Redox-cycling compounds, including endogenously produced phenazine antibiotics, induce expression of the efflux pump MexGHIOpmD in the opportunistic pathogen Pseudomonas aeruginosa. Previous studies of P. aeruginosa virulence, physiology, and biofilm development have focused on the blue phenazine pyocyanin and the yellow phenazine-1-carboxylic acid (PCA). In P. aeruginosa phenazine biosynthesis, conversion of PCA to pyocyanin is presumed to proceed through the intermediate 5-methylphenazine-1-carboxylate (5-Me-PCA), a reactive compound that has eluded detection in most laboratory samples. Here, we apply electrochemical methods to directly detect 5-Me-PCA and find that it is transported by MexGHIOpmD in P. aeruginosa strain PA14 planktonic and biofilm cells. We also show that 5-Me-PCA is sufficient to fully induce MexGHI-OpmD expression and that it is required for wild-type colony biofilm morphogenesis. These physiological effects are consistent with the high redox potential of 5-Me-PCA, which distinguishes it from other well-studied P. aeruginosa phenazines. Our observations highlight the importance of this compound, which was previously overlooked due to the challenges associated with its detection, in the context of P. aeruginosa gene expression and multicellular behavior. This study constitutes a unique demonstration of efflux-based selfresistance, controlled by a simple circuit, in a Gram-negative pathogen.phenazine | RND efflux | MexGHI-OpmD | biofilm | antibiotic
Highlights d P. aeruginosa-infected macrophages produce itaconate d Itaconate generates membrane stress in P. aeruginosa d Itaconate leads to decreased LPS, but increased EPS, to promote biofilm formation d The EPS-itaconate axis thwarts immune clearance enabling chronic infection
Diverse organisms secrete redox-active antibiotics, which can be used as extracellular electron shuttles by resistant microbes. Shuttlemediated metabolism can support survival when substrates are available not locally but rather at a distance. Such conditions arise in multicellular communities, where the formation of chemical gradients leads to resource limitation for cells at depth. In the pathogenic bacterium Pseudomonas aeruginosa PA14, antibiotics called phenazines act as oxidants to balance the intracellular redox state of cells in anoxic biofilm subzones. PA14 colony biofilms show a profound morphogenic response to phenazines resulting from electron acceptor-dependent inhibition of ECM production. This effect is reminiscent of the developmental responses of some eukaryotic systems to redox control, but for bacterial systems its mechanistic basis has not been well defined. Here, we identify the regulatory protein RmcA and show that it links redox conditions to PA14 colony morphogenesis by modulating levels of bis-(3′,5′)-cyclic-dimeric-guanosine (c-di-GMP), a second messenger that stimulates matrix production, in response to phenazine availability. RmcA contains four Per-Arnt-Sim (PAS) domains and domains with the potential to catalyze the synthesis and degradation of c-di-GMP. Our results suggest that phenazine production modulates RmcA activity such that the protein degrades c-di-GMP and thereby inhibits matrix production during oxidizing conditions. RmcA thus forms a mechanistic link between cellular redox sensing and community morphogenesis analogous to the functions performed by PAS-domain-containing regulatory proteins found in complex eukaryotes.W hen microbial cells cannot import or are physically separated from metabolic electron donors or acceptors, diffusible compounds can act as electron carriers and support survival on these substrates (1, 2). These conditions arise in the presence of poised-potential electrodes or insoluble minerals, such as iron oxides (3-5), and in multicellular communities (biofilms) where the formation of chemical gradients leads to oxidant limitation for cells at depth (6-9). Diverse microbes secrete redoxactive compounds with the capacity to function as electron shuttles (10-12). In the pathogenic bacterium Pseudomonas aeruginosa PA14, electron-shuttling antibiotics called phenazines support survival on poised-potential electrodes and balance the intracellular redox state of cells in anoxic biofilm subzones (1, 9).Similar to those formed by many species of microbes, colonies of PA14 develop intricate wrinkle structures on agar-solidified growth media (13). Phenazines profoundly alter PA14 colony morphogenesis, inhibiting the onset of wrinkle formation and changing the organization of wrinkles (12) (Fig. 1A). Modeling of resource availability within colonies suggests that the earlier increase in the colony surface area-to-volume ratio in phenazine-null (Δphz) mutants maximizes access to oxygen for cells that would otherwise become limited for oxidant (14). Measurements of...
Pseudomonas species and other aerobic bacteria have a biotin-independent malonate decarboxylase that is crucial for their utilization of malonate as the sole carbon and energy source. The malonate decarboxylase holoenzyme contains four subunits, having an acyl-carrier protein (MdcC subunit) with a distinct prosthetic group, as well as decarboxylase (MdcD–MdcE) and acyl-carrier protein transferase (MdcA) catalytic activities. Here we report the crystal structure of a Pseudomonas malonate decarboxylase hetero-tetramer, as well as biochemical and functional studies based on the structural information. We observe a malonate molecule in the active site of MdcA and we also determine the structure of malonate decarboxylase with CoA in the active site of MdcD–MdcE. Both structures provide molecular insights into malonate decarboxylase catalysis. Mutations in the hetero-tetramer interface can abolish holoenzyme formation. Mutations in the hetero-tetramer interface and the active sites can abolish Pseudomonas aeruginosa growth in a defined medium with malonate as the sole carbon source.
Voltage-gated calcium (Cav) channels and protein kinase C (PKC) isozymes are involved in insulin secretion. In addition, Cavβ, one of the auxiliary subunits of Cav channels, also regulate the secretion of insulin as knockout of Cavβ3 (β3−/−) subunits in mice led to efficient glucose homeostasis and increased insulin levels. We examined if other types of Cavβ subunits also have similar properties. In this regard, we used small interfering RNA (siRNA) of these subunits (20 μg each) to down regulate them and examined blood glucose, serum insulin and PKC translocation in isolated pancreatic β cells of mice. While the down regulation of Cavβ2 and β3 subunits increased serum insulin levels and caused efficient glucose homeostasis, the down regulation of Cavβ1 and β4 subunits failed to affect both these parameters. Examination of PKC isozymes in the pancreatic β-cells of Cavβ2 or β3 siRNA injected mice showed that three PKC isozymes, viz., PKCα, βII and θ translocated to the membrane. This suggests that when present, Cavβ2 and β3 subunits inhibited PKC activation. Among these three isozymes, only PKCα siRNA inhibited insulin and increased glucose concentrations. It is possible that the activation of PKCs βII and θ are not sufficient for the release of insulin and PKCα is the mediator of insulin secretion under the control of Cavβ subunits. Since Cavβ subunits are present intracellularly, it is possible that they i) inhibited the translocation of PKC isozymes to the membrane and ii) decreased the interaction between Cav channels and PKC isozymes and thus the secretion of insulin.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.