Exploring the Localization of Siderophore-Mediated Cargo Delivery in Gram-Negative Bacteria Using 3-Hydroxypyridin-4(1H)-one-Fluorescein Probes.

The design of siderophore-antibiotic conjugates is a promising strategy to overcome drug resistance in negative bacteria. However, accumulating studies have shown that only those antibiotics acting on the cell wall or cell membrane multiply their antibacterial effects when coupled with siderophores, while antibiotics acting on targets in the cytoplasm of bacteria do not show an obvious enhancement of their antibacterial effects when coupled with siderophores. To explore the causes of this phenomenon, we synthesized several conjugate probes using 3-hydroxypyridin-4(1H)-ones as siderophores and replacing the antibiotic cargo with 5-carboxyfluorescein (5-FAM) or malachite green (MG) cargo. By monitoring changes in the fluorescence intensity of FAM conjugate 20 in bacteria, the translocation of the conjugate across the outer membranes of Gram-negative pathogens was confirmed. Further, the use of the fluorogen activating protein(FAP)/MG system revealed that 3-hydroxypyridin-4(1H)-one-MG conjugate 26 was ultimately distributed mainly in the periplasm rather than being translocated into the cytosol of Escherichia coli and Pseudomonas aeruginosa PAO1. Additional mechanistic studies suggested that the uptake of the conjugate involved the siderophore-dependent iron transport pathway and the 3-hydroxypyridin-4(1H)-ones siderophore receptor-dependent mechanism. Meanwhile, we demonstrated that the conjugation of 3-hydroxypyridin-4(1H)-ones to the fluorescein 5-FAM can reduce the possibility of the conjugates crossing the membrane layers of mammalian Vero cells by passive diffusion, and the advantages of the mono-3-hydroxypyridin-4(1H)-ones as a delivery vehicle in the design of conjugates compared to the tri-3-hydroxypyridin-4(1H)-ones. Overall, this work reveals the localization rules of 3-hydroxypyridin-4(1H)-ones as siderophores to deliver the cargo into Gram-negative bacteria. It provides a theoretical basis for the subsequent design of siderophore-antibiotic conjugates, especially based on 3-hydroxypyridin-4(1H)-ones as siderophores.

Biosensor that Detects Stress Caused by Periplasmic Proteins.

Escherichia coli is often used as a factory to produce recombinant proteins. In many cases, the recombinant protein needs disulfide bonds to fold and function correctly. These proteins are genetically fused to a signal peptide so that they are secreted to the oxidizing environment of the periplasm (where the enzymes required for disulfide bond formation exist). Currently, it is difficult to determine in vivo whether a recombinant protein is efficiently secreted from the cytoplasm and folded in the periplasm or if there is a bottleneck in one of these steps because cellular capacity has been exceeded. To address this problem, we have developed a biosensor that detects cellular stress caused by (1) inefficient secretion of proteins from the cytoplasm and (2) aggregation of proteins in the periplasm. We demonstrate how the fluorescence fingerprint obtained from the biosensor can be used to identify induction conditions that do not exceed the capacity of the cell and therefore do not cause cellular stress. These induction conditions result in more effective biomass and in some cases higher titers of soluble recombinant proteins.

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

The survival and virulence of Gram-negative bacteria require proper biogenesis and maintenance of the outer membrane (OM), which is densely packed with ß-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 kinetic 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.

Scs system links copper and redox homeostasis in bacterial pathogens.

The bacterial envelope is an essential compartment involved in metabolism and metabolites transport, virulence, and stress defense. Its roles become more evident when homeostasis is challenged during host-pathogen interactions. In particular, the presence of free radical groups and excess copper in the periplasm causes noxious reactions, such as sulfhydryl group oxidation leading to enzymatic inactivation and protein denaturation. In response to this, canonical and accessory oxidoreductase systems are induced, performing quality control of thiol groups, and therefore contributing to restoring homeostasis and preserving survival under these conditions. Here, we examine recent advances in the characterization of the Dsb-like, Salmonella-specific Scs system. This system includes the ScsC/ScsB pair of Cu+-binding proteins with thiol-oxidoreductase activity, an alternative ScsB-partner, the membrane-linked ScsD, and a likely associated protein, ScsA, with a role in peroxide resistance. We discuss the acquisition of the scsABCD locus and its integration into a global regulatory pathway directing envelope response to Cu stress during the evolution of pathogens that also harbor the canonical Dsb systems. The evidence suggests that the canonical Dsb systems cannot satisfy the extra demands that the host-pathogen interface imposes to preserve functional thiol groups. This resulted in the acquisition of the Scs system by Salmonella. We propose that the ScsABCD complex evolved to connect Cu and redox stress responses in this pathogen as well as in other bacterial pathogens.

A role for the ATP-dependent DNA ligase lig E of Neisseria gonorrhoeae in biofilm formation.

BACKGROUND: The ATP-dependent DNA ligase Lig E is present as an accessory DNA ligase in numerous proteobacterial genomes, including many disease-causing species. Here we have constructed a genomic Lig E knock-out in the obligate human pathogen Neisseria gonorrhoeae and characterised its growth and infection phenotype. RESULTS: This demonstrates that N. gonorrhoeae Lig E is a non-essential gene and its deletion does not cause defects in replication or survival of DNA-damaging stressors. Knock-out strains were partially defective in biofilm formation on an artificial surface as well as adhesion to epithelial cells. In addition to in vivo characterisation, we have recombinantly expressed and assayed N. gonorrhoeae Lig E and determined the crystal structure of the enzyme-adenylate engaged with DNA substrate in an open non-catalytic conformation. CONCLUSIONS: These findings, coupled with the predicted extracellular/ periplasmic location of Lig E indicates a role in extracellular DNA joining as well as providing insight into the binding dynamics of these minimal DNA ligases.

Aberrant Topologies of Bacterial Membrane Proteins Revealed by High Sensitivity Fluorescence Labelling.

The cytoplasmic membrane compartmentalises the bacterial cell into cytoplasm and periplasm. Proteins located in this membrane have a defined topology that is established during their biogenesis. However, the accuracy of this fundamental biosynthetic process is unknown. We developed compartment-specific fluorescence labelling methods with up to single-molecule sensitivity. Application of these methods to the single and multi-spanning membrane proteins of the Tat protein transport system revealed rare topogenesis errors. This methodology also detected low level soluble protein mislocalization from the cytoplasm to the periplasm. This study shows that it is possible to uncover rare errors in protein localization by leveraging the high sensitivity of fluorescence methods.

Proteomics from compartment-specific APEX2 labeling in Mycobacterium tuberculosis reveals Type VII secretion substrates in the cell wall.

The cell wall of mycobacteria plays a key role in interactions with the environment. Its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites, from ions to lipids to proteins. Identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that although chemical labeling of live cells did not exclusively label surface proteins, protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis accurately identified the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

Single-protein Diffusion in the Periplasm of Escherichia coli.

The width of the periplasmic space of Gram-negative bacteria is only about 25-30 nm along the long axis of the cell, which affects free diffusion of (macro)molecules. We have performed single-particle displacement measurements and diffusion simulation studies to determine the impact of confinement on the apparent mobility of proteins in the periplasm of Escherichia coli. The diffusion of a reporter protein and of OsmY, an osmotically regulated periplasmic protein, is characterized by a fast and slow component regardless of the osmotic conditions. The diffusion coefficient of the fast fraction increases upon osmotic upshift, in agreement with a decrease in macromolecular crowding of the periplasm, but the mobility of the slow (immobile) fraction is not affected by the osmotic stress. We observe that the confinement created by the inner and outer membranes results in a lower apparent diffusion coefficient, but this can only partially explain the slow component of diffusion in the particle displacement measurements, suggesting that a fraction of the proteins is hindered in its mobility by large periplasmic structures. Using particle-based simulations, we have determined the confinement effect on the apparent diffusion coefficient of the particles for geometries akin the periplasmic space of Gram-negative bacteria.

Approaches for high-throughput quantification of periplasmic recombinant proteins.

The Gram-negative periplasm is a convenient location for the accumulation of many recombinant proteins including biopharmaceutical products. It is the site of disulphide bond formation, required by some proteins (such as antibody fragments) for correct folding and function. It also permits simpler protein release and downstream processing than cytoplasmic accumulation. As such, targeting of recombinant proteins to the E. coli periplasm is a key strategy in biologic manufacture. However, expression and translocation of each recombinant protein requires optimisation including selection of the best signal peptide and growth and production conditions. Traditional methods require separation and analysis of protein compositions of periplasmic and cytoplasmic fractions, a time- and labour-intensive method that is difficult to parallelise. Therefore, approaches for high throughput quantification of periplasmic protein accumulation offer advantages in rapid process development.

A critical role of the periplasm in copper homeostasis in Gram-negative bacteria.

Copper is essential for life, but is toxic in excess. Copper homeostasis is achieved in the cytoplasm and the periplasm as a unique feature of Gram-negative bacteria. Especially, it has become clear the role of the periplasm and periplasmic proteins regarding whole-cell copper homeostasis. Here, we addressed the role of the periplasm and periplasmic proteins in copper homeostasis using a Systems Biology approach integrating experiments with models. Our analysis shows that most of the copper-bound molecules localize in the periplasm but not cytoplasm, suggesting that Escherichia coli utilizes the periplasm to sense the copper concentration in the medium and sequester copper ions. In particular, a periplasmic multi-copper oxidase CueO and copper-responsive transcriptional factor CusS contribute both to protection against Cu(I) toxicity and to incorporating copper into the periplasmic components/proteins. We propose that Gram-negative bacteria have evolved mechanisms to sense and store copper in the periplasm to expand their living niches.

The role of glutathione in periplasmic redox homeostasis and oxidative protein folding in Escherichia coli.

The thiol redox balance in the periplasm of E. coli depends on the DsbA/B pair for oxidative power and the DsbC/D system as its complement for isomerization of non-native disulfides. While the standard redox potentials of those systems are known, the in vivo "steady state" redox potential imposed onto protein thiol disulfide pairs in the periplasm remains unknown. Here, we used genetically encoded redox probes (roGFP2 and roGFP-iL), targeted to the periplasm, to directly probe the thiol redox homeostasis in this compartment. These probes contain two cysteine residues that are virtually completely reduced in the cytoplasm, but once exported into the periplasm, can form a disulfide bond, a process that can be monitored by fluorescence spectroscopy. Even in the absence of DsbA, roGFP2, exported to the periplasm, was almost fully oxidized, suggesting the presence of an alternative system for the introduction of disulfide bonds into exported proteins. However, the absence of DsbA shifted the steady state periplasmic thiol-redox potential from -228 mV to a more reducing -243 mV and the capacity to re-oxidize periplasmic roGFP2 after a reductive pulse was significantly decreased. Re-oxidation in a DsbA strain could be fully restored by exogenous oxidized glutathione (GSSG), while reduced GSH accelerated re-oxidation of roGFP2 in the WT. In line, a strain devoid of endogenous glutathione showed a more reducing periplasm, and was significantly worse in oxidatively folding PhoA, a native periplasmic protein and substrate of the oxidative folding machinery. PhoA oxidative folding could be enhanced by the addition of exogenous GSSG in the WT and fully restored in a ΔdsbA mutant. Taken together this suggests the presence of an auxiliary, glutathione-dependent thiol-oxidation system in the bacterial periplasm.

Systematic conformation-to-phenotype mapping via limited deep sequencing of proteins.

Non-native conformations drive protein-misfolding diseases, complicate bioengineering efforts, and fuel molecular evolution. No current experimental technique is well suited for elucidating them and their phenotypic effects. Especially intractable are the transient conformations populated by intrinsically disordered proteins. We describe an approach to systematically discover, stabilize, and purify native and non-native conformations, generated in vitro or in vivo, and directly link conformations to molecular, organismal, or evolutionary phenotypes. This approach involves high-throughput disulfide scanning (HTDS) of the entire protein. To reveal which disulfides trap which chromatographically resolvable conformers, we devised a deep-sequencing method for double-Cys variant libraries of proteins that precisely and simultaneously locates both Cys residues within each polypeptide. HTDS of the abundant E. coli periplasmic chaperone HdeA revealed distinct classes of disordered hydrophobic conformers with variable cytotoxicity depending on where the backbone was cross-linked. HTDS can bridge conformational and phenotypic landscapes for many proteins that function in disulfide-permissive environments.

Highly efficient export of a disulfide-bonded protein to the periplasm and medium by the Tat pathway using CyDisCo in Escherichia coli.

High-value heterologous proteins produced in Escherichia coli that contain disulfide bonds are almost invariably targeted to the periplasm via the Sec pathway as it, among other advantages, enables disulfide bond formation and simplifies downstream processing. However, the Sec system cannot transport complex or rapidly folding proteins, as it only transports proteins in an unfolded state. The Tat system also transports proteins to the periplasm, and it has significant potential as an alternative means of recombinant protein production because it transports fully folded proteins. Most of the studies related to Tat secretion have used the well-studied TorA signal peptide that is Tat-specific, but this signal peptide also tends to induce degradation of the protein of interest, resulting in lower yields. This makes it difficult to use Tat in the industry. In this study, we show that a model disulfide bond-containing protein, YebF, can be exported to the periplasm and media at a very high level by the Tat pathway in a manner almost completely dependent on cytoplasmic disulfide formation, by other two putative Tat SPs: those of MdoD and AmiC. In contrast, the TorA SP exports YebF at a low level.

The Periplasmic Tail-Specific Protease, Tsp, Is Essential for Secondary Differentiation in Chlamydia trachomatis.

The obligate intracellular human pathogen Chlamydia trachomatis (Ctr) undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms: the elementary body (EB) and the reticulate body (RB). The EB is the smaller, infectious, nondividing form which initiates infection of a susceptible host cell, whereas the RB is the larger, non-infectious form which replicates within a membrane-bound vesicle called an inclusion. The mechanism(s) which drives differentiation between these developmental forms is poorly understood. Bulk protein turnover is likely required for chlamydial differentiation given the significant differences in the protein repertoires and functions of the EB and RB. We hypothesize that periplasmic protein turnover is also critical for the reorganization of an RB into an EB, referred to as secondary differentiation. Ct441 is a periplasmic protease ortholog of tail-specific proteases (i.e., Tsp, Prc) and is expressed in Ctr during secondary differentiation. We investigated the effect of altering Tsp expression on developmental cycle progression. Through assessment of bacterial morphology and infectious progeny production, we found that both overexpression and CRISPR interference/dCas9 (CRISPRi)-mediated knockdown of Tsp negatively impacted chlamydial development through different mechanisms. We also confirmed that catalytic activity is required for the negative effect of overexpression and confirmed the effect of the mutation in in vitro assays. Electron microscopic assessments during knockdown experiments revealed a defect in EB morphology, directly linking Tsp function to secondary differentiation. These data implicate Ct441/Tsp as a critical factor in secondary differentiation. IMPORTANCE The human pathogen Chlamydia trachomatis is the leading cause of preventable infectious blindness and bacterial sexually transmitted infections worldwide. This pathogen has a unique developmental cycle that alternates between distinct forms. However, the key processes of chlamydial development remain obscure. Uncovering the mechanisms of differentiation between its metabolically and functionally distinct developmental forms may foster the discovery of novel Chlamydia-specific therapeutics and limit development of resistant bacterial populations derived from the clinical use of broad-spectrum antibiotics. In this study, we investigate chlamydial tail-specific protease (Tsp) and its function in chlamydial growth and development. Our work implicates Tsp as essential to chlamydial developmental cycle progression and indicates that Tsp is a potential drug target for Chlamydia infections.

Cell wall proteomics in live Mycobacterium tuberculosis uncovers exposure of ESX substrates to the periplasm.

The cell wall of mycobacteria plays a key role in interactions with the environment and its ability to act as a selective filter is crucial to bacterial survival. Proteins in the cell wall enable this function by mediating the import and export of diverse metabolites from ions to lipids to proteins. Accurately identifying cell wall proteins is an important step in assigning function, especially as many mycobacterial proteins lack functionally characterized homologues. Current methods for protein localization have inherent limitations that reduce accuracy. Here we showed that protein tagging by the engineered peroxidase APEX2 within live Mycobacterium tuberculosis enabled the accurate identification of the cytosolic and cell wall proteomes. Our data indicate that substrates of the virulence-associated Type VII ESX secretion system are exposed to the Mtb periplasm, providing insight into the currently unknown mechanism by which these proteins cross the mycobacterial cell envelope.

Physiological and regulatory convergence between osmotic and nutrient stress responses in microbes.

Bacterial cells are regularly confronted with simultaneous changes in environmental nutrient supply and osmolarity. Despite the importance of osmolarity and osmoregulation in bacterial physiology, the relationship between the cellular response to osmotic perturbations and other stresses has remained largely unexplored. Bacteria cultured in hyperosmotic conditions and bacteria experiencing nutrient stress exhibit similar physiological changes, including metabolic shutdown, increased protein instability, dehydration, and condensation of chromosomal DNA. In this review, we highlight overlapping molecular players between osmotic and nutrient stresses. These connections between two seemingly disparate stress response pathways reinforce the importance of central carbon metabolism as a control point for diverse aspects of homeostatic regulation. We identify important open questions for future research, emphasizing the pressing need to develop and exploit new methods for probing how osmolarity affects phylogenetically diverse species.

Specific N-terminal amino acids potentiate the periplasmic expression of single-chain variable fragments in Escherichia coli.

Single-chain variable fragments (ScFvs) are important in therapy, diagnosis and research because of their elevated antigen affinity and low immunogenicity. At present, high-yield scFv expression in Escherichia coli is limited by insoluble aggregation in the reducing environment of the cytoplasm or low yields in the periplasm. Here we achieved increased expression of scFvs in the periplasm by inserting optimal amino acids between the signal peptide and scFv. We constructed an expression library with three random amino acids at the scFv N-terminus, screened this library with a single-step colony assay and identified the specific sequences that boosted periplasmic expression of scFvs.

Formation of Fibrils by the Periplasmic Molecular Chaperone HdeB from Escherichia coli.

The molecular chaperones HdeA and HdeB of the Escherichia coli (E. coli) periplasm protect client proteins from acid denaturation through a unique mechanism that utilizes their acid denatured states to bind clients. We previously demonstrated that the active, acid-denatured form of HdeA is also prone to forming inactive, amyloid fibril-like aggregates in a pH-dependent, reversible manner. In this study, we report that HdeB also displays a similar tendency to form fibrils at low pH. HdeB fibrils were observed at pH < 3 in the presence of NaCl. Similar to HdeA, HdeB fibrils could be resolubilized by a simple shift to neutral pH. In the case of HdeB, however, we found that after extended incubation at low pH, HdeB fibrils were converted into a form that could not resolubilize at pH 7. Fresh fibrils seeded from these "transformed" fibrils were also incapable of resolubilizing at pH 7, suggesting that the transition from reversible to irreversible fibrils involved a specific conformational change that was transmissible through fibril seeds. Analyses of fibril secondary structure indicated that HdeB fibrils retained significant alpha helical content regardless of the conditions under which fibrils were formed. Fibrils that were formed from HdeB that had been treated to remove its intrinsic disulfide bond also were incapable of resolubilizing at pH 7, suggesting that certain residual structures that are retained in acid-denatured HdeB are important for this protein to recover its soluble state from the fibril form.

Lack of Specificity in Geobacter Periplasmic Electron Transfer.

Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are utilized depending on redox potential, and at least five cytochrome conduits span the outer membrane. Because G. sulfurreducens produces 5 structurally similar triheme periplasmic cytochromes (PpcABCDE) that differ in expression level, midpoint potential, and heme biochemistry, many hypotheses propose distinct periplasmic carriers could be used for specific redox potentials, terminal acceptors, or growth conditions. Using a panel of marker-free single, quadruple, and quintuple mutants, little support for these models could be found. Three quadruple mutants containing only one paralog (PpcA, PpcB, and PpcD) reduced Fe(III) citrate and Fe(III) oxide at the same rate and extent, even though PpcB and PpcD were at much lower periplasmic levels than PpcA. Mutants containing only PpcC and PpcE showed defects, but these cytochromes were nearly undetectable in the periplasm. When expressed sufficiently, PpcC and PpcE supported wild-type Fe(III) reduction. PpcA and PpcE from G. metallireducens similarly restored metal respiration in G. sulfurreducens. PgcA, an unrelated extracellular triheme c-type cytochrome, also participated in periplasmic electron transfer. While triheme cytochromes were important for metal reduction, sextuple ΔppcABCDE ΔpgcA mutants grew near wild-type rates with normal cyclic voltammetry profiles when using anodes as electron acceptors. These results reveal broad promiscuity in the periplasmic electron transfer network of metal-reducing Geobacter and suggest that an as-yet-undiscovered periplasmic mechanism supports electron transfer to electrodes. IMPORTANCE Many inner and outer membrane cytochromes used by Geobacter for electron transfer to extracellular acceptors have specific functions. How these are connected by periplasmic carriers remains poorly understood. G. sulfurreducens contains multiple triheme periplasmic cytochromes with unique biochemical properties and expression profiles. It is hypothesized that each could be involved in a different respiratory pathway, depending on redox potential or energy needs. Here, we show that Geobacter periplasmic cytochromes instead show evidence of being highly promiscuous. Any of 6 triheme cytochromes supported similar growth with soluble or insoluble metals, but none were required when cells utilized electrodes. These findings fail to support many models of Geobacter electron transfer, and question why these organisms produce such an array of periplasmic cytochromes.

Bacterial Outer Membrane Polysaccharide Export (OPX) Proteins Occupy Three Structural Classes with Selective ß-Barrel Porin Requirements for Polymer Secretion.

Secretion of high-molecular-weight polysaccharides across the bacterial envelope is ubiquitous, as it enhances prokaryotic survival in (a)biotic settings. Such polymers are often assembled by Wzx/Wzy- or ABC transporter-dependent schemes implicating outer membrane (OM) polysaccharide export (OPX) proteins in cell-surface polymer translocation. In the social predatory bacterium Myxococcus xanthus, the exopolysaccharide (EPS) pathway WzaX, major spore coat (MASC) pathway WzaS, and biosurfactant polysaccharide (BPS) pathway WzaB were herein found to be truncated OPX homologues of Escherichia coli Wza lacking OM-spanning α-helices. Comparative genomics across all bacteria (>91,000 OPX proteins identified and analyzed), complemented with cryo-electron tomography cell-envelope analyses, revealed such "truncated" WzaX/S/B architecture to be the most common among three defined OPX-protein structural classes independent of periplasm thickness. Fold recognition and deep learning revealed the conserved M. xanthus proteins MXAN_7418/3226/1916 (encoded beside wzaX/S/B, respectively) to be integral OM ß-barrels, with structural homology to the poly-N-acetyl-d-glucosamine synthase-dependent pathway porin PgaA. Such bacterial porins were identified near numerous genes for all three OPX protein classes. Interior MXAN_7418/3226/1916 ß-barrel electrostatics were found to match properties of their associated polymers. With MXAN_3226 essential for MASC export, and MXAN_7418 herein shown to mediate EPS translocation, we have designated this new secretion machinery component "Wzp" (i.e., Wz porin), with the final step of M. xanthus EPS/MASC/BPS secretion across the OM now proposed to be mediated by WzpX/S/B (i.e., MXAN_7418/3226/1916). Importantly, these data support a novel and widespread secretion paradigm for polysaccharide biosynthesis pathways in which those containing OPX components that cannot span the OM instead utilize ß-barrel porins to mediate polysaccharide transport across the OM. IMPORTANCE Diverse bacteria assemble and secrete polysaccharides that alter their physiologies through modulation of motility, biofilm formation, and host immune system evasion. Most such pathways require outer membrane (OM) polysaccharide export (OPX) proteins for sugar-polymer transport to the cell surface. In the prototypic Escherichia coli Group-1-capsule biosynthesis system, eight copies of this canonical OPX protein cross the OM with an α-helix, forming a polysaccharide-export pore. Herein, we instead reveal that most OPX proteins across all bacteria lack this α-helix, raising questions as to the manner by which most secreted polysaccharides actually exit cells. In the model developmental bacterium Myxococcus xanthus, we show this process to depend on OPX-coupled OM-spanning ß-barrel porins, with similar porins encoded near numerous OPX genes in diverse bacteria. Knowledge of the terminal polysaccharide secretion step will enable development of antimicrobial compounds targeted to blocking polymer export from outside the cell, thus bypassing any requirements for antimicrobial compound uptake by the cell.