Multiple Pathways Impact Swarming Motility of Pseudomonas fluorescens Pf0-1

Abstract

Swarming motility in pseudomonads typically requires both a functional flagellum and production/secretion of a biosurfactant. Published work has shown that the wild-type Pseudomonas fluorescens Pf0-1 is swarming-deficient due to a point mutation in the gacA gene, which until recently, was thought to inactivate rather than attenuate the Gac/Rsm pathway. As a result, little is known about the underlying mechanisms that regulate swarming motility by P. fluorescens Pf0-1. Here, we demonstrate that a ΔrsmA ΔrsmE ΔrsmI mutant, which phenotypically mimics Gac/Rsm pathway overstimulation, is proficient at swarming motility. RsmA and RsmE appear to play a key role in this regulation. Transposon mutagenesis of the ΔrsmA ΔrsmE ΔrsmI mutant identified multiple factors that impact swarming motility, including pathways involved in flagellar synthesis and biosurfactant production/secretion. We find that loss of genes linked to biosurfactant Gacamide A biosynthesis or secretion impact swarming motility, as does loss of the alternative sigma factor FliA, which results in a defect in flagellar function. Collectively, these findings provide evidence that P. fluorescens Pf0-1 can swarm if the Gac/Rsm pathway is activated, highlight the regulatory complexity of swarming motility in this strain, and demonstrate that the cyclic lipopeptide Gacamide A is utilized as a biosurfactant for swarming motility.

Keywords: Pseudomonas fluorescens; biosurfactant; flagellum; regulation; swarming.

FIG 1. A strain deficient for the Rsm proteins swarms.

(A) Swim zone (in millimeters) of the WT strain, ΔfleQ single mutant, ΔrsmA ΔrsmE ΔrsmI triple mutant, and a ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant after toothpick inoculation on KA minimal medium supplemented with 0.3% agar followed by 24h growth at 30°C. (B) Biosurfactant zone (in millimeters) of the WT strain, ΔfleQ single mutant, ΔrsmA ΔrsmE ΔrsmI triple mutant, and a ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C. (C) Swarm zone (in millimeters) of the WT strain, ΔfleQ single mutant, ΔrsmA ΔrsmE ΔrsmI triple mutant, and a ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C then 24h growth at room temperature. Statistical significance for this figure was determined using one-way ANOVA with Tukey’s multiple comparisons tests. ****, P<0.0001. All error bars represent standard deviation.

FIG 2. A strain deficient for both RsmA and RsmE shows swarming motility.

(A) Swarm zone (in millimeters) of the WT strain, ΔfleQ single mutant, ΔrsmA single mutant, ΔfleQ ΔrsmA double mutant, ΔrsmE single mutant, ΔfleQ ΔrsmE double mutant, ΔrsmI single mutant, ΔfleQ ΔrsmI double mutant, ΔrsmA ΔrsmE ΔrsmI triple mutant, and a ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C then 24h growth at room temperature. (B) Swarm zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE double mutant, ΔfleQ ΔrsmA ΔrsmE triple mutant, ΔrsmA ΔrsmI double mutant, ΔfleQ ΔrsmA ΔrsmI triple mutant, ΔrsmE ΔrsmI double mutant, and ΔfleQ ΔrsmE ΔrsmI triple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C then 24h growth at room temperature. (C) Swarm zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI mutant, ΔrsmA ΔrsmE ΔrsmI mutant + pMQ72-rsmA, and ΔfleQ ΔrsmA ΔrsmE ΔrsmI mutant + pMQ72-rsmA after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C then 24h growth at room temperature. (D) Swarm zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI mutant, ΔrsmA ΔrsmE ΔrsmI mutant + pMQ72-rsmE, and ΔfleQ ΔrsmA ΔrsmE ΔrsmI mutant + pMQ72-rsmE after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C then 24h growth at room temperature. Statistical significance for this figure was determined using one-way ANOVA with Tukey’s multiple comparisons tests. ****, P<0.0001. All error bars represent standard deviation. All error bars represent standard deviation.

FIG 3. Summary of genetic loci identified by transposon mutagenesis.

Stacked bar charts categorizing the candidates (n=108) identified from the transposon mutagenesis. Candidates were phenotypically sub-grouped based on swarming motility into hyper-swarming (n= 18) or swarm-deficient (n=90). The swarm-deficient group was then sub-grouped based on a loss of biosurfactant production (n=25) or a defect in swimming motility (n=82), with a subset of candidates present in both groups.

FIG 4. Loss of components of the biosurfactant operon effect swarming motility through the altered biosurfactant levels.

(A) Biosurfactant zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔgamA quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔgamA quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C. (B) Swarm zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔgamA quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔgamA quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C then 24h growth at room temperature. (C) Biosurfactant zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔgamB quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔgamB quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C. (D) Swarm zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔgamB quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔgamB quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C then 24h growth at room temperature. (E) Biosurfactant zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔgamC quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔgamC quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C. (F) Swarm zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔgamC quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔgamC quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C then 24h growth at room temperature. Statistical significance for this figure was determined using one-way ANOVA with Tukey’s multiple comparisons tests. ****, P<0.0001. All error bars represent standard deviation. All error bars represent standard deviation.

FIG 5. Loss of any component of the biosurfactant secretion system results in a swarm motility defect.

(A) Biosurfactant zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔpleA quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔpleA quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C. (B) Swarm zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔpleA quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔpleA quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% followed by 24h growth at 30°C then 24h growth at room temperature. (C) Biosurfactant zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔpleB quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔpleB quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C. (D) Swarm zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔpleB quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔpleB quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C then 24h growth at room temperature. (E) Biosurfactant zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔpleC quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔpleC quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C. (F) Swarm zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔpleC quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔpleC quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C then 24h growth at room temperature. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons tests. ****, P<0.0001. All error bars represent standard deviation.

FIG 6. A FliA-deficient strain has a motility defect likely due to loss of flagellar function.

(A) Swim zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔfliA quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔfliA quintuple mutant after toothpick inoculation on KA minimal medium supplemented with 0.3% agar followed by 24h growth at 30°C. (B) Swarm zone (in millimeters) of the ΔrsmA ΔrsmE ΔrsmI triple mutant, ΔfleQ ΔrsmA ΔrsmE ΔrsmI quadruple mutant, ΔrsmA ΔrsmE ΔrsmI ΔfliA quadruple mutant, and the ΔfleQ ΔrsmA ΔrsmE ΔrsmI ΔfliA quintuple mutant after inoculation of 2.5µl of overnight culture on the surface of KA minimal medium supplemented with 0.5% agar followed by 24h growth at 30°C then 24h growth at room temperature. 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.