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.

AmrZ Regulates Swarming Motility Through Cyclic di-GMP-Dependent Motility Inhibition and Controlling Pel Polysaccharide Production in Pseudomonas aeruginosa PA14

“…However, to understand the specificity and generality of these receptors in c-di-GMP signal transmission will require the identification of individual c-di-GMP signaling pathway of the multiple DGCs and PDEs encoded in P. aeruginosa genome. In another study, Lewis and colleagues performed a screen for proteins involved in c-di-GMP-mediated response to ethanol which led to the identification of a few candidates including GcbA and FlgZ (Lewis et al, 2019); however, the relationship between the two proteins was not investigated in this research, and our findings may provide an explanation for this screening result. Unlike FlgZ, there is limited information available about PA14_56180, but our results highlight a role for PA14_56180 as a GcbA c-di-GMP receptor affecting swarming motility of P. aeruginosa .…”

Conditional antagonism in co-cultures of Pseudomonas aeruginosa and Candida albicans: An intersection of ethanol and phosphate signaling distilled from dual-seq transcriptomics

“…Like many fermentative organisms, C. albicans produces ethanol. Ethanol is a biologicallyactive metabolite which, at sub-inhibitory concentrations, modulates P. aeruginosa behavior in multiple ways: it induces activity of the sigma factor AlgU through DksA and ppGpp signaling [31]; it promotes Pel matrix production through the Wsp system [29]; it decreases flagellarmediated motility through a surface-sensing pathway [29,32]; it affects programs known to contribute to P. aeruginosa virulence [29,33]; and it fuels fungal antagonism [29]. The broad effects of ethanol apply to many contexts and the response of P. aeruginosa to C. albicans-produced ethanol can serve as a model for how P. aeruginosa may respond to other fermentative fungi and bacteria.…”

“…Ethanol is a biologically-active metabolite which, at sub-inhibitory concentrations, modulates P . aeruginosa behavior in multiple ways: it induces activity of the sigma factor AlgU through DksA and ppGpp signaling [ 31 ]; it promotes Pel matrix production through the Wsp system [ 29 ]; it decreases flagellar-mediated motility through a surface-sensing pathway [ 29 , 32 ]; it affects programs known to contribute to P . aeruginosa virulence [ 29 , 33 ]; and it fuels fungal antagonism [ 29 ].…”

Unraveling Pseudomonas aeruginosa and Candida albicans Communication in Coinfection Scenarios: Insights Through Network Analysis

“…In the context of infections where P. aeruginosa coexists with C. albicans , exogenous fungal-produced ethanol may alter phenazine production and promote biofilm development on biotic and abiotic surfaces. In addition, ethanol enhances bacterial Pel matrix production and represses surface motility ( Lewis et al, 2019 ). Its production is continuously stimulated by the enhanced P. aeruginosa biofilm formation and production of antifungal phenazines, in a positive feedback loop ( Chen et al, 2014 ).…”