| Filamentous fungi and bacteria often form mix-species biofilms in many different contexts ranging from natural rhizosphere to the lungs of cystic fibrosis (CF) patients. One way these microbes interact is through the production and secretion of small molecule secondary metabolites. Traditionally, these microbial metabolites are viewed as toxins that inhibit the growth of competing microorganisms. The knowledge of the signaling effect of secreted secondary metabolites is still limited. Here, we show that these "toxins" have can act as signal species stimulating fungal asexual sporulation (conidiation) via oxidative stress regulation, and morphology modulators affecting fungal biofilm formation and morphogenesis. Specifically, in the co-culture biofilms, Pseudomonas aeruginosa phenazine-derived metabolites differentially modulated Aspergillus fumigatus development, shifting from weak vegetative growth to induced conidiation along a decreasing phenazine gradient, correlated with the induction of oxidative stress through phenazine redox cycling. Further, phenazine conidial signaling was conserved in A. nidulans, and mediated by several oxidative stress regulators, including NapA (a homolog of AP-1-like bZIP transcription factor that is essential for the response to oxidative stress in humans, yeast, and filamentous fungi), AtfA (a homolog of Atf1 bZIP transcription factor that mediates fungal response to environmental stresses), and NoxR (a key component in NADPH oxidase system regulating ROS generation). Lastly, the morphological altering effect of bioconversion phenazine 1-methoxyphenazine (1-Me-PHZ) was characterized. 1-Me-PHZ induced the formation of "spike" structures through NapA related pathway, associated with the suppression of ammonia secretion and consequently delayed extracellular alkalization. The morphological altering effect of 1-Me-PHZ was highly dependent on ambient pH and oxygen conditions. In summary, our data demonstrate the versatile roles of phenazines in modulating multiple processes during Aspergillus biofilm development. Our results can provide important implications in understanding the redox-active secondary metabolite mediated interspecies interactions in polymicrobial biofilms. This work provides a foundation for interspecies signaling in environmental and clinical biofilms involving bacteria and fungi. |
