Surface sensing and lateral subcellular localization of WspA, the receptor in a chemosensory-like system leading to c-di-GMP production

Abstract

Pseudomonas aeruginosa responds to growth on agar surfaces to produce cyclic-di-GMP, which stimulates biofilm formation. This is mediated by an alternative cellular function chemotaxis-like system called Wsp. The receptor protein WspA, is bioinformatically indistinguishable from methyl-accepting chemotaxis proteins. However, unlike standard chemoreceptors, WspA does not form stable clusters at cell poles. Rather, it forms dynamic clusters at both polar and lateral subcellular locations. To begin to study the mechanism of Wsp signal transduction in response to surfaces, we carried out a structure-function study of WspA and found that its C-terminus is important for its lateral subcellular localization and function. When this region was replaced with that of a chemoreceptor for amino acids, WspA became polarly localized. In addition, introduction of mutations in the C-terminal region of WspA that rendered this protein able to form more stable receptor-receptor interactions, also resulted in a WspA protein that was less capable of activating signal transduction. Receptor chimeras with a WspA C-terminus and N-terminal periplasmic domains from chemoreceptors that sense amino acids or malate responded to surfaces to produce c-di-GMP. Thus, the amino acid sequence of the WspA periplasmic region did not need to be conserved for the Wsp system to respond to surfaces.

Fig. 1. Schematic model of the Wsp system

(A) Genetic organization of the wsp locus in PAO1. (B) Model of the Wsp protein complex. The chemotaxis counterparts of the Wsp proteins are indicated in parentheses. WspA, the receptor, detects a signal associated with growth on a surface. WspB and WspD are CheW-like proteins. WspE is a CheA-like histidine kinase with an additional receiver domain (REC) at the C-terminus. WspR is a REC-GGDEF domain protein that has diguanylate cyclase activity. WspC is a predicted CheR-like methyltransferase with a C-terminal tetratricopeptide repeat (TPR) domain and WspF is a predicted CheB-like methylesterase. In our model, detection of surface growth by WspA results in phosphorylation of WspR. Phosphorylated WspR synthesizes c-di-GMP, which triggers biofilm formation.

Fig. 2. Subcellular localization of YFP-tagged (A) WspA and the chemoreceptors (B) PctA and (C) PctB

(i) Phase contrast image, (ii) YFP image, (iii) Composite data showing the subcellular localization of clusters in a population of cells. The cell length and width represents the average cell size, the cluster intensity is represented by the locus intensity heat map, the brightest cluster in each cell was plotted. The proportion (%) of clusters found in the old (left) or new (right) cell pole or laterally localized is indicated.

Fig. 3. Subcellular localization of WspA-YFP in wild-type (A) and mutant backgrounds (B–D)

(i) Phase contrast image, (ii) YFP image, (iii) Composite data showing the subcellular localization of clusters in a population of cells. The cell length and width represents the average cell size, the cluster intensity is represented by the locus intensity heat map, the brightest cluster in each cell was plotted. The proportion (%) of clusters found in the old (left) or new (right) cell pole or laterally localized is shown.

Fig. 4. Construction and subcellular localization of chemoreceptor – WspA chimeras

(A) Schematic of the topology of the P. aeruginosa chemoreceptors PctA, PctB, PctC, PA2652 and WspA. (B) Subcellular localization of WspA-PctA and (C) PctA-WspA chimeras. (i) Phase contrast image, (ii) fluorescent image, (iii) Composite data showing the subcellular localization of clusters in a population of cells. The cell length and width represents the average cell size, the cluster intensity is represented by the locus intensity heat map, the brightest cluster in each cell was plotted. The proportion (%) of clusters found in the old (left) or new (right) cell pole or laterally localized is shown.

Fig. 5. Functional analysis of chemoreceptor-WspA chimeras

(A) and (C) ability of each chemoreceptor-WspA chimera to phosphorylate WspR during liquid (white bars) and surface (black bars) growth, measured as percentage of cells in the population that have at least one WspR-YFP cluster. (B) and (D) colony morphology of each strain. (A) and (B) wild-type background encoding WspA or chemoreceptor-WspA variants. (C) and (D) ΔwspF background encoding WspA or chemoreceptor-WspA variants.

Fig. 6. Identification of WspA amino acids that may participate in stabilization of trimer interactions

ClustalW alignment of the trimer interaction region. Likely trimer interaction residues (bold and underlined) were identified by comparison to those identified in Tsr (Ames et al., 2002). * identical,: highly conserved,. weakly conserved. Arrows show amino acids of WspA that have non-conservative changes compared to PctA.

Fig. 7. Localization of WspA-YFP and WspA- 5-YFP

(A) E. coli RP437 (chemotaxis proficient) strain background, (B) HCB437 (chemotaxis genes deleted) strain background. (i) phase contrast, (ii) YFP.

Fig. 8. Subcellular localization of (A) WspA-YFP and (B) WspA-5-YFP in PAO1

(i) Phase contrast, (ii) YFP (iii) Composite data showing the subcellular localization of clusters in a population of cells. The cell length and width represents the average cell size, the cluster intensity is represented by the locus intensity heat map, the brightest cluster in each cell was plotted. The proportion (%) of clusters found in the old (left) or new (right) cell pole or laterally localized is shown.

Fig. 9. Functional analysis of WspA and WspA-5

(A) Ability of WspA/WspA-5 to phosphorylate WspR, measured by percentage of cells in the population that have at least one WspR-YFP cluster. Liquid grown cells: white bars; surface grown cells: black bars. (B) Colony morphology of corresponding strains. Vector control strains were ΔwspA attB::vector control and ΔwspFΔwspA attB::vector control, these strains did not have any WspR-YFP clusters and had smooth colony morphology (data not shown).