP in the wild-type strain of P. aeruginosa, where flagellar motor and chemotaxis complex colocalize in cells. 145 cells with labeled flagella were observed, all of which exhibited consistent colocalization. White arrows point to individuals about to be divided, and the yellow dashed box marks the cell outline. The scale bar is 1 μm.
A. Localization of CheY-EYFP in the ΔflhF strain of P. aeruginosa. CheY-EYFP is no longer robustly distributed at the single cell pole. The yellow dashed box marks the cell outline. B. The merged imaging of flagellar filaments and CheY-EYFP in the ΔflhF strain of P. aeruginosa, flagellar motor and chemotaxis complex still colocalize in cells. 101 cells with labeled flagella were observed, all of which exhibited consistent colocalization. The yellow dashed box marks the cell outline. The scale bar is 1 μm.
A. Localization of CheY-EYFP in various P. aeruginosa strains. The scale bar is 10 μm. B. Western blot analysis was performed to detect CheY expression in various P. aeruginosa strains. β-actin was used as the housekeeping protein. C. Occurrence probability of obvious chemotaxis complex in P. aeruginosa wild-type and several mutant strains. The proportion of individuals with obvious chemotaxis complex decreased significantly in the motor-incomplete strains (ΔfliF and ΔfliG), and this value was further reduced after flhF knockout (ΔfliFΔflhF). The ΔfliF and ΔmotAΔmotCD strains have a similar chemotaxis complex occurrence probability as the wild-type strain. The number of cells analyzed for each strain (from left to right) were: 372, 221, 234, 323, 672, and 242. ‘***’: significant difference (P-value<0.0001), ‘ns.’: no significant difference (P-value>0.05).
A. The evolution of cell aggregation as the intracellular CheY concentration increases by induction with higher concentrations of arabinose. The scale bar is 10 μm. B. Quantitative characterization of intracellular c-di-GMP levels at different CheY concentrations. From top to bottom, they correspond to the ΔcheY strain (N=198), wildtype strain (N=249) and CheY overexpression strain (N=228), respectively.
A. The construction mode of the chemotaxis network and flagellar motor throughout the complete cell growth cycle. B. The proximal growth mode of the P. aeruginosa flagellar motor and receptor clusters will effectively regulate the spatial range of CheY action, thus avoiding unintentional cross-pathway regulation.
Strains and plasmids used in this study.
Distribution of chemotaxis complexes in multiple strains within representative large fields.
Red dotted circles indicate cells where the chemotaxis complex is located at the mid-cell position.
Co-localization of CheY-EYFP and CheA-ECFP in P. aeruginosa.
CheA-ECFP is shown in blue (left), CheY-EYFP in yellow (center), and the merged image is shown in the right panel.
Simultaneous observation of the chemotaxis complex and flagellar filaments in the wild-type strain, shown in a representative large field.
Distribution statistics of the chemotaxis complex in the wild-type strain and the flhF mutant.
The distribution patterns are categorized into three types: precise-polar, near-polar, and mid-cell localization. In the wild-type strain, 98.1% of cells exhibit precise-polar localization of the chemotaxis complex, with the remainder showing near-polar distribution. In the flhF mutant, the proportion of cells with precise-polar localization decreases to 85.5%, and mid-cell localization appears in approximately 5% of cells. The flhF-fliF double mutant displays a distribution pattern similar to that of the wild-type strain.
Knockout of cheA did not affect lagellar assembly efficiency of P. aeruginosa.
Localization of CheY-EYFP in the ΔflgI strain of P. aeruginosa.
The phenotype is similar to that of the wild-type strain.
Intracellular CheY levels induced by different arabinose concentrations, measured by western blot analysis, showing clear differences across the concentration gradient.
β-actin was used as the housekeeping protein.
