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Journal of Bacteriology Jun 2023The outer membranes (OM) of Gram-negative bacteria contain a class of proteins (TBDTs) that require energy for the import of nutrients and to serve as receptors for... (Review)
Review
The outer membranes (OM) of Gram-negative bacteria contain a class of proteins (TBDTs) that require energy for the import of nutrients and to serve as receptors for phages and protein toxins. Energy is derived from the proton motif force (pmf) of the cytoplasmic membrane (CM) through the action of three proteins, namely, TonB, ExbB, and ExbD, which are located in the CM and extend into the periplasm. The leaky phenotype of exbB exbD mutants is caused by partial complementation by homologous tolQ tolR. TonB, ExbB, and ExbD are genuine components of an energy transmission system from the CM into the OM. Mutant analyses, cross-linking experiments, and most recently X-ray and cryo-EM determinations were undertaken to arrive at a model that describes the energy transfer from the CM into the OM. These results are discussed in this paper. ExbB forms a pentamer with a pore inside, in which an ExbD dimer resides. This complex harvests the energy of the pmf and transmits it to TonB. TonB interacts with the TBDT at the TonB box, which triggers a conformational change in the TBDT that releases bound nutrients and opens the pore, through which nutrients pass into the periplasm. The structurally altered TBDT also changes the interactions of its periplasmic signaling domain with anti-sigma factors, with the consequence being that the sigma factors initiate transcription.
Topics: Escherichia coli Proteins; Escherichia coli; Membrane Proteins; Cell Membrane; Biological Transport; Bacterial Proteins
PubMed: 37219427
DOI: 10.1128/jb.00035-23 -
BioRxiv : the Preprint Server For... Aug 2023Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. phage P22 has four such systems...
Many temperate phages encode prophage-expressed functions that interfere with superinfection of the host bacterium by external phages. phage P22 has four such systems that are expressed from the prophage in a lysogen that are encoded by the (repressor), , , and genes. Here we report that the P22-encoded SieA protein is the only phage protein required for exclusion by the SieA system, and that it is an inner membrane protein that blocks DNA injection by P22 and its relatives, but has no effect on infection by other tailed phage types. The P22 virion injects its DNA through the host cell membranes and periplasm via a conduit assembled from three "ejection proteins" after their release from the virion. Phage P22 mutants were isolated that overcome the SieA block, and they have amino acid changes in the C-terminal regions of the gene and encoded ejection proteins. Three different single amino acid changes in these proteins are required to obtain nearly full resistance to SieA. Hybrid P22 phages that have phage HK620 ejection protein genes are also partially resistant to SieA. There are three sequence types of extant phage-encoded SieA proteins that are less than 30% identical to one another, yet comparison of two of these types found no differences in target specificity. Our data are consistent with a model in which the inner membrane protein SieA interferes with the assembly or function of the periplasmic gp20 and membrane-bound gp16 DNA delivery conduit.
PubMed: 37645741
DOI: 10.1101/2023.08.15.553423 -
Nature May 2021Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year. Specialized protein...
Mycobacterium tuberculosis is the cause of one of the most important infectious diseases in humans, which leads to 1.4 million deaths every year. Specialized protein transport systems-known as type VII secretion systems (T7SSs)-are central to the virulence of this pathogen, and are also crucial for nutrient and metabolite transport across the mycobacterial cell envelope. Here we present the structure of an intact T7SS inner-membrane complex of M. tuberculosis. We show how the 2.32-MDa ESX-5 assembly, which contains 165 transmembrane helices, is restructured and stabilized as a trimer of dimers by the MycP protease. A trimer of MycP caps a central periplasmic dome-like chamber that is formed by three EccB dimers, with the proteolytic sites of MycP facing towards the cavity. This chamber suggests a central secretion and processing conduit. Complexes without MycP show disruption of the EccB periplasmic assembly and increased flexibility, which highlights the importance of MycP for complex integrity. Beneath the EccB-MycP chamber, dimers of the EccC ATPase assemble into three bundles of four transmembrane helices each, which together seal the potential central secretion channel. Individual cytoplasmic EccC domains adopt two distinctive conformations that probably reflect different secretion states. Our work suggests a previously undescribed mechanism of protein transport and provides a structural scaffold to aid in the development of drugs against this major human pathogen.
Topics: Cryoelectron Microscopy; Cytosol; Models, Molecular; Mycobacterium tuberculosis; Periplasm; Protein Domains; Protein Multimerization; Protein Stability; Tuberculosis; Type VII Secretion Systems
PubMed: 33981042
DOI: 10.1038/s41586-021-03517-z -
Research in Microbiology 2019FtsEX is a member of a small subclass of ABC transporters that uses mechano-transmission to perform work in the periplasm. FtsEX controls periplasmic peptidoglycan (PG)... (Review)
Review
FtsEX is a member of a small subclass of ABC transporters that uses mechano-transmission to perform work in the periplasm. FtsEX controls periplasmic peptidoglycan (PG) hydrolase activities in many Gram negative and positive organisms to ensure the safe separation of daughter cells during division. In these organisms FtsEX localizes to the Z ring and uses its ATPase activity to regulate its periplasmic effectors. In Escherichia coli, FtsEX also participates in building the divisome and coordinates PG synthesis with PG hydrolysis. This review discusses studies that are beginning to elucidate the mechanisms of FtsEX's various roles in cell division.
Topics: ATP-Binding Cassette Transporters; Biomechanical Phenomena; Cell Cycle Proteins; Cell Division; Cell Wall; Cystic Fibrosis Transmembrane Conductance Regulator; Escherichia coli; Escherichia coli Proteins; N-Acetylmuramoyl-L-alanine Amidase; Peptidoglycan
PubMed: 31376483
DOI: 10.1016/j.resmic.2019.07.003 -
Frontiers in Microbiology 2022Several new structures of three types of protein complexes, obtained by cryo-electron microscopy (cryo-EM) and published between 2019 and 2021, identify a new family of... (Review)
Review
Several new structures of three types of protein complexes, obtained by cryo-electron microscopy (cryo-EM) and published between 2019 and 2021, identify a new family of natural molecular wheels, the "5:2 rotary motors." These span the cytoplasmic membranes of bacteria, and their rotation is driven by ion flow into the cell. They consist of a pentameric wheel encircling a dimeric axle within the cytoplasmic membrane of both Gram-positive and gram-negative bacteria. The axles extend into the periplasm, and the wheels extend into the cytoplasm. Rotation of these wheels has never been observed directly; it is inferred from the symmetry of the complexes and from the roles they play within the larger systems that they are known to power. In particular, the new structure of the stator complex of the Bacterial Flagellar Motor, MotAB, is consistent with a "wheels within wheels" model of the motor. Other 5:2 rotary motors are believed to share the core rotary function and mechanism, driven by ion-motive force at the cytoplasmic membrane. Their structures diverge in their periplasmic and cytoplasmic parts, reflecting the variety of roles that they perform. This review focuses on the structures of 5:2 rotary motors and their proposed mechanisms and functions. We also discuss molecular rotation in general and its relation to the rotational symmetry of molecular complexes.
PubMed: 35992645
DOI: 10.3389/fmicb.2022.948383 -
Contact (Thousand Oaks (Ventura County,... 2023In eukaryotic cells, nonvesicular lipid transport between organelles is mediated by lipid-transfer proteins. Recently, a new class of these lipid transporters has been... (Review)
Review
In eukaryotic cells, nonvesicular lipid transport between organelles is mediated by lipid-transfer proteins. Recently, a new class of these lipid transporters has been described to facilitate the bulk of inter-organelle lipid transport at contact sites by forming bridge-like structures with a hydrophobic groove through which lipids travel. Because their predicted structure is composed of repeating β-groove (RBG) domains, they have been named the RBG protein superfamily. Early studies on RBG proteins VPS13 and ATG2 recognized the resemblance of their predicted structures to that of the bacterial Lpt system, which transports newly synthesized lipopolysaccharides (LPS) between the inner and the outer membranes (IMs and OMs) of Gram-negative bacteria. In these didermic bacteria, the IMs and OMs are separated by an aqueous periplasmic compartment that is traversed by a bridge-like structure built with β-jelly roll domains from several Lpt proteins that provides a hydrophobic groove for LPS molecules to travel across the periplasm. Despite structural and functional similarities between RBG proteins and the Lpt system, the bacterial AsmA-like protein family has recently emerged as the likely ancestor of RBG proteins and long sought-after transporters that facilitate the transfer of phospholipids from the IM to the OM. Here, we review our current understanding of the structure and function of bacterial AsmA-like proteins, mainly focusing on recent studies that have led to the proposal that AsmA-like proteins mediate the bulk of phospholipid transfer between the IMs and OMs.
PubMed: 37455811
DOI: 10.1177/25152564231185931 -
Microbiology (Reading, England) Oct 2023Multidrug efflux pumps are molecular machines that sit in the bacterial cell membrane and pump molecules out from either the periplasm or cytoplasm to outside the cell....
Multidrug efflux pumps are molecular machines that sit in the bacterial cell membrane and pump molecules out from either the periplasm or cytoplasm to outside the cell. While involved in a variety of biological roles, they are primarily known for their contribution to antibiotic resistance by limiting the intracellular accumulation of antimicrobial compounds within bacteria. These transporters are often overexpressed in clinical isolates, leading to multidrug-resistant phenotypes. Efflux pumps are classified into several families based on their structure and understanding the characteristics of each family is important for the development of novel therapies to restore antibiotic potency.
Topics: Humans; Cytoplasm; Periplasm; Cell Membrane; Anti-Bacterial Agents; Membrane Transport Proteins
PubMed: 37787650
DOI: 10.1099/mic.0.001370 -
Microbiology Spectrum Feb 2022Porphyromonas gingivalis is an important human pathogen and also a model organism for the Bacteroidetes phylum. O-glycosylation has been reported in this phylum with...
Porphyromonas gingivalis is an important human pathogen and also a model organism for the Bacteroidetes phylum. O-glycosylation has been reported in this phylum with findings that include the O-glycosylation motif, the structure of the O-glycans in a few species, and an extensive O-glycoproteome analysis in Tannerella forsythia. However, O-glycosylation has not yet been confirmed in P. gingivalis. We therefore used glycoproteomics approaches including partial deglycosylation with trifluoromethanesulfonic acid as well as both HILIC and FAIMS based glycopeptide enrichment strategies leading to the identification of 257 putative glycosylation sites in 145 glycoproteins. The sequence of the major O-glycan was elucidated to be HexNAc-HexNAc(P-Gro-[Ac])-dHex-Hex-HexA-Hex(dHex). Western blot analyses of mutants lacking the glycosyltransferases PGN_1134 and PGN_1135 demonstrated their involvement in the biosynthesis of the glycan while mass spectrometry analysis of the truncated O-glycans suggested that PGN_1134 and PGN_1135 transfer the two HexNAc sugars. Interestingly, a strong bias against the O-glycosylation of abundant proteins exposed to the cell surface such as abundant T9SS cargo proteins, surface lipoproteins, and outer membrane β-barrel proteins was observed. In contrast, the great majority of proteins associated with the inner membrane or periplasm were glycosylated irrespective of their abundance. The P. gingivalis O-glycosylation system may therefore function to establish the desired physicochemical properties of the periplasm. Porphyromonas gingivalis is an oral pathogen primarily associated with severe periodontal disease and further associated with rheumatoid arthritis, dementia, cardiovascular disease, and certain cancers. Protein glycosylation can be important for a variety of reasons including protein function, solubility, protease resistance, and thermodynamic stability. This study has for the first time demonstrated the presence of O-linked glycosylation in this organism by determining the basic structure of the O-glycans and identifying 257 glycosylation sites in 145 proteins. It was found that most proteins exposed to the periplasm were O-glycosylated; however, the abundant surface exposed proteins were not. The O-glycans consisted of seven monosaccharides and a glycerol phosphate with 0-2 acetyl groups. These glycans are likely to have a stabilizing role to the proteins that bear them and must be taken into account when the proteins are produced in heterologous organisms.
Topics: Amino Acid Motifs; Bacterial Proteins; Carbohydrate Sequence; Glycoproteins; Glycosylation; Humans; Polysaccharides; Porphyromonas gingivalis
PubMed: 34985300
DOI: 10.1128/spectrum.01502-21 -
Journal of Bacteriology Dec 2022Reduction of extracellular acceptors requires electron transfer across the periplasm. In Geobacter sulfurreducens, three separate cytoplasmic membrane cytochromes are...
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 similarly restored metal respiration in G. sulfurreducens. PgcA, an unrelated extracellular triheme -type cytochrome, also participated in periplasmic electron transfer. While triheme cytochromes were important for metal reduction, sextuple Δ Δ 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 and suggest that an as-yet-undiscovered periplasmic mechanism supports electron transfer to electrodes. Many inner and outer membrane cytochromes used by 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 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 electron transfer, and question why these organisms produce such an array of periplasmic cytochromes.
Topics: Geobacter; Periplasm; Ferric Compounds; Electrons; Electron Transport; Cytochromes; Oxidation-Reduction
PubMed: 36383007
DOI: 10.1128/jb.00322-22 -
MBio Oct 2022Bacteria have evolved many different signal transduction systems to sense and respond to changing environmental conditions. Signal integration is mainly achieved by...
Bacteria have evolved many different signal transduction systems to sense and respond to changing environmental conditions. Signal integration is mainly achieved by signal recognition at extracytosolic ligand-binding domains (LBDs) of receptors. Hundreds of different LBDs have been reported, and our understanding of their sensing properties is growing. Receptors must function over a range of environmental pH values, but there is little information available on the robustness of sensing as a function of pH. Here, we have used isothermal titration calorimetry to determine the pH dependence of ligand recognition by nine LBDs that cover all major LBD superfamilies, of periplasmic solute-binding proteins, and cytosolic LBDs. We show that periplasmic LBDs recognize ligands over a very broad pH range, frequently stretching over eight pH units. This wide pH range contrasts with a much narrower pH response range of the cytosolic LBDs analyzed. Many LBDs must be dimeric to bind ligands, and analytical ultracentrifugation studies showed that the LBD of the Tar chemoreceptor forms dimers over the entire pH range tested. The pH dependences of Pseudomonas aeruginosa motility and chemotaxis were bell-shaped and centered at pH 7.0. Evidence for pH robustness of signaling was obtained by Förster Resonance Energy Transfer (FRET) measurements of the chemotaxis pathway responses in Escherichia coli. Bacteria have evolved several strategies to cope with extreme pH, such as periplasmic chaperones for protein refolding. The intrinsic pH resistance of periplasmic LBDs appears to be another strategy that permits bacteria to survive under adverse conditions. Demonstration of the pH robustness of extracytoplasmic sensing reveals a previously undescribed evolutionary mechanism that enables bacteria to monitor environmental changes under changing conditions. This mechanism includes the maintenance of the dimeric state of four-helixbundle ligand-binding domains (LBDs). The construction of biosensors is a rapidly growing field of research, and their use to monitor the progression of the COVID-19 pandemic has impressively demonstrated their usefulness. LBDs represent an enormous reservoir of binding modules that can be used to create novel biosensors. Among ligands recognized by LBDs are neurotransmitters, hormones, and quorum-sensing signals. The demonstration that extracytosolic LBDs bind their signals over a wide range of pH values will facilitate the design of biosensors that function under highly variable conditions of acidity and alkalinity.
Topics: Humans; Ligands; Bacterial Proteins; Protein Binding; Pandemics; COVID-19; Chemotaxis; Bacteria; Escherichia coli; Hormones; Hydrogen-Ion Concentration
PubMed: 36154178
DOI: 10.1128/mbio.01650-22