Substrates Control Multimerization and Activation of the Multi-Domain ATPase Motor of Type VII Secretion.

Mycobacterium tuberculosis and Staphylococcus aureus secrete virulence factors via type VII protein secretion (T7S), a system that intriguingly requires all of its secretion substrates for activity. To gain insights into T7S function, we used structural approaches to guide studies of the putative translocase EccC, a unique enzyme with three ATPase domains, and its secretion substrate EsxB. The crystal structure of EccC revealed that the ATPase domains are joined by linker/pocket interactions that modulate its enzymatic activity. EsxB binds via its signal sequence to an empty pocket on the C-terminal ATPase domain, which is accompanied by an increase in ATPase activity. Surprisingly, substrate binding does not activate EccC allosterically but, rather, by stimulating its multimerization. Thus, the EsxB substrate is also an integral T7S component, illuminating a mechanism that helps to explain interdependence of substrates, and suggests a model in which binding of substrates modulates their coordinate release from the bacterium.

Pseudomonas aeruginosa PumA acts on an endogenous phenazine to promote self-resistance.

The activities of critical metabolic and regulatory proteins can be altered by exposure to natural or synthetic redox-cycling compounds. Many bacteria, therefore, possess mechanisms to transport or transform these small molecules. The opportunistic pathogen Pseudomonas aeruginosa PA14 synthesizes phenazines, redox-active antibiotics that are toxic to other organisms but have beneficial effects for their producer. Phenazines activate the redox-sensing transcription factor SoxR and thereby induce the transcription of a small regulon, including the operon mexGHI-opmD, which encodes an efflux pump that transports phenazines, and PA14_35160 (pumA), which encodes a putative monooxygenase. Here, we provide evidence that PumA contributes to phenazine resistance and normal biofilm development, particularly during exposure to or production of strongly oxidizing N-methylated phenazines. We show that phenazine resistance depends on the presence of residues that are conserved in the active sites of other putative and characterized monooxygenases found in the antibiotic producer Streptomyces coelicolor. We also show that during biofilm growth, PumA is required for the conversion of phenazine methosulfate to unique phenazine metabolites. Finally, we compare ∆mexGHI-opmD and ∆pumA strains in assays for colony biofilm morphogenesis and SoxR activation, and find that these deletions have opposing phenotypic effects. Our results suggest that, while MexGHI-OpmD-mediated efflux has the effect of making the cellular phenazine pool more reducing, PumA acts on cellular phenazines to make the pool more oxidizing. We present a model in which these two SoxR targets function simultaneously to control the biological activity of the P. aeruginosa phenazine pool.

Phenazine oxidation by a distal electrode modulates biofilm morphogenesis.

Microbes living in biofilms, dense assemblages of cells, experience limitation for resources such as oxygen when cellular consumption outpaces diffusion. The pathogenic bacterium Pseudomonas aeruginosa has strategies for coping with hypoxia that support cellular redox balancing in biofilms; these include (1) increasing access to oxygen by forming wrinkles in the biofilm surface and (2) electrochemically reducing endogenous compounds called phenazines, which can shuttle electrons to oxidants available at a distance. Phenazine-mediated extracellular electron transfer (EET) has been shown to support survival for P. aeruginosa cells in anoxic liquid cultures, but the physiological relevance of EET over a distance for P. aeruginosa biofilms has remained unconfirmed. Here, we use a custom-built electrochemistry setup to show that phenazine-mediated electron transfer at a distance inhibits wrinkle formation in P. aeruginosa biofilms. This result demonstrates that phenazine-dependent EET to a distal oxidant affects biofilm morphogenesis.