Structural insights into the mechanism of protein transport by the Type 9 Secretion System translocon.

Secretion systems are protein export machines that enable bacteria to exploit their environment through the release of protein effectors. The Type 9 Secretion System (T9SS) is responsible for protein export across the outer membrane (OM) of bacteria of the phylum Bacteroidota. Here we trap the T9SS of Flavobacterium johnsoniae in the process of substrate transport by disrupting the T9SS motor complex. Cryo-EM analysis of purified substrate-bound T9SS translocons reveals an extended translocon structure in which the previously described translocon core is augmented by a periplasmic structure incorporating the proteins SprE, PorD and a homologue of the canonical periplasmic chaperone Skp. Substrate proteins bind to the extracellular loops of a carrier protein within the translocon pore. As transport intermediates accumulate on the translocon when energetic input is removed, we deduce that release of the substrate-carrier protein complex from the translocon is the energy-requiring step in T9SS transport.

Interbacterial competition mediated by the type VIIb secretion system.

Successful occupancy of a given niche requires the colonising bacteria to interact extensively with the biotic and abiotic environment, including other resident microbes. Bacteria have evolved a range of protein secretion machines for this purpose with eleven such systems identified to date. The type VIIb secretion system (T7SSb) is utilised by Bacillota to secrete a range of protein substrates, including antibacterial toxins targeting closely related strains, and the system as a whole has been implicated in a range of activities such as iron acquisition, intercellular signalling, host colonisation and virulence. This review covers the components and secretion mechanism of the T7SSb, the substrates of these systems and their roles in Gram-positive bacteria, with a focus on interbacterial competition.

Three small partner proteins facilitate the type VII-dependent secretion of an antibacterial nuclease.

IMPORTANCE: Staphylococcus aureus is an opportunistic human pathogen associated with severe infections and antimicrobial resistance. S. aureus strains utilize a type VII secretion system to secrete toxins targeting competitor bacteria, likely facilitating colonization. EsaD is a nuclease toxin secreted by the type VII secretion system in many strains of S. aureus as well as other related bacterial species. Here, we identify three small proteins of previously unknown function as export factors, required for efficient secretion of EsaD. We show that these proteins bind to the transport domain of EsaD, forming a complex with a striking cane-like conformation.

Three small partner proteins facilitate the type VII-dependent secretion export of an antibacterial nuclease.

The type VIIb protein secretion system (T7SSb) plays a role in interbacterial competition in Gram-positive Firmicute bacteria and secretes various toxic effector proteins. The mechanism of secretion and the roles of numerous conserved genes within T7SSb gene clusters remain unknown. EsaD is a nuclease toxin secreted by the Staphylococcus aureus T7SSb, which forms a complex with its cognate immunity protein, EsaG, and chaperone EsaE. Encoded upstream of EsaD are three small secreted proteins, EsxB, EsxC and EsxD. Here we show that EsxBCD bind to the transport domain of EsaD and function as EsaD export factors. We report the first structural information for a complete T7SSb substrate pre-secretion complex. Cryo-EM of the EsaDEG trimer and the EsaDEG-EsxBCD hexamer shows that incorporation of EsxBCD confers a conformation comprising a flexible globular cargo domain attached to a long narrow shaft that is likely to be crucial for efficient toxin export.

New insights into the Tat protein transport cycle from characterizing the assembled Tat translocon.

The twin-arginine protein translocation (Tat) system transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane of chloroplasts. The Tat translocation site is transiently assembled by the recruitment of multiple TatA proteins to a substrate-activated TatBC receptor complex in a process requiring the protonmotive force. The ephemeral nature of the Tat translocation site has so far precluded its isolation. We now report that detergent solubilization of membranes during active transport allows the recovery of receptor complexes that are associated with elevated levels of TatA. We apply this biochemical analysis in combination with live cell fluorescence imaging to Tat systems trapped in the assembled state. We resolve sub-steps in the Tat translocation cycle and infer that TatA assembly precedes the functional interaction of TatA with a polar cluster site on TatC. We observe that dissipation of the protonmotive force releases TatA oligomers from the assembled translocation site demonstrating that the stability of the TatA oligomer does not depend on binding to the receptor complex and implying that the TatA oligomer is assembled at the periphery of the receptor complex. This work provides new insight into the Tat transport cycle and advances efforts to isolate the active Tat translocon.

Structure and mechanism of the proton-driven motor that powers type 9 secretion and gliding motility.

Three classes of ion-driven protein motors have been identified to date: ATP synthase, the bacterial flagellar motor and a proton-driven motor that powers gliding motility and the type 9 protein secretion system in Bacteroidetes bacteria. Here, we present cryo-electron microscopy structures of the gliding motility/type 9 protein secretion system motors GldLM from Flavobacterium johnsoniae and PorLM from Porphyromonas gingivalis. The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM/PorM proteins are positioned inside a ring of five GldL/PorL proteins. Mutagenesis and single-molecule tracking identify protonatable amino acid residues in the transmembrane domain of the complex that are important for motor function. Our data provide evidence for a mechanism in which proton flow results in rotation of the periplasm-spanning GldM/PorM dimer inside the intra-membrane GldL/PorL ring to drive processes at the bacterial outer membrane.

Commensal Escherichia coli are a reservoir for the transfer of XDR plasmids into epidemic fluoroquinolone-resistant Shigella sonnei.

Despite the sporadic detection of fluoroquinolone-resistant Shigella in Asia in the early 2000s and the subsequent global spread of ciprofloxacin-resistant (cipR) Shigella sonnei from 2010, fluoroquinolones remain the recommended therapy for shigellosis1-7. The potential for cipR S. sonnei to develop resistance to alternative second-line drugs may further limit future treatment options8. Here, we aim to understand the evolution of novel antimicrobial resistant (AMR) S. sonnei variants after introduction into Vietnam. We found that cipR S. sonnei displaced the resident ciprofloxacin-susceptible (cipS) lineage while rapidly acquiring additional resistance to multiple alternative antimicrobial classes. We identified several independent acquisitions of extensively drug-resistant/multidrug-resistant-inducing plasmids, probably facilitated by horizontal transfer from commensals in the human gut. By characterizing commensal Escherichia coli from Shigella-infected and healthy children, we identified an extensive array of AMR genes and plasmids, including an identical multidrug-resistant plasmid isolated from both S. sonnei and E. coli in the gut of a single child. We additionally found that antimicrobial usage may impact plasmid transfer between commensal E. coli and S. sonnei. These results suggest that, in a setting with high antimicrobial use and a high prevalence of AMR commensals, cipR S. sonnei may be propelled towards pan-resistance by adherence to outdated international treatment guidelines.

Evolution of mitochondrial TAT translocases illustrates the loss of bacterial protein transport machines in mitochondria.

BACKGROUND: Bacteria and mitochondria contain translocases that function to transport proteins across or insert proteins into their inner and outer membranes. Extant mitochondria retain some bacterial-derived translocases but have lost others. While BamA and YidC were integrated into general mitochondrial protein transport pathways (as Sam50 and Oxa1), the inner membrane TAT translocase, which uniquely transports folded proteins across the membrane, was retained sporadically across the eukaryote tree. RESULTS: We have identified mitochondrial TAT machinery in diverse eukaryotic lineages and define three different types of eukaryote-encoded TatABC-derived machineries (TatAC, TatBC and TatC-only). Here, we investigate TatAC and TatC-only machineries, which have not been studied previously. We show that mitochondria-encoded TatAC of the jakobid Andalucia godoyi represent the minimal functional pathway capable of substituting for the Escherichia coli TatABC complex and can transport at least one substrate. However, selected TatC-only machineries, from multiple eukaryotic lineages, were not capable of supporting the translocation of this substrate across the bacterial membrane. Despite the multiple losses of the TatC gene from the mitochondrial genome, the gene was never transferred to the cell nucleus. Although the major constraint preventing nuclear transfer of mitochondrial TatC is likely its high hydrophobicity, we show that in chloroplasts, such transfer of TatC was made possible due to modifications of the first transmembrane domain. CONCLUSIONS: At its origin, mitochondria inherited three inner membrane translocases Sec, TAT and Oxa1 (YidC) from its bacterial ancestor. Our work shows for the first time that mitochondrial TAT has likely retained its unique function of transporting folded proteins at least in those few eukaryotes with TatA and TatC subunits encoded in the mitochondrial genome. However, mitochondria, in contrast to chloroplasts, abandoned the machinery multiple times in evolution. The overall lower hydrophobicity of the Oxa1 protein was likely the main reason why this translocase was nearly universally retained in mitochondrial biogenesis pathways.

An investigation into the Omp85 protein BamK in hypervirulent Klebsiella pneumoniae, and its role in outer membrane biogenesis.

Members of the Omp85 protein superfamily have important roles in Gram-negative bacteria, with the archetypal protein BamA being ubiquitous given its essential function in the assembly of outer membrane proteins. In some bacterial lineages, additional members of the family exist and, in most of these cases, the function of the protein is unknown. We detected one of these Omp85 proteins in the pathogen Klebsiella pneumoniae B5055, and refer to the protein as BamK. Here, we show that bamK is a conserved element in the core genome of Klebsiella, and its expression rescues a loss-of-function ∆bamA mutant. We developed an E. coli model system to measure and compare the specific activity of BamA and BamK in the assembly reaction for the critical substrate LptD, and find that BamK is as efficient as BamA in assembling the native LptDE complex. Comparative structural analysis revealed that the major distinction between BamK and BamA is in the external facing surface of the protein, and we discuss how such changes may contribute to a mechanism for resistance against infection by bacteriophage.

In vivo experiments do not support the charge zipper model for Tat translocase assembly.

The twin-arginine translocase (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat translocation site is formed by substrate-triggered oligomerization of the protein TatA. Walther and co-workers have proposed a structural model for the TatA oligomer in which TatA monomers self-assemble using electrostatic 'charge zippers' (Cell (2013) 132: 15945). This model was supported by in vitro analysis of the oligomeric state of TatA variants containing charge-inverting substitutions. Here we have used live cell assays of TatA assembly and function in Escherichia coli to re-assess the roles of the charged residues of TatA. Our results do not support the charge zipper model. Instead, we observe that substitutions of charged residues located in the TatA amphipathic helix lock TatA in an assembled state, suggesting that these charged residues play a critical role in the protein translocation step that follows TatA assembly.

A signal sequence suppressor mutant that stabilizes an assembled state of the twin arginine translocase.

The twin-arginine protein translocation (Tat) system mediates transport of folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. The Tat system of Escherichia coli is made up of TatA, TatB, and TatC components. TatBC comprise the substrate receptor complex, and active Tat translocases are formed by the substrate-induced association of TatA oligomers with this receptor. Proteins are targeted to TatBC by signal peptides containing an essential pair of arginine residues. We isolated substitutions, locating to the transmembrane helix of TatB that restored transport activity to Tat signal peptides with inactivating twin arginine substitutions. A subset of these variants also suppressed inactivating substitutions in the signal peptide binding site on TatC. The suppressors did not function by restoring detectable signal peptide binding to the TatBC complex. Instead, site-specific cross-linking experiments indicate that the suppressor substitutions induce conformational change in the complex and movement of the TatB subunit. The TatB F13Y substitution was associated with the strongest suppressing activity, even allowing transport of a Tat substrate lacking a signal peptide. In vivo analysis using a TatA-YFP fusion showed that the TatB F13Y substitution resulted in signal peptide-independent assembly of the Tat translocase. We conclude that Tat signal peptides play roles in substrate targeting and in triggering assembly of the active translocase.

Assembling the Tat protein translocase.

The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes.

The TatC component of the twin-arginine protein translocase functions as an obligate oligomer.

The Tat protein export system translocates folded proteins across the bacterial cytoplasmic membrane and the plant thylakoid membrane. The Tat system in Escherichia coli is composed of TatA, TatB and TatC proteins. TatB and TatC form an oligomeric, multivalent receptor complex that binds Tat substrates, while multiple protomers of TatA assemble at substrate-bound TatBC receptors to facilitate substrate transport. We have addressed whether oligomerisation of TatC is an absolute requirement for operation of the Tat pathway by screening for dominant negative alleles of tatC that inactivate Tat function in the presence of wild-type tatC. Single substitutions that confer dominant negative TatC activity were localised to the periplasmic cap region. The variant TatC proteins retained the ability to interact with TatB and with a Tat substrate but were unable to support the in vivo assembly of TatA complexes. Blue-native PAGE analysis showed that the variant TatC proteins produced smaller TatBC complexes than the wild-type TatC protein. The substitutions did not alter disulphide crosslinking to neighbouring TatC molecules from positions in the periplasmic cap but abolished a substrate-induced disulphide crosslink in transmembrane helix 5 of TatC. Our findings show that TatC functions as an obligate oligomer.

Live cell imaging shows reversible assembly of the TatA component of the twin-arginine protein transport system.

The twin-arginine translocation (Tat) machinery transports folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of chloroplasts. It has been inferred that the Tat translocation site is assembled on demand by substrate-induced association of the protein TatA. We tested this model by imaging YFP-tagged TatA expressed at native levels in living Escherichia coli cells in the presence of low levels of the TatA paralogue TatE. Under these conditions the TatA-YFP fusion supports full physiological Tat transport activity. In agreement with the TatA association model, raising the number of transport-competent substrate proteins within the cell leads to an increase in the number of large TatA complexes present. Formation of these complexes requires both a functional TatBC substrate receptor and the transmembrane proton motive force (PMF). Removing the PMF causes TatA complexes to dissociate, except in strains with impaired Tat transport activity. Based on these observations we propose that TatA assembly reaches a critical point at which oligomerization can be reversed only by substrate transport. In contrast to TatA-YFP, the oligomeric states of TatB-YFP and TatC-YFP fusions are not affected by substrate or the PMF, although TatB-YFP oligomerization does require TatC.

A small Tim homohexamer in the relict mitochondrion of Cryptosporidium.

The apicomplexan parasite Cryptosporidium parvum possesses a mitosome, a relict mitochondrion with a greatly reduced metabolic capability. This mitosome houses a mitochondrial-type protein import apparatus, but elements of the protein import pathway have been reduced, and even lost, through evolution. The small Tim protein family is a case in point. The genomes of C. parvum and related species of Cryptosporidium each encode just one small Tim protein, CpTimS. This observation challenged the tenet that small Tim proteins are always found in pairs as α3ß3 hexamers. We show that the atypical CpTimS exists as a relatively unstable homohexamer, shedding light both on the early evolution of the small Tim protein family and on small Tim hexamer formation in contemporary eukaryotes.

Minor modifications and major adaptations: the evolution of molecular machines driving mitochondrial protein import.

Bacterial endosymbionts gave rise to mitochondria in a process that depended on the acquisition of protein import pathways. Modification and in some cases major re-tooling of the endosymbiont's cellular machinery produced these pathways, establishing mitochondria as organelles common to all eukaryotic cells. The legacy of this evolutionary tinkering can be seen in the homologies and structural similarities between mitochondrial protein import machinery and modern day bacterial proteins. Comparative analysis of these systems is revealing both possible routes for the evolution of the mitochondrial membrane translocases and a greater understanding of the mechanisms behind mitochondrial protein import. This article is part of a Special Issue entitled Protein translocation across or insertion into membranes.

Mitochondrial ATP-independent chaperones.

Mitochondria possess a dedicated-chaperone system in the intermembrane space, the small Tims that are ubiquitous in all eukaryotes from yeast to man. They escort membrane proteins to the outer or the inner membrane for proper insertion. These mitochondrial chaperones do not require external energy to perform their function and have structural similarities to other ATP-independent chaperones. Here, we discuss their structural properties and how these relate to their chaperoning function in the mitochondrial intermembrane space.

Mammalian OS-9 is upregulated in response to endoplasmic reticulum stress and facilitates ubiquitination of misfolded glycoproteins.

Proteins that fail to fold or assemble with partner subunits are selectively removed from the endoplasmic reticulum (ER) via the ER-associated degradation (ERAD) pathway. Proteins selected for ERAD are polyubiquitinated and retrotranslocated into the cytosol for degradation by the proteasome. Although it is unclear how proteins are initially identified by the ERAD system in mammalian cells, OS-9 was recently proposed to play a key role in this process. Here we show that OS-9 is upregulated in response to ER stress and is associated both with components of the ERAD machinery and with ERAD substrates. Using RNA interference, we show that OS-9 is required for efficient ubquitination of glycosylated ERAD substrates, suggesting that it helps transfer misfolded proteins to the ubiquitination machinery. We also find that OS-9 binds to a misfolded nonglycosylated protein destined for ERAD, but not to the properly folded wild-type protein. Surprisingly, however, OS-9 is not required for ubiquitination or degradation of this nonglycosylated ERAD substrate. We propose a model in which OS-9 recognises terminally misfolded proteins via polypeptide-based rather than glycan-based signals, but is only required for transferring those bearing N-glycans to the ubiquitination machinery.