When nutrients in their environment are exhausted, bacterial cells become arrested for growth. During these periods, a primary challenge is maintaining cellular integrity with a reduced capacity for renewal or repair. Here, we show that the heat-shock protease FtsH is generally required for growth arrest survival of Pseudomonas aeruginosa, and that this requirement is independent of a role in regulating lipopolysaccharide synthesis, as has been suggested for Escherichia coli. We find that ftsH interacts with diverse genes during growth and overlaps functionally with the other heat-shock protease-encoding genes hslVU, lon, and clpXP to promote survival during growth arrest. Systematic deletion of the heat-shock protease-encoding genes reveals that the proteases function hierarchically during growth arrest, with FtsH and ClpXP having primary, nonredundant roles, and HslVU and Lon deploying a secondary response to aging stress. This hierarchy is partially conserved during growth at high temperature and alkaline pH, suggesting that heat, pH, and growth arrest effectively impose a similar type of proteostatic stress at the cellular level. In support of this inference, heat and growth arrest act synergistically to kill cells, and protein aggregation appears to occur more rapidly in protease mutants during growth arrest and correlates with the onset of cell death. Our findings suggest that protein aggregation is a major driver of aging and cell death during growth arrest, and that coordinated activity of the heat-shock response is required to ensure ongoing protein quality control in the absence of growth.
Classifying microorganisms as “obligate” aerobes has colloquially implied death without air, leading to the erroneous assumption that, without oxygen, they are unable to survive. However, over the past few decades, more than a few obligate aerobes have been found to possess anaerobic energy conservation strategies that sustain metabolic activity in the absence of growth or at very low growth rates. Similarly, studies emphasizing the aerobic prowess of certain facultative aerobes have sometimes led to underrecognition of their anaerobic capabilities. Yet an inescapable consequence of the affinity both obligate and facultative aerobes have for oxygen is that the metabolism of these organisms may drive this substrate to scarcity, making anoxic survival an essential skill. To illustrate this, we highlight the importance of anaerobic survival strategies for Pseudomonas aeruginosa and Streptomyces coelicolor, representative facultative and obligate aerobes, respectively. Included among these strategies, we describe a role for redox-active secondary metabolites (RAMs), such as phenazines made by P. aeruginosa, in enhancing substrate-level phosphorylation. Importantly, RAMs are made by diverse bacteria, often during stationary phase in the absence of oxygen, and can sustain anoxic survival. We present a hypothesis for how RAMs may enhance or even unlock energy conservation pathways that facilitate the anaerobic survival of both RAM producers and nonproducers.
Rab-GTPases and their interacting partners are key regulators of secretory vesicle trafficking, docking, and fusion to the plasma membrane in neurons and neuroendocrine cells. Where and how these proteins are positioned and organized with respect to the vesicle and plasma membrane are unknown. Here, we use correlative super-resolution light and platinum replica electron microscopy to map Rab-GTPases (Rab27a and Rab3a) and their effectors (Granuphilin-a, Rabphilin3a, and Rim2) at the nanoscale in 2D. Next, we apply a targetable genetically-encoded electron microscopy labeling method that uses histidine based affinity-tags and metal-binding gold-nanoparticles to determine the 3D axial location of these exocytic proteins and two SNARE proteins (Syntaxin1A and SNAP25) using electron tomography. Rab proteins are distributed across the entire surface and t-SNARE proteins at the base of docked vesicles. We propose that the circumferential distribution of Rabs and Rab-effectors could aid in the efficient transport, capture, docking, and rapid fusion of calcium-triggered exocytic vesicles in excitable cells.
Phenazines are redox‐active secondary metabolites produced by diverse bacteria including the opportunistic pathogen Pseudomonas aeruginosa. Extracellular electron transfer via phenazines enhances anaerobic survival by serving as an electron sink for glucose catabolism. However, the specific phenazine reductase(s) used to support this catabolism are unknown. Because electron transport chain components have been previously implicated in phenazine reduction, we sought to determine which of them possess phenazine reductase activity. We show that phenazine‐1‐carboxamide (PCN) and pyocyanin (PYO) are reduced at the highest rate by cells and are localized to the cell envelope while reduced. Using a coupled genetic and biochemical approach, we show that phenazine reductase activity in membrane fractions is attributable to the three NADH dehydrogenases present in P. aeruginosa and that their order of phenazine reductase activity is Nqr > Nuo > Ndh. In mutants possessing only one functional NADH dehydrogenase, whole cell reduction rates of PCN, but not PYO, recapitulate the pattern of biochemical results, implying that PYO reduction is predominantly occurring in the cytosol. Lastly, we show that ubiquinone rapidly and non‐enzymatically oxidizes reduced phenazines, demonstrating that phenazines have the capability to serve in a redox loop between the NADH and ubiquinone pools, a finding that carries bioenergetic implications.
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