Light-mediated decreases in cyclic di-GMP levels are potentiated by pyocyanin and inhibit structure formation inPseudomonas aeruginosabiofilms

Abstract

25Light is known to trigger regulatory responses in diverse organisms including slime molds, animals, plants, and phototrophic bacteria. However, light-dependent processes in nonphototrophic bacteria, and those of pathogens in particular, have received comparatively little research attention. In this study, we examined the impact of light on multicellular development in Pseudomonas aeruginosa, a leading cause of biofilm-based bacterial 30 infections, using a colony morphology assay. In this assay, P. aeruginosa strain PA14 grown in the dark forms vertical structures (i.e., "wrinkles") on the third day of incubation.We found that growth in blue light inhibited wrinkle formation until the fifth day and that this required the phenazine pyocyanin, a redox-active metabolite produced by PA14.Light-dependent inhibition of wrinkling was also correlated with low levels of cyclic di-35 GMP (c-di-GMP), consistent with the role of this signal in stimulating biofilm matrix production. Though phenazine-null biofilms also showed lower levels of c-di-GMP and subtle effects on wrinkling when grown in the light, their overall levels of c-di-GMP were higher than those of the wild type. This indicates that phenazines and light simultaneously promote c-di-GMP degradation such that c-di-GMP is pushed to a minimum level, yielding 40 a pronounced macroscopic phenotype. A screen of enzymes with the potential to catalyze c-di-GMP synthesis or degradation identified four proteins that contribute to lightdependent inhibition of biofilm wrinkling. Together, these results provide a foundation for understanding the significance of light-dependent regulation in P. aeruginosa.3 Importance 45 The response to light in non-phototrophic bacteria (i.e., chemotrophs) is relatively understudied in comparison to light-mediated behavior in eukaryotes and phototrophic bacteria. Though they do not depend on light for growth, chemotrophic bacteria could benefit from sensing this cue when it correlates with other parameters that are important for metabolism. In this paper, we describe light-dependent effects on a cellular signal that 50 controls the development of multicellular assemblages, called biofilms, in Pseudomonas aeruginosa. We found that light at intensities that are not harmful to human cells inhibited biofilm maturation. P. aeruginosa is a leading cause of chronic lung infections in people with cystic fibrosis and of hospital-acquired infections. As P. aeruginosa's recalcitrance to treatment is attributed in part to its facile formation of biofilms, this study provides 55 insight into a mechanism that could be inhibited via new therapeutic tools, such as photodynamic therapy. 75 light inhibits exopolysaccharide excretion, biofilm development, and virulence via a bacteriophytochrome that binds a bilin cofactor (3). In Caulobacter crescentus, Bacillus subtilis, and Brucella abortus, photosensory proteins link the effects of blue light on FMN cofactors to regulation of adhesion, the general stress response, and pathogenicity (4-6). Furthermore...

Extracellular DNA promotes efficient extracellular electron transfer by pyocyanin in Pseudomonas aeruginosa biofilms

“…Figure 4I shows that both WT biofilms relying solely on endogenous PYO and Dphz* biofilms + exogenous PYO generate robust current over many days, while Dphz* alone does not. The daily periodic rise in current likely reflects the impact of slight temperature or light fluctuations in the room on the cells' metabolic activity over the course of the experiment (Kahl et al, 2016(Kahl et al, , 2019. Differences in current magnitude between the WT and Dphz* + PYO runs are likely due to differences in PYO abundance in the biofilms and/or efficiency of cellular PYO reduction.…”

Extracellular DNA Promotes Efficient Extracellular Electron Transfer by Pyocyanin in Pseudomonas aeruginosa Biofilms

“…Figure 4I shows that both WT biofilms relying solely on endogenous PYO and Dphz biofilms + exogenous PYO generate robust current over many days, whereas Dphz alone does not. The daily periodic rise in current likely reflects the impact of slight temperature or light fluctuations in the room on the cells' metabolic activity over the course of the experiment (Kahl et al, 2016(Kahl et al, , 2019. Differences in current magnitude between the WT and Dphz + PYO runs are likely due to stochastic differences in PYO abundance in the biofilms and/or efficiency of cellular PYO reduction.…”