Multicomponent efflux complexes constitute a primary mechanism for Gram-negative bacteria to expel toxic molecules for survival. As these complexes traverse the periplasm and link inner and outer membranes, it remains unclear how they operate efficiently without compromising periplasmic plasticity. Combining single-molecule superresolution imaging and genetic engineering, we study in living Escherichia coli cells the tripartite efflux complex CusCBA of the resistance-nodulation-division family that is essential for bacterial resistance to drugs and toxic metals. We find that CusCBA complexes are dynamic structures and shift toward the assembled form in response to metal stress. Unexpectedly, the periplasmic adaptor protein CusB is a key metal-sensing element that drives the assembly of the efflux complex ahead of the transcription activation of the cus operon for defending against metals. This adaptor protein-mediated dynamic pump assembly allows the bacterial cell for efficient efflux upon cellular demand while still maintaining periplasmic plasticity; this could be broadly relevant to other multicomponent efflux systems.multicomponent efflux complex | substrate-responsive dynamic assembly | periplasmic adaptor protein | metal sensing | single-molecule tracking B acteria are often exposed to harsh environments, including high metal ion concentrations and toxic organic molecules. Efflux of metal ions helps bacteria maintain appropriate intracellular concentrations of essential metals while removing toxic ones (1-6). Efflux of organic molecules, including antibiotics, is a key mechanism for bacterial multidrug resistance (7-14). The tripartite resistance-nodulation-division (RND) family efflux pumps confer major clinically relevant drug resistance in Gram-negative bacteria such as Escherichia coli and the infectious Pseudomonas aeruginosa (7-14). They are composed of a proton-motive-force-driven inner-membrane pump, a periplasmic adaptor protein, and an outer-membrane channel. Once assembled, these pumps traverse the cell periplasm, providing a direct extrusion pathway from the periplasm (and cytoplasm) to the outside of the cell. However, these direct pathways also tightly link the inner and outer membranes, which, if overly stable, would impede the periplasm's plasticity and ability to respond dynamically to external and internal stimuli to buffer the cell from changes in its surroundings (15).How can tripartite efflux pumps operate without compromising the dynamic nature of the periplasm? One possibility is that these efflux complexes are dynamic structures and assemble only in the presence of their substrates. This mechanism has been hypothesized for the E. coli HlyBD-TolC complex (16), in which HlyB is a ATP-binding cassette superfamily efflux pump. However, experimental validation of this mechanism, as well as its relevance to the RND family efflux pumps, remains elusive, partly due to the difficulty in studying two membrane proteins together with a periplasmic protein under physiologically relevant conditi...
Physical forces have a profound effect on growth, morphology, locomotion, and survival of organisms. At the level of individual cells, the role of mechanical forces is well recognized in eukaryotic physiology, but much less is known about prokaryotic organisms. Recent findings suggest an effect of physical forces on bacterial shape, cell division, motility, virulence, and biofilm initiation, but it remains unclear how mechanical forces applied to a bacterium are translated at the molecular level. In Gram-negative bacteria, multicomponent protein complexes can form rigid links across the cell envelope and are therefore subject to physical forces experienced by the cell. Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and use single-molecule tracking to show that octahedral shear (but not hydrostatic) stress within the cell envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used byEscherichia colito resist copper and silver toxicity. By promoting disassembly of this protein complex, mechanical forces within the cell envelope make the bacteria more susceptible to metal toxicity. These findings demonstrate that mechanical forces can inhibit the function of cell envelope protein assemblies in bacteria and suggest the possibility that other multicomponent, transenvelope efflux complexes may be sensitive to mechanical forces including complexes involved in antibiotic resistance, cell division, and translocation of outer membrane components. By modulating the function of proteins within the cell envelope, mechanical stress has the potential to regulate multiple processes required for bacterial survival and growth.
19 20 21 22 Physical forces have long been recognized for their effects on the growth, 23 morphology, locomotion, and survival of eukaryotic organisms 1 . Recently, mechanical 24 forces have been shown to regulate processes in bacteria, including cell division 2 , motility 3 , 25 virulence 4 , biofilm initiation 5,6 , and cell shape 7,8 , although it remains unclear how 26 mechanical forces in the cell envelope lead to changes in molecular processes. In Gram-27 negative bacteria, multicomponent protein complexes that form rigid links across the cell 28 envelope directly experience physical forces and mechanical stresses applied to the cell. 29Here we manipulate tensile and shear mechanical stress in the bacterial cell envelope and 30 use single-molecule tracking to show that shear (but not tensile) stress within the cell 31 envelope promotes disassembly of the tripartite efflux complex CusCBA, a system used by 32 E. coli to resist copper and silver toxicity, thereby making bacteria more susceptible to 33 metal toxicity. These findings provide the first demonstration that mechanical forces, such 34 as those generated during colony overcrowding or bacterial motility through submicron 35 pores, can inhibit the contact and function of multicomponent complexes in bacteria. As 36 multicomponent, trans-envelope efflux complexes in bacteria are involved in many 37 processes including antibiotic resistance 9 , cell division 10 , and translocation of outer 38 membrane components 11 , our findings suggest that the mechanical environment may 39 regulate multiple processes required for bacterial growth and survival. 40
The unprecedented 2020 COVID‐19 pandemic prompted the rapid shift to online learning in higher education. For courses particularly in the natural sciences, this shift posed a challenge to adapt complex course material that relied on in‐person demonstrations and hands‐on laboratory experiences to successfully be mastered. Online learning continues grow in these fields where it was once limited. In this piece, we describe the rejuvenation of our problem‐based learning Introduction to Biochemistry for students in a virtual environment. We utilized Zoom for engaging discussions between student‐led discussion groups focused on their reading of primary literature, adapted labs with course content to enable them to be performed at home, and utilized the online format to connect with a scientist whose work we studied. In particular, our course centered around the mechanisms of antibiotics, beginning with Pasteur’s discovery of fermentation and ending with present day papers deconvoluting the synthesis of bacterial peptidoglycan and how different antibiotics inhibit this pathway. Throughout the course, we were able to utilize the online format and at home labs to connect science to everyday life in different ways than traditional classroom learning. Students overall were engaged and enjoyed how this course was different than other online courses. Our observations provide guidance for how to improve future online‐based science courses, particularly problem‐based learning ones, to make them most effective for successful student learning.
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