Spatial integration of sensory input and motor output in Pseudomonas aeruginosa chemotaxis through colocalized distribution
- Research Center of Translational Medicine, Central Hospital Affiliated to Shandong First Medical University, China
- Hefei National Research Center for Physical Sciences at the Microscale and Department of Physics, University of Science and Technology of China, China
- Research Center of Translational Medicine, Jinan Central Hospital, Shandong University, China
- Department of Critical Care Medicine, Zibo Central Hospital Affiliated to Binzhou Medical University, China
Figures
Figure 1 with 3 supplements
Spatial distribution of the chemotaxis complex and flagellar motor in wild-type P. aeruginosa.
(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 3x 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 an 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.
Figure 1—figure supplement 1
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.
Figure 1—figure supplement 2
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.
Figure 1—figure supplement 3
Simultaneous observation of the chemotaxis complex and flagellar filaments in the wild-type strain, shown in a representative large field.
Figure 2 with 1 supplement
Spatial distribution of the chemotaxis complex and flagellar motor in ΔflhF mutant.
(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.
Figure 2—figure supplement 1
Quantitative statistics of chemotactic complex distribution in wild-type and flhF-related mutants.
(A) 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. (B) Schematic diagram of the classification of chemotactic complex locations. (C) Cells with different chemotactic complex distribution patterns. Red arrows correspond to mid-cell type, yellow arrows correspond to near-polar type, and green arrows correspond to precise-polar type.
Figure 3 with 2 supplements
Characterization of chemotactic complex distribution in several mutants with incomplete flagellar motor.
(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 ΔflhF 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).
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Figure 3—source data 1
PDF file containing original western blots for Figure 3B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97514/elife-97514-fig3-data1-v1.zip
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Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3B.
- https://cdn.elifesciences.org/articles/97514/elife-97514-fig3-data2-v1.zip
Figure 3—figure supplement 1
Knockout of cheA did not affect flagellar assembly efficiency of P. aeruginosa.
Figure 3—figure supplement 2
Localization of CheY-EYFP in the ΔflgI strain of P. aeruginosa.
The phenotype is similar to that of the wild-type strain.
Figure 4 with 2 supplements
Increased CheY content elevates intracellular c-di-GMP levels, leading to cell aggregation.
(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), wild-type strain (N=249), and CheY overexpression strain (N=228), respectively.
Figure 4—figure supplement 1
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.
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Figure 4—figure supplement 1—source data 1
PDF file containing original western blots for Figure 4—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/97514/elife-97514-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/97514/elife-97514-fig4-figsupp1-data2-v1.zip
Figure 4—figure supplement 2
Co-overexpressing CheY and the phosphodiesterase (PDE) YhjH from E. coli mitigates cell aggregation caused by CheY overexpression in P. aeruginosa.
Figure 5
The potential physiological significance of this study.
(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.
Tables
Table 1
Strains and plasmids used in this study.
| Strain, Plasmid | Genotype, phenotype, and description | Source |
|---|---|---|
| Strains | ||
| P. aeruginosa | ||
| PAO1 | wild-type strain | Fan Jin Group |
| PAO1 fliCT394C | Replacement of chromosomal fliC in PAO1 | Tian et al., 2022 |
| PAO1 fliCT394CcheY-eyfp | yfp fusions at the N-terminus of cheY in PAO1 fliCT394C | This work |
| PAO1 fliCT394C cheY-eyfp ΔflhF | Nonpolar flhF deletion in PAO1 fliCT394C cheY-eyfp | This work |
| PAO1 fliCT394C cheY-eyfp ΔfliF | Nonpolar fliF deletion in PAO1 fliCT394C cheY-eyfp | This work |
| PAO1 fliCT394C cheY-eyfp ΔfliG | Nonpolar fliG deletion in PAO1 fliCT394C cheY-eyfp | This work |
| PAO1 fliCT394C cheY-eyfp ΔflhF ΔfliF | Nonpolar fliF deletion in PAO1 fliCT394C cheY-eyfp ΔflhF | This work |
| PAO1 fliCT394C cheY-eyfp ΔmotA ΔmotCD | Nonpolar motAB and motCD deletion in PAO1 fliCT394C cheY-eyfp | This work |
| PAO1 fliCT394C ΔcheA | Nonpolar cheA deletion in PAO1 fliCT394C | This work |
| PAO1 fliCT394C cheY-eyfp ΔflgI | Nonpolar flgI deletion in PAO1 fliCT394C cheY-eyfp | This work |
| E. coli | ||
| Top10 | F-mcrA Δ(mrr-hsRMS-mcrBC)Φ80lacZΔM15 ΔlacX74 recA1 araD139 Δ(araleu)7697 galU galK rpsL(NalR) endA1 nupG | Invitrogen |
| Plasmids | ||
| pex18gm | oriT+sacB+; gene replacement vector with MCS from pUC18; Gmr | Fan Jin Group |
| flhF-pex18gm | In-frame deletion of flhF cloned into pex18gm; Gmr | This work |
| fliF-pex18gm | In-frame deletion of fliF cloned into pex18gm; Gmr | This work |
| fliG-pex18gm | In-frame deletion of fliG cloned into pex18gm; Gmr | This work |
| motA-pex18gm | In-frame deletion of motA cloned into pex18gm; Gmr | This work |
| motCD-pex18gm | In-frame deletion of motCD cloned into pex18gm; Gmr | Wu et al., 2021 |
| cheA-pex18gm | In-frame deletion of cheA cloned into pex18gm; Gmr | This work |
| flgI-pex18gm | In-frame deletion of flgI cloned into pex18gm; Gmr | This work |
| cheY-eyfp-pex18gm | eyfp fusions cheY cloned into pex18gm; Gmr | This work |
| cheY-pJN105 | cheY overexpression vector in pJN105, cheY expression is controlled by PBAD promoter; Gmr | This work |
| cheY-pME6032 | cheY overexpression vector in pME6032, cheY expression is controlled by Plac promoter; Tetr | This work |
| yhjH-pME6032 | yhjH overexpression vector in pME6032, yhjH expression is controlled by Plac promoter; Tetr | This work |
| pCdrA-gfp | pUCP22-NotI based cyclic di-GMP level reporter, Gmr | Rybtke et al., 2012 |
| cheY-eyfp-pJN105 | eyfp fusions cheY cloned into pJN105, cheY-eyfp expression is controlled by PBAD promoter; Gmr | This work |
| cheA-ecfp-pJN105 | ecfp fusions cheA cloned into pJN105, cheA-ecfp expression is controlled by PBAD promoter; Gmr | This work |
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Spatial integration of sensory input and motor output in Pseudomonas aeruginosa chemotaxis through colocalized distribution
eLife 13:RP97514.
https://doi.org/10.7554/eLife.97514.4
