Ethanol Decreases Pseudomonas aeruginosa Flagellar Motility through the Regulation of Flagellar Stators

Abstract

Pseudomonas aeruginosa frequently encounters microbes that produce ethanol. Low concentrations of ethanol reduced P. aeruginosa swim zone area by up to 45% in soft agar. The reduction of swimming by ethanol required the flagellar motor proteins MotAB and two PilZ domain proteins (FlgZ and PilZ). PilY1 and the type 4 pilus alignment complex (comprising PilMNOP) were previously implicated in MotAB regulation in surface-associated cells and were required for ethanol-dependent motility repression. As FlgZ requires the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) to represses motility, we screened mutants lacking genes involved in c-di-GMP metabolism and found that mutants lacking diguanylate cyclases SadC and GcbA were less responsive to ethanol. The double mutant was resistant to its effects. As published previously, ethanol also represses swarming motility, and the same genes required for ethanol effects on swimming motility were required for its regulation of swarming. Microscopic analysis of single cells in soft agar revealed that ethanol effects on swim zone area correlated with ethanol effects on the portion of cells that paused or stopped during the time interval analyzed. Ethanol increased c-di-GMP in planktonic wild-type cells but not in ΔmotAB or ΔsadC ΔgcbA mutants, suggesting c-di-GMP plays a role in the response to ethanol in planktonic cells. We propose that ethanol produced by other microbes induces a regulated decrease in P. aeruginosa motility, thereby promoting P. aeruginosa colocalization with ethanol-producing microbes. Furthermore, some of the same factors involved in the response to surface contact are involved in the response to ethanol.IMPORTANCE Ethanol is an important biologically active molecule produced by many bacteria and fungi. It has also been identified as a potential marker for disease state in cystic fibrosis. In line with previous data showing that ethanol promotes biofilm formation by Pseudomonas aeruginosa, here we report that ethanol reduces swimming motility using some of the same proteins involved in surface sensing. We propose that these data may provide insight into how microbes, via their metabolic byproducts, can influence P. aeruginosa colocalization in the context of infection and in other polymicrobial settings.

Keywords: Ethanol; Pseudomonas aeruginosa; c-di-GMP; motility; stator.

FIG 1

Ethanol decreases swim zone area in a dose-dependent manner independent of changing growth, increasing matrix, or ethanol catabolism. (A) Swim zone area of wild-type P. aeruginosa PA14 in M63 medium solidified with 0.3% agar (soft agar) and supplemented with 0, 0.25, 0.5, 0.75, 1, 2, or 5% ethanol. Hashed bars (2% and 5% ethanol) depict ethanol concentration where growth was affected, and the arrow indicates the ethanol concentration used in this study. (B) Growth curves of wild-type P. aeruginosa PA14 in liquid M63 medium without (blue) and with (black) 1% ethanol grown for 24 h at 37°C with OD₆₀₀ measured at the indicated time points. No significant differences were observed until the late 24-h time point, as measured by two-way ANOVA with multiple comparisons (P value of <0.05 is significant). (C) Swim zone area of wild-type P. aeruginosa PA14 (WT) and the ΔpelA (Pel defective mutant) and ΔwspR mutants in soft agar without (gray) and with (black) 1% ethanol. (D) Swim zone area of WT and the ΔalgD and ΔalgU mutants in soft agar without (gray) and with (black) 1% ethanol. (E) Swim zone area of WT and the ΔexaA mutant (ethanol catabolic mutant) in soft agar without (gray) and with (black) 1% ethanol. All swim zones were measured after 18 to 20 h. All error bars indicate standard deviations, n = 4 replicates. The same lowercase letters indicate samples that are not significantly different and different lowercase letters indicate significant differences (P < 0.05), as determined by two-way ANOVA with multiple comparisons.

FIG 2

Ethanol effects on swimming motility require the MotAB flagellar stator complex. Swim zone areas of wild-type P. aeruginosa PA14 (WT) and the ΔflgK, ΔmotAB, and ΔmotCD mutants in soft agar without (gray) and with (black) 1% ethanol after 18 to 20 h. Error bars indicate standard deviations, n = 4 replicates. Each sample was statistically compared to every other sample; the same lowercase letters indicate samples that are not significantly different and different lowercase letters indicate significant differences (P < 0.05) as determined by two-way ANOVA with multiple comparisons.

FIG 3

PilZ domain proteins, PilZ and FlgZ, are required for ethanol effects on swim zone area. (A) Swim zone areas for wild-type P. aeruginosa PA14 (WT) and the ΔflgZ, ΔPA14_00130, ΔPA14_60970, ΔPA14_27930, ΔPA14_56180, Δalg44, ΔPA14_25420, and ΔpilZ mutants in soft agar without and with 1% ethanol (EtOH) after 18 to 20 h. Floating bars represent the minimum, maximum, and mean values from 4 replicates. Shaded boxes represent the ethanol samples that are significantly different from their controls (P < 0.05) as determined by one-way ANOVA with multiple comparisons. Arrows indicate the candidate mutants that were analyzed further. (B) WT and the ΔpilZ, ΔflgZ, and ΔpilZ ΔflgZ mutants in soft agar without (gray) and with (black) 1% ethanol after 18 to 20 h. Error bars indicate standard deviations, n = 4 replicates. Each sample was statistically compared to every other sample; the same lowercase letters indicate samples that are not significantly different and different letters indicate significant differences (P < 0.05) as determined by two-way ANOVA with multiple comparisons.

FIG 4

Ethanol increases cyclic di-GMP levels. (A and C) Quantification of cyclic di-GMP levels in wild-type P. aeruginosa PA14 (WT) and the ΔwspR, ΔmotAB, and ΔmotCD mutants (A) and WT and the ΔsadC ΔgcbA mutant (C), exposed for 16 h to 1% ethanol (black) or medium with no ethanol (gray). Bars depict the averages of normalized values (n = 5 replicates). Error bars indicate standard deviations. (B) Swim zone areas of WT and the ΔsadC, ΔgcbA, and ΔsadC ΔgcbA mutants in soft agar without (gray) and with (black) 1% ethanol measured at 18 to 20 h. Error bars indicate standard deviations, n = 4 replicates. Each sample was statistically compared to every other sample by two-way ANOVA with multiple comparisons. P values are indicated. ns, not significantly different.

FIG 5

PilY1 and PilMNOP, but not T4P activity, are necessary for ethanol effects on swim zone area. (A) Schematic of the PilY1 protein showing the amino acid positions of the signal sequence (SS), von Willebrand A factor domain (vWA), calcium-binding domain (red), and the PilC domain (green). (B) Swim zone areas of wild-type P. aeruginosa PA14 (WT) and the ΔpilY1 and ΔpilMNOP mutants in soft agar without (gray) and with (black) 1% ethanol measured after 18 to 20 h. Error bars indicate standard deviations, n = 4 replicates. The hashed bar indicates mutant that swam more in ethanol than in control cultures. (C) Swim zone areas of WT and the ΔpilY1, ΔpilY1 mutants with an empty vector (EV), with a plasmid-borne pilY1 (PpilY1), or pilY1 with the vWA domain deleted (pilY1-ΔvWA) and placed at the native pilY1 locus in soft agar without (gray) and with (black) 1% ethanol measured after growth for 18 to 20 h; 0.05% arabinose was added to the medium. Error bars indicate standard deviations, n = 4 replicates. Hashed bars indicate mutants that swam more in ethanol than in control cultures. (D) Swim zone areas of WT and the ΔpilA (pilus-deficient mutant) mutant in soft agar without (gray) and with (black) 1% ethanol after 18 to 20 h. Error bars indicate standard deviations, n = 4 replicates. (E) Twitch zone areas of WT and the ΔpilY1 (twitching deficient) mutant in medium without (gray) and with (black) 1% ethanol after 40 h. Error bars depict standard deviations, n = 4 replicates and repeated in more than four separate experiments. Pictures at the top show representative twitch zones from the indicated sample. Each sample was statistically compared to every other sample; the same lowercase letters indicate samples that are not significantly different and different lowercase letters indicate significant differences (P < 0.05) as determined by two-way ANOVA with multiple comparisons.

FIG 6

Ethanol decreases the number of continuously motile cells in soft agar, in a manner dependent on MotAB, FlgZ, PilZ, PilY1, and PilMNOP. (A) Schematic of agar motility assay treated with water (control) or 1% ethanol in soft agar. A sample was mixed well, 250 μl was pipetted onto a glass slide, and staples were used to create a chamber using a glass coverslip. The sample was then incubated at room temperature for 30 min and then imaged as outlined in Materials and Methods. (B) Agar motility assay of wild-type P. aeruginosa PA14 (WT) and the ΔsadC ΔgcbA, ΔpilZ ΔflgZ, ΔmotAB, ΔpilY1, and ΔpilMNOP mutants in soft agar without (Ctrl) and with (+ EtOH) 1% ethanol after 30 min. Three time-lapse (8 s) movies of the cells in the agar matrix, for each sample, were captured. Shown is a box and whiskers plot of the average population percentages of the continuously motile subpopulation in each movie. Error bars represent the minimum to maximum data points, n ≥ 6 replicate movies. Shaded boxes represent the ethanol samples that are significantly different from their controls. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant as determined by one-way ANOVA with multiple comparisons.

FIG 7

Ethanol effects on swarming motility repression require the same components needed for motility repression in soft agar. Representative images of swarming motility assays of wild-type P. aeruginosa PA14 (WT) and ΔmotAB mutant (A), WT and the ΔflgZ, ΔpilZ, and ΔpilZ ΔflgZ mutants (B), WT and the ΔsadC, ΔgcbA, and ΔsadC ΔgcbA mutants (C), WT, the ΔpilY1 mutant, and the ΔpilY1 mutant with an empty vector (EV) or with a plasmid that enables arabinose-inducible expression pilY1 (PpilY1) or pilY1 without a vWA domain (pilY1-ΔvWA) (D), and WT and ΔpilMNOP mutant (E) on M8 medium with 0.5% agar (swarm agar) without and with 1% ethanol and grown for 16 h. Images are representative of observed phenotypes, n = 4 replicates per experiment, and each experiment was performed 3 to 5 times.

FIG 8

Model for the effects of ethanol on Pseudomonas aeruginosa motility. We propose that in the absence of ethanol (No EtOH), P. aeruginosa remains motile with a bias toward a MotCD-dominant stator. As shown on the right, ethanol (depicted as red curvy lines) leads to decreased motility through a mechanism that requires the MotAB stator complex, PilY1, and PilVWX and PilMNOP protein complexes. We propose that PilZ and FlgZ, two PilZ domain proteins, may aid in MotCD delocalization from the motor. Two diguanylate cyclases, SadC and GcbA, also participate in ethanol-induced motility repression. As we show, ethanol may interact with the bacterial membrane, directly affect proteins, such as PilY1, or have other indirect effects such as the increase in c-di-GMP observed in this pathway.