Tunable force transduction through the Escherichia coli cell envelope.

The outer membrane (OM) of Gram-negative bacteria is not energised and so processes requiring a driving force must connect to energy-transduction systems in the inner membrane (IM). Tol (Tol-Pal) and Ton are related, proton motive force- (PMF-) coupled assemblies that stabilise the OM and import essential nutrients, respectively. Both rely on proton-harvesting IM motor (stator) complexes, which are homologues of the flagellar stator unit Mot, to transduce force to the OM through elongated IM force transducer proteins, TolA and TonB, respectively. How PMF-driven motors in the IM generate mechanical work at the OM via force transducers is unknown. Here, using cryoelectron microscopy, we report the 4.3Å structure of the Escherichia coli TolQR motor complex. The structure reaffirms the 5:2 stoichiometry seen in Ton and Mot and, with motor subunits related to each other by 10 to 16° rotation, supports rotary motion as the default for these complexes. We probed the mechanism of force transduction to the OM through in vivo assays of chimeric TolA/TonB proteins where sections of their structurally divergent, periplasm-spanning domains were swapped or replaced by an intrinsically disordered sequence. We find that TolA mutants exhibit a spectrum of force output, which is reflected in their respective abilities to both stabilise the OM and import cytotoxic colicins across the OM. Our studies demonstrate that structural rigidity of force transducer proteins, rather than any particular structural form, drives the efficient conversion of PMF-driven rotary motions of 5:2 motor complexes into physiologically relevant force at the OM.

Porin threading drives receptor disengagement and establishes active colicin transport through Escherichia coli OmpF.

Bacteria deploy weapons to kill their neighbours during competition for resources and to aid survival within microbiomes. Colicins were the first such antibacterial system identified, yet how these bacteriocins cross the outer membrane (OM) of Escherichia coli is unknown. Here, by solving the structures of translocation intermediates via cryo-EM and by imaging toxin import, we uncover the mechanism by which the Tol-dependent nuclease colicin E9 (ColE9) crosses the bacterial OM. We show that threading of ColE9's disordered N-terminal domain through two pores of the trimeric porin OmpF causes the colicin to disengage from its primary receptor, BtuB, and reorganises the translocon either side of the membrane. Subsequent import of ColE9 through the lumen of a single OmpF subunit is driven by the proton-motive force, which is delivered by the TolQ-TolR-TolA-TolB assembly. Our study answers longstanding questions, such as why OmpF is a better translocator than OmpC, and reconciles the mechanisms by which both Tol- and Ton-dependent bacteriocins cross the bacterial outer membrane.

Complex Formation between Two Biosynthetic Enzymes Modifies the Allosteric Regulatory Properties of Both: AN EXAMPLE OF MOLECULAR SYMBIOSIS.

Allostery, where remote ligand binding alters protein function, is essential for the control of metabolism. Here, we have identified a highly sophisticated allosteric response that allows complex control of the pathway for aromatic amino acid biosynthesis in the pathogen Mycobacterium tuberculosis. This response is mediated by an enzyme complex formed by two pathway enzymes: chorismate mutase (CM) and 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS). Whereas both enzymes are active in isolation, the catalytic activity of both enzymes is enhanced, and in particular that of the much smaller CM is greatly enhanced (by 120-fold), by formation of a hetero-octameric complex between CM and DAH7PS. Moreover, on complex formation M. tuberculosis CM, which has no allosteric response on its own, acquires allosteric behavior to facilitate its own regulatory needs by directly appropriating and partly reconfiguring the allosteric machinery that provides a synergistic allosteric response in DAH7PS. Kinetic and analytical ultracentrifugation experiments demonstrate that allosteric binding of phenylalanine specifically promotes hetero-octameric complex dissociation, with concomitant reduction of CM activity. Together, DAH7PS and CM from M. tuberculosis provide exquisite control of aromatic amino acid biosynthesis, not only controlling flux into the start of the pathway, but also directing the pathway intermediate chorismate into either Phe/Tyr or Trp biosynthesis.

Force-Generation by the Trans-Envelope Tol-Pal System.

The Tol-Pal system spans the cell envelope of Gram-negative bacteria, transducing the potential energy of the proton motive force (PMF) into dissociation of the TolB-Pal complex at the outer membrane (OM), freeing the lipoprotein Pal to bind the cell wall. The primary physiological role of Tol-Pal is to maintain OM integrity during cell division through accumulation of Pal molecules at division septa. How the protein complex couples the PMF at the inner membrane into work at the OM is unknown. The effectiveness of this trans-envelope energy transduction system is underscored by the fact that bacteriocins and bacteriophages co-opt Tol-Pal as part of their import/infection mechanisms. Mechanistic understanding of this process has been hindered by a lack of structural data for the inner membrane TolQ-TolR stator, of its complexes with peptidoglycan (PG) and TolA, and of how these elements combined power events at the OM. Recent studies on the homologous stators of Ton and Mot provide a starting point for understanding how Tol-Pal works. Here, we combine ab initio protein modeling with previous structural data on sub-complexes of Tol-Pal as well as mutagenesis, crosslinking, co-conservation analysis and functional data. Through this composite pooling of in silico, in vitro, and in vivo data, we propose a mechanism for force generation in which PMF-driven rotary motion within the stator drives conformational transitions within a long TolA helical hairpin domain, enabling it to reach the TolB-Pal complex at the OM.

Lipids mediate supramolecular outer membrane protein assembly in bacteria.

ß Barrel outer membrane proteins (OMPs) cluster into supramolecular assemblies that give function to the outer membrane (OM) of Gram-negative bacteria. How such assemblies form is unknown. Here, through photoactivatable cross-linking into the Escherichia coli OM, coupled with simulations, and biochemical and biophysical analysis, we uncover the basis for OMP clustering in vivo. OMPs are typically surrounded by an annular shell of asymmetric lipids that mediate higher-order complexes with neighboring OMPs. OMP assemblies center on the abundant porins OmpF and OmpC, against which low-abundance monomeric ß barrels, such as TonB-dependent transporters, are packed. Our study reveals OMP-lipid-OMP complexes to be the basic unit of supramolecular OMP assembly that, by extending across the entire cell surface, couples the requisite multifunctionality of the OM to its stability and impermeability.

Toxin import through the antibiotic efflux channel TolC.

Bacteria often secrete diffusible protein toxins (bacteriocins) to kill bystander cells during interbacterial competition. Here, we use biochemical, biophysical and structural analyses to show how a bacteriocin exploits TolC, a major outer-membrane antibiotic efflux channel in Gram-negative bacteria, to transport itself across the outer membrane of target cells. Klebicin C (KlebC), a rRNase toxin produced by Klebsiella pneumoniae, binds TolC of a related species (K. quasipneumoniae) with high affinity through an N-terminal, elongated helical hairpin domain common amongst bacteriocins. The KlebC helical hairpin opens like a switchblade to bind TolC. A cryo-EM structure of this partially translocated state, at 3.1 Å resolution, reveals that KlebC associates along the length of the TolC channel. Thereafter, the unstructured N-terminus of KlebC protrudes beyond the TolC iris, presenting a TonB-box sequence to the periplasm. Association with proton-motive force-linked TonB in the inner membrane drives toxin import through the channel. Finally, we demonstrate that KlebC binding to TolC blocks drug efflux from bacteria. Our results indicate that TolC, in addition to its known role in antibiotic export, can function as a protein import channel for bacteriocins.

Structural Analysis of the Menangle Virus P Protein Reveals a Soft Boundary between Ordered and Disordered Regions.

The paramyxoviral phosphoprotein (P protein) is the non-catalytic subunit of the viral RNA polymerase, and coordinates many of the molecular interactions required for RNA synthesis. All paramyxoviral P proteins oligomerize via a centrally located coiled-coil that is connected to a downstream binding domain by a dynamic linker. The C-terminal region of the P protein coordinates interactions between the catalytic subunit of the polymerase, and the viral nucleocapsid housing the genomic RNA. The inherent flexibility of the linker is believed to facilitate polymerase translocation. Here we report biophysical and structural characterization of the C-terminal region of the P protein from Menangle virus (MenV), a bat-borne paramyxovirus with zoonotic potential. The MenV P protein is tetrameric but can dissociate into dimers at sub-micromolar protein concentrations. The linker is globally disordered and can be modeled effectively as a worm-like chain. However, NMR analysis suggests very weak local preferences for alpha-helical and extended beta conformation exist within the linker. At the interface between the disordered linker and the structured C-terminal binding domain, a gradual disorder-to-order transition occurs, with X-ray crystallographic analysis revealing a dynamic interfacial structure that wraps the surface of the binding domain.

Colicin-Mediated Transport of DNA through the Iron Transporter FepA.

Colicins are protein antibiotics deployed by Escherichia coli to eliminate competing strains. Colicins frequently exploit outer membrane (OM) nutrient transporters to penetrate the selectively permeable bacterial cell envelope. Here, by applying live-cell fluorescence imaging, we were able to monitor the entry of the pore-forming toxin colicin B (ColB) into E. coli and localize it within the periplasm. We further demonstrate that single-stranded DNA coupled to ColB can also be transported to the periplasm, emphasizing that the import routes of colicins can be exploited to carry large cargo molecules into bacteria. Moreover, we characterize the molecular mechanism of ColB association with its OM receptor FepA by applying a combination of photoactivated cross-linking, mass spectrometry, and structural modeling. We demonstrate that complex formation is coincident with large-scale conformational changes in the colicin. Thereafter, active transport of ColB through FepA involves the colicin taking the place of the N-terminal half of the plug domain that normally occludes this iron transporter. IMPORTANCE Decades of excessive use of readily available antibiotics has generated a global problem of antibiotic resistance and, hence, an urgent need for novel antibiotic solutions. Bacteriocins are protein-based antibiotics produced by bacteria to eliminate closely related competing bacterial strains. Bacteriocin toxins have evolved to bypass the complex cell envelope in order to kill bacterial cells. Here, we uncover the cellular penetration mechanism of a well-known but poorly understood bacteriocin called colicin B that is active against Escherichia coli. Moreover, we demonstrate that the colicin B-import pathway can be exploited to deliver conjugated DNA cargo into bacterial cells. Our work leads to a better understanding of the way bacteriocins, as potential alternative antibiotics, execute their mode of action as well as highlighting how they might even be exploited in the genomic manipulation of Gram-negative bacteria.

A method for analyzing the composition of viral nucleoprotein complexes, produced by heterologous expression in bacteria.

Viral genomes are protected and organized by virally encoded packaging proteins. Heterologous production of these proteins often results in formation of particles resembling the authentic viral capsid or nucleocapsid, with cellular nucleic acids packaged in place of the viral genome. Quantifying the total protein and nucleic acid content of particle preparations is a recurrent biochemical problem. We describe a method for resolving this problem, developed when characterizing particles resembling the Menangle Virus nucleocapsid. The protein content was quantified using the biuret assay, which is largely independent of amino acid composition. Bound nucleic acids were quantified by determining the phosphorus content, using inductively coupled plasma mass spectrometry. Estimates for the amount of RNA packaged within the particles were consistent with the structurally-characterized packaging mechanism. For a bacterially-produced nucleoprotein complex, phosphorus usually provides a unique elemental marker of bound nucleic acids, hence this method of analysis should be routinely applicable.