The regulator FleQ both transcriptionally and post-transcriptionally regulates the level of RTX adhesins of Pseudomonas fluorescens

Abstract

Biofilm formation by the Gram-negative, Gammaproteobacteria Pseudomonas fluorescens relies on the repeats-in-toxin adhesins LapA and MapA in the cytoplasm, secretion of these adhesins through their respective type 1 secretion systems, and retention at the cell surface. Published work has shown that retention of the adhesins occurs via a post-translational mechanism involving the cyclic-di-GMP receptor LapD and the protease LapG. However, little is known about the underlying mechanisms that regulate the level of these adhesins. Here, we demonstrate that the master regulator FleQ modulates biofilm formation by both transcriptionally and post-transcriptionally regulating LapA and MapA. We find that a ΔfleQ mutant has a biofilm formation defect compared to the wild-type (WT) strain, which is attributed in part to a decrease in LapA and MapA abundance in the cell, despite the ΔfleQ mutant having increased levels of lapA and mapA transcripts compared to the WT strain. Through transposon mutagenesis and subsequent genetic analysis, we found that overstimulation of the Gac/Rsm pathway partially rescues biofilm formation in the ΔfleQ mutant background. Collectively, these findings provide evidence that FleQ regulates biofilm formation by both transcriptionally regulating the expression of the lapA and mapA genes and post-transcriptionally regulating the abundance of LapA and MapA, and that activation of the Gac/Rsm pathway can post-transcriptionally enhance biofilm formation by P. fluorescens. IMPORTANCE Biofilm formation is a highly coordinated process that bacteria undergo to colonize a variety of surfaces. For Pseudomonas fluorescens, biofilm formation requires the production and localization of repeats-in-toxin adhesins to the cell surface. To date, little is known about the underlying mechanisms that regulate biofilm formation by P. fluorescens. Here, we identify FleQ as a key regulator of biofilm formation that modulates both gene expression and abundance of LapA and MapA through both a transcriptional and post-transcriptional mechanism. We provide further evidence implicating activation of the Gac/Rsm system in FleQ-dependent regulation of biofilm formation. Together, our findings uncover evidence for a dual mechanism of transcriptional and post-transcriptional regulation of the LapA and MapA adhesins.

Keywords: Pseudomonas fluorescens; RTX adhesins; biofilm; regulation.

Fig 1

A FleQ-deficient strain has a biofilm formation defect due to a decrease in adhesin abundance. (A) Quantification of the biofilm formed by the WT strain, ΔfleQ mutant, and a ΔfleQ mutant complemented with the WT fleQ gene at the att site measured at an optical density of 550 nm (OD₅₅₀) after 16 h of growth in KA minimal medium. Statistical significance was determined using an unpaired t-test. ****, P < 0.0001. (B) Swim zone (in millimeters) of the WT strain, ΔfleQ mutant, and a ΔfleQ mutant complemented with the WT fleQ gene at the att site after toothpick inoculation on KA medium supplemented with 0.3% agar after 24 h of growth at 30°C. Statistical significance was determined using an unpaired t-test. ****, P < 0.0001. (C) Gene expression of lapA, lapE, mapA, and mapE genes in the ΔfleQ mutant relative to the WT strain via quantitative reverse transcription PCR, after 16 h of growth on KA medium supplemented with 1.5% agar (KA agar), using the 2^-ΔΔCt method, commonly referred to as the Livak method (31). For statistical significance, paired t-tests were conducted for each gene between the WT strain and ΔfleQ mutant. *, P < 0.05; **, P < 0.01. (D and F) Quantification of cell surface-associated LapA after 16 h of growth on KA agar (D) or MapA after 24 h of growth on KA agar (F) for the WT strain and ΔfleQ mutant using ImageJ by measuring the mean gray value of each spot using a pre-defined region of interest (ROI) and subtracting the background, which was determined by measuring the mean gray value of a section of the blot without any sample. Representative images are included above each graph. Statistical significance was determined using unpaired t-tests. *, P < 0.05; ***, P < 0.001. (E,G) Quantification of LapA after 16 h of growth on KA agar (E) or MapA after 24 h of growth on KA medium (G) from whole cell lysates that were prepared from 25 mL of cultures, concentrated to 100 µL in 3 mg/mL lysozyme with sonication, and quantified for total protein using the bicinchoninic acid (BCA) assay. Twenty-five micrograms (E) or 50 µg (G) of total protein was resolved on a 7.5% TGX gel and then blotted for LapA or MapA, respectively. Representative images are included above each graph. Statistical significance was determined using unpaired t-tests. *, P < 0.05; **, P < 0.01. All error bars represent standard deviation.

Fig 2

A FleQ-deficient strain produces adhesin sufficient to restore biofilm formation in a lapG mutant. (A) Quantification of the biofilm formed for the WT and the ΔfleQ, ΔlapG, and ΔfleQΔlapG mutant strains measured at OD₅₅₀ after 24 h of growth in KA minimal medium. This time point was chosen because both LapA and MapA are detected at the cell surface at this time point. (B and C) Quantification of cell surface-associated LapA after 16 h of growth on KA agar (B) or MapA after 24 h of growth on KA agar (C) in the WT, ΔfleQ, ΔlapG, and ΔfleQΔlapG strains as described in Fig. 1. Representative images are included above each graph. (D) Swim zone (in millimeters) for the WT, ΔfleQ, ΔlapG, and ΔfleQΔlapG strains after toothpick inoculation on KA supplemented with 0.3% agar and 24 h of growth at 30°C. Statistical significance for this figure was determined using one-way analyses of variance (ANOVAs) with Tukey’s multiple comparisons tests. **, P < 0.01; ***, P < 0.001. All error bars represent standard deviation.

Fig 3

Mutagenizing the FleQ consensus sequences in the lapA promoter has a transcriptional effect that is masked in the ΔfleQ mutant. (A) Schematic showing the divergent lapA/lapE promoter. FleQ binding Box 1 and Box 2 are illustrated, and the respective sites of the point mutations are highlighted in bold. The lapA and lapE start codons are highlighted in bold and direction of translation is indicated by an arrow. The previously published FleQ binding consensus sequence and amino acid conservation of the two predicted boxes can be found in Baraquet and Harwood (29). (B) Quantification of the biofilm formed for the WT and the ΔfleQ mutant with a mutated FleQ binding Box 1 or Box 2, or the WT sequence at OD₅₅₀ after 24 h of growth in KA minimal medium. (C) Swim zone (in millimeters) of WT and ΔfleQ strains with a mutated FleQ binding Box 1 or Box 2, or the WT sequence after toothpick inoculation on KA medium supplemented with 0.3% agar and 24 h of growth at 30°C. Statistical significance for this figure was determined using one-way ANOVAs with Tukey’s multiple comparisons tests. **, P < 0.01. All error bars represent standard deviation.

Fig 4

Identifying other factors that contribute to FleQ-dependent regulation of adhesins. (A) Schematic showing the location of FleQ-deficient transposon mutants that restore biofilm formation. Triangles indicate insertion into the genome and the arrow above each triangle indicates directionality of the Ptac promoter. Genomic coordinates are included for reference. (B) Quantification of the biofilm formed by the WT strain, the ΔfleQ mutant and the indicated ΔfleQ derivatives at OD₅₅₀ after 24 h of growth in KA minimal medium. (C) Swim zone (in millimeters) measurements of the WT strain, the ΔfleQ mutant, and ΔfleQ derivatives after toothpick inoculation on KA medium supplemented with 0.3% agar and 24 h of growth at 30°C. Statistical significance for this figure was determined using one-way ANOVAs with Tukey’s multiple comparisons tests. ****, P < 0.0001. All error bars represent standard deviation.

Fig 5

Modulation of the Gac/Rsm system increases biofilm formation in a WT and FleQ-deficient strain. (A) Quantification of the biofilm formed by the WT, ΔfleQ, ΔrsmAΔrsmEΔrsmI, and ΔfleQ ΔrsmAΔrsmEΔrsmI strains measured at OD₅₅₀ after 24 h of growth in KA minimal medium. (B) Swim zone (in millimeters) of the WT, ΔfleQ, ΔrsmAΔrsmEΔrsmI, and ΔfleQ ΔrsmAΔrsmEΔrsmI strains after toothpick inoculation on KA medium supplemented with 0.3% agar and 24 h of growth at 30°C. (C) Quantification of cell surface-associated LapA after 16 h of growth on KA agar as described in Fig. 1. Representative images are included above each graph. Statistical significance for this figure was determined using one-way ANOVAs with Tukey’s multiple comparisons tests. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. All error bars represent standard deviation.

Fig 6

Overexpressing the small regulatory RNA RsmZ in a FleQ-deficient increases biofilm formation. (A) Quantification of the biofilm formed by the ΔfleQ strain with and without pMQ123, pMQ123-RsmX, pMQ123-RsmY, or pMQ123-RsmZ measured at OD₅₅₀ after 24 h of growth in KA minimal medium with or without 1 mM IPTG for induction. (B) Quantification of the biofilm formed by the ΔfleQΔrsmAΔrsmI strain with and without the pMQ123-RsmZ construct measured at OD₅₅₀ after 24 h of growth in KA minimal medium with or without 1 mM IPTG for induction. (C) Quantification of cell surface-associated LapA after 16 h of growth on KA agar with or without 1 mM IPTG for induction as described in Fig. 1. Statistical significance for this figure was determined using two-way ANOVAs with Tukey’s multiple comparisons tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. All error bars represent standard deviation.

Fig 7

The Gac/Rsm system contributes to LapA abundance. (A) Quantification of the biofilm formed by the WT, ΔfleQ, ΔgacA, and ΔfleQΔgacA strains measured at OD₅₅₀ after 24 h of growth in KA minimal medium. (B) Quantification of cell surface-associated LapA after 16 h of growth on KA agar as described in Fig. 1. Statistical significance for this figure was determined using one-way ANOVAs with Tukey’s multiple comparisons tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All error bars represent standard deviation. (C) Swim zone (in millimeters) of the WT, ΔfleQ, ΔgacA, and ΔfleQΔgacA strains after toothpick inoculation on KA supplemented with 0.3% agar and 24 h of growth at 30°C.

Fig 8

Replacing the native lapA promoter with a non-native FleQ-independent promoter partially restores biofilm formation in a FleQ-deficient strain. (A) Quantification of the biofilm formed by the WT and ΔfleQ strains with the native lapA or P_lac promoter measured at OD₅₅₀ after 24 h of growth in KA minimal medium. (B) Quantification of cell surface-associated LapA after 16 h of growth on KA agar as described in Fig. 1. (C) Swim zone (in millimeters) of the WT and ΔfleQ strains with the native lapA or P_lac promoter strains after toothpick inoculation on KA supplemented with 0.3% agar and 24 h of growth at 30°C. Statistical significance for this figure was determined using one-way ANOVAs with Tukey’s multiple comparisons tests. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. All error bars represent standard deviation.