Broth versus Surface-Grown Cells: Differential Regulation of RsmY/Z Small RNAs in Pseudomonas aeruginosa by the Gac/HptB System

Abstract

Two-component systems are capable of profoundly affecting genetic regulation in bacteria by detecting environmental stimuli, allowing them to quickly adapt. In Pseudomonas aeruginosa, the small RNAs (sRNAs) RsmY and RsmZ are under the control of the GacS/A system. They have been described as ones of the major key players in the control of planktonic and surface-associated behaviors. Genetic regulation by these sRNAs is achieved by the titration of the negative post-transcriptional regulator RsmA which affects the expression of over 500 genes. There is increasing evidence pinpointing the importance of RsmY and RsmZ in the planktonic-sessile P. aeruginosa lifestyles switch control. Using swarming motility as a model, we show here that these sRNA are differentially regulated depending on the selected growth conditions (i.e., planktonic versus surface grown-cells). Also, we report that opposite to planktonically grown cells, rsmZ regulation does not implicate the response regulator GacA in swarming cells. Furthermore, we present data indicating that RsmY/Z expression influence swarming motility via the protein HptB which acts as a negative regulator of these sRNAs and that they do not strictly converge to RsmA as previously reported.

Keywords: HptB; broth-surface; genetic regulation; histidine phosphotransfer protein; small RNAs; surface motility; swarming.

FIGURE 1

A mutation in the hptB gene affects swarming motility. (A) Swarming motility phenotypes of the wild-type PA14 strain and the ΔhptB mutant. (B) Swimming motility assay in various media containing 0.25% agar. (C) Rhamnolipids quantification in planktonic cultures. (D) Rhamnolipids quantification on swarming plates. Error bars represent the standard error of the mean for experiments carried out at least twice with a minimum of three replicates per experiment. Student’s t-test analysis was applied on two independent experiments with ^∗∗p < 0.01; ns, not significant.

FIGURE 2

Swarming motility is linked to sRNA expression. (A) Expression of the sRNAs RsmY and RsmZ in the ΔhptB mutant strain grown in broth or on a surface (swarming condition) determined by qRT-PCR. (B) Swarming motility of various sRNA mutants. (C) Time-lapse analysis of the ΔrsmY/Z mutants. Error bars represent the standard error of the mean for experiments carried out at least twice with three biological replicates per experiment. Student’s t-test analysis was applied on two independent experiments with ^∗∗∗p < 0.001; ns, not significant. For (C), the data correspond to one single plate per strain. Gene expression variation is shown as relative expression variation (log₂) to the wild-type PA14 strain.

FIGURE 3

Swarming deficiency of the ΔhptB mutant is due to sRNA overexpression. (A) Swarming motility assessment of various double and triple mutants. Shown are the average representative swarming phenotype of various strains. (B) Swarming motility surface coverage of the simple ΔhptB mutant, the double ΔhptBrsmY/Z mutants, the triple ΔhptBrsmYZ mutant and ΔrsmYZ double mutant. Student’s t-test analysis was done on the ΔrsmYZ double mutant and the triple ΔhptBrsmYZ mutant based on two independent experiments (ns, not significant). Error bars represent the standard deviation of three technical replicates.

FIGURE 4

The expression of sRNA is dependent of growth conditions. (A) Expression of rsmY and rsmZ determined by qRT-PCR in various genetic backgrounds grown in broth. (B) Expression of rsmY and rsmZ of strains grown on a surface (swarming). Error bars represent the standard deviation of experiments carried out using three biological replicates. Student’s t-test analysis was based on two independent experiments (^∗p < 0.05; ^∗∗p < 0.01). Gene expression variation is shown as relative expression variation (log₂) to the wild-type PA14 strain.

FIGURE 5

Model for broth-surface differential sRNA regulation. We propose a regulation model where the control of the expression of rsmY and rsmZ by HptB is under the exclusive control of the GacS/GacA system and converges to the post-transcriptional regulator RsmA for both planktonic and swarming cells. However, in cells grown on a surface such as in swarming motility, the regulation of both rsmY and rsmZ is differential and does not strictly mediate their output via RsmA. The obtained data in our study indicates that other key-players allows for regulation of rsmZ by HptB and does not implicate the Gac system. This regulation most likely involves other membrane sensors that can modulate the activity of the HptB protein. Presented model integrates previously published data (Hsu et al., 2008; Brencic et al., 2009; Bordi et al., 2010). Full arrows represent direct positive regulation. Dashed arrows represent indirect positive regulation. Dashed bars represent indirect negative control. Full bars represent direct negative regulation. ? = unknown contribution. X = Unknown regulating factor.