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1. Structural bases for F plasmid conjugation and F pilus biogenesis in Escherichia coli

   https://doi.org/10.1073/pnas.1904428116  

   Hu, Bo; Khara, Pratick; Christie, Peter J. (July 2019, Proceedings of the National Academy of Sciences)

   null (Ed.)

   Bacterial conjugation systems are members of the large type IV secretion system (T4SS) superfamily. Conjugative transfer of F plasmids residing in the Enterobacteriaceae was first reported in the 1940s, yet the architecture of F plasmid-encoded transfer channel and its physical relationship with the F pilus remain unknown. We visualized F-encoded structures in the native bacterial cell envelope by in situ cryoelectron tomography (CryoET). Remarkably, F plasmids encode four distinct structures, not just the translocation channel or channel-pilus complex predicted by prevailing models. The F1 structure is composed of distinct outer and inner membrane complexes and a connecting cylinder that together house the envelope-spanning translocation channel. The F2 structure is essentially the F1 complex with the F pilus attached at the outer membrane (OM). Remarkably, the F3 structure consists of the F pilus attached to a thin, cell envelope-spanning stalk, whereas the F4 structure consists of the pilus docked to the OM without an associated periplasmic density. The traffic ATPase TraC is configured as a hexamer of dimers at the cytoplasmic faces of the F1 and F2 structures, where it respectively regulates substrate transfer and F pilus biogenesis. Together, our findings present architectural renderings of the DNA conjugation or “mating” channel, the channel–pilus connection, and unprecedented pilus basal structures. These structural snapshots support a model for biogenesis of the F transfer system and allow for detailed comparisons with other structurally characterized T4SSs. 

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2. A team of chaperones play to win in the bacterial periplasm

   https://doi.org/10.1016/j.tibs.2024.03.015  

   Devlin, Taylor; Fleming, Karen G (April 2024, Trends in Biochemical Sciences)

   The survival and virulence of Gram-negative bacteria require proper biogenesis and maintenance of the outer membrane (OM), which is densely packed with à-barrel OM proteins (OMPs). Before reaching the OM, precursor unfolded OMPs (uOMPs) must cross the whole cell envelope. A network of periplasmic chaperones and proteases maintains unfolded but folding-competent conformations of these membrane proteins in the aqueous periplasm while simultaneously preventing off-pathway aggregation. These periplasmic proteins utilize different strategies, including conformational heterogeneity, oligomerization, multivalency, and ki- netic partitioning, to perform and regulate their functions. Redundant and unique characteristics of the individual periplasmic players synergize to create a protein quality control team capable responding to changing environmental stresses. 

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3. Periplasmic stress contributes to a trade-off between protein secretion and cell growth in  Escherichia coli Nissle 1917

   https://doi.org/10.1093/synbio/ysad013  

   Emani, Sivaram Subaya; Kan, Anton; Storms, Timothy; Bonanno, Shanna; Law, Jade; Ray, Sanhita; Joshi, Neel S. (July 2023, Synthetic Biology)

   Abstract Maximizing protein secretion is an important target in the design of engineered living systems. In this paper, we characterize a trade-off between cell growth and per-cell protein secretion in the curli biofilm secretion system of Escherichia coli Nissle 1917. Initial characterization using 24-h continuous growth and protein production monitoring confirms decreased growth rates at high induction, leading to a local maximum in total protein production at intermediate induction. Propidium iodide (PI) staining at the endpoint indicates that cellular death is a dominant cause of growth reduction. Assaying variants with combinatorial constructs of inner and outer membrane secretion tags, we find that diminished growth at high production is specific to secretory variants associated with periplasmic stress mediated by outer membrane secretion and periplasmic accumulation of protein containing the outer membrane transport tag. RNA sequencing experiments indicate upregulation of known periplasmic stress response genes in the highly secreting variant, further implicating periplasmic stress in the growth–secretion trade-off. Overall, these results motivate additional strategies for optimizing total protein production and longevity of secretory engineered living systems Graphical Abstract 

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4. Computationally exploring the mechanism of bacteriophage T7 gp4 helicase translocating along ssDNA

   https://doi.org/10.1073/pnas.2202239119  

   Jin, Shikai; Bueno, Carlos; Lu, Wei; Wang, Qian; Chen, Mingchen; Chen, Xun; Wolynes, Peter G.; Gao, Yang (August 2022, Proceedings of the National Academy of Sciences)

   Bacteriophage T7 gp4 helicase has served as a model system for understanding mechanisms of hexameric replicative helicase translocation. The mechanistic basis of how nucleoside 5′-triphosphate hydrolysis and translocation of gp4 helicase are coupled is not fully resolved. Here, we used a thermodynamically benchmarked coarse-grained protein force field, Associative memory, Water mediated, Structure and Energy Model (AWSEM), with the single-stranded DNA (ssDNA) force field 3SPN.2C to investigate gp4 translocation. We found that the adenosine 5′-triphosphate (ATP) at the subunit interface stabilizes the subunit–subunit interaction and inhibits subunit translocation. Hydrolysis of ATP to adenosine 5′-diphosphate enables the translocation of one subunit, and new ATP binding at the new subunit interface finalizes the subunit translocation. The LoopD2 and the N-terminal primase domain provide transient protein–protein and protein–DNA interactions that facilitate the large-scale subunit movement. The simulations of gp4 helicase both validate our coarse-grained protein–ssDNA force field and elucidate the molecular basis of replicative helicase translocation. 

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5. Conserved lipid-facing basic residues promote the insertion of the porin OmpC into the E. coli outer membrane

   https://doi.org/10.1128/mbio.03319-24  

   Peterson, Janine H; Yang, Lixinhao; Gumbart, James C; Bernstein, Harris D (March 2025, mBio)

   Trent, M Stephen; Konovalova, Anna (Ed.)

   ABSTRACT Almost all integral membrane proteins that reside in the outer membrane (OM) of gram-negative bacteria contain a closed amphipathic β sheet (“β barrel”) that serves as a membrane anchor. The membrane integration of β barrel structures is catalyzed by a highly conserved heterooligomer called thebarrelassemblymachine (BAM). Although charged residues that are exposed to the lipid bilayer are infrequently found in outer membrane protein β barrels, the β barrels of OmpC/OmpF-type trimeric porins produced by Enterobacterales contain multiple conserved lipid-facing basic residues located near the extracellular side of the OM. Here, we show that these residues are required for the efficient insertion of theEscherichia coliOmpC protein into the OMin vivo. We found that the mutation of multiple basic residues to glutamine or alanine slowed insertion and reduced insertion efficiency. Furthermore, molecular dynamics simulations provided evidence that the basic residues promote the formation of hydrogen bonds and salt bridges with lipopolysaccharide, a unique glycolipid located exclusively in the outer leaflet of the OM. Taken together, our results support a model in which hydrophilic interactions between OmpC and LPS help to anchor the protein in the OM when the local environment is perturbed by BAM during membrane insertion and suggest a surprising role for membrane lipids in the insertion reaction.IMPORTANCEThe assembly (folding and membrane insertion) of bacterial outer membrane proteins (OMPs) is an essential cellular process that is a potential target for novel antibiotics. A heterooligomer called thebarrelassemblymachine (BAM) plays a major role in catalyzing OMP assembly. Here, we show that a group of highly conserved lipid-facing basic residues inEscherichia coliOmpC, a member of a major family of abundant OMPs known as trimeric porins, is required for the efficient integration of the protein into the outer membrane (OM). Based on our work and previous studies, we propose that the basic residues form interactions with a unique OM lipid (lipopolysaccharide) that promotes the insertion reaction. Our results provide strong evidence that interactions between specific membrane lipids and at least a subset of OMPs are required to supplement the activity of BAM and facilitate the integration of the proteins into the membrane. 

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