Spatial heterogeneity in biofilm metabolism elicited by local control of phenazine methylation

Abstract

ABSTRACTWithin biofilms, gradients of electron acceptors such as oxygen stimulate the formation of physiological subpopulations. This heterogeneity can enable cross-feeding and promote drug resilience, features of the multicellular lifestyle that make biofilm-based infections difficult to treat. The pathogenic bacteriumPseudomonas aeruginosaproduces pigments called phenazines that can support metabolic activity in hypoxic/anoxic biofilm subzones, but these compounds also include methylated derivatives that are toxic to their producer under some conditions. Here, we uncover roles for the global regulators RpoS and Hfq/Crc in controlling the beneficial and detrimental effects of methylated phenazines in biofilms. Our results indicate that RpoS controls phenazine methylation by modulating activity of the carbon catabolite repression pathway, in which the Hfq/Crc complex inhibits translation of the phenazine methyltransferase PhzM. We find that RpoS indirectly inhibits expression of CrcZ, a small RNA that binds to and sequesters Hfq/Crc, specifically in the oxic subzone ofP. aeruginosabiofilms. Deletion ofrpoSorcrctherefore leads to overproduction of methylated phenazines, which we show leads to increased metabolic activity—an apparent beneficial effect—in hypoxic/anoxic subpopulations within biofilms. However, we also find that biofilms lacking Crc show increased sensitivity to an exogenously added methylated phenazine, indicating that the increased metabolic activity in this mutant comes at a cost. Together, these results suggest that complex regulation of PhzM allowsP. aeruginosato simultaneously exploit the benefits and limit the toxic effects of methylated phenazines.Significance StatementP. aeruginosacauses biofilm-based infections and is known for its production of colorful phenazine derivatives. Among these the methylated phenazines are the most toxic and can cause condition-dependent damage to their producer. In this study, we show that methylated phenazines also have a beneficial effect in that they specifically support metabolic activity at depth inP. aeruginosabiofilms, where oxygen limitation would otherwise stall metabolism. We describe a new link betweenP. aeruginosaglobal regulators that control methylated phenazine production in a manner that limits their toxicity while simultaneously enabling their contribution to metabolism. These results expand our understanding of the strategies that enableP. aeruginosasurvival in multicellular structures, which is key to its success during chronic host colonization.