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Nature Communications Mar 2022Peptidoglycan hydrolases contribute to the generation of helical cell shape in Campylobacter and Helicobacter bacteria, while cytoskeletal or periskeletal proteins...
Peptidoglycan hydrolases contribute to the generation of helical cell shape in Campylobacter and Helicobacter bacteria, while cytoskeletal or periskeletal proteins determine the curved, vibrioid cell shape of Caulobacter and Vibrio. Here, we identify a peptidoglycan hydrolase in the vibrioid-shaped predatory bacterium Bdellovibrio bacteriovorus which invades and replicates within the periplasm of Gram-negative prey bacteria. The protein, Bd1075, generates cell curvature in B. bacteriovorus by exerting LD-carboxypeptidase activity upon the predator cell wall as it grows inside spherical prey. Bd1075 localizes to the outer convex face of B. bacteriovorus; this asymmetric localization requires a nuclear transport factor 2-like (NTF2) domain at the protein C-terminus. We solve the crystal structure of Bd1075, which is monomeric with key differences to other LD-carboxypeptidases. Rod-shaped Δbd1075 mutants invade prey more slowly than curved wild-type predators and stretch invaded prey from within. We therefore propose that the vibrioid shape of B. bacteriovorus contributes to predatory fitness.
Topics: Bdellovibrio; Bdellovibrio bacteriovorus; Cell Wall; Peptidoglycan; Periplasm
PubMed: 35314810
DOI: 10.1038/s41467-022-29007-y -
Biochimica Et Biophysica Acta Aug 2014Bacteriocins are a diverse group of ribosomally synthesized protein antibiotics produced by most bacteria. They range from small lanthipeptides produced by lactic acid... (Review)
Review
Bacteriocins are a diverse group of ribosomally synthesized protein antibiotics produced by most bacteria. They range from small lanthipeptides produced by lactic acid bacteria to much larger multi domain proteins of Gram negative bacteria such as the colicins from Escherichia coli. For activity bacteriocins must be released from the producing cell and then bind to the surface of a sensitive cell to instigate the import process leading to cell death. For over 50years, colicins have provided a working platform for elucidating the structure/function studies of bacteriocin import and modes of action. An understanding of the processes that contribute to the delivery of a colicin molecule across two lipid membranes of the cell envelope has advanced our knowledge of protein-protein interactions (PPI), protein-lipid interactions and the role of order-disorder transitions of protein domains pertinent to protein transport. In this review, we provide an overview of the arrangement of genes that controls the synthesis and release of the mature protein. We examine the uptake processes of colicins from initial binding and sequestration of binding partners to crossing of the outer membrane, and then discuss the translocation of colicins through the cell periplasm and across the inner membrane to their cytotoxic site of action. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.
Topics: Bacteriocins; Cell Membrane; Colicins; Escherichia coli; Membrane Proteins; Periplasm; Periplasmic Proteins; Protein Structure, Tertiary; Protein Transport; Structure-Activity Relationship
PubMed: 24746518
DOI: 10.1016/j.bbamcr.2014.04.010 -
3 Biotech Jun 2016Disulfide bonds occurred in majority of secreted protein. Formation of correct disulfide bonds are must for achieving native conformation, solubility and activity.... (Review)
Review
Disulfide bonds occurred in majority of secreted protein. Formation of correct disulfide bonds are must for achieving native conformation, solubility and activity. Production of recombinant proteins containing disulfide bond for therapeutic, diagnostic and various other purposes is a challenging task of research. Production of such proteins in the reducing cytosolic compartment of E. coli usually ends up in inclusion bodies formation. Refolding of inclusion bodies can be difficult, time and labor consuming and uneconomical. Translocation of these proteins into the oxidative periplasmic compartment provides correct environment to undergo proper disulfide bonds formation and thus achieving native conformation. However, not all proteins can be efficiently translocated to the periplasm with the help of bacterial signal peptides. Therefore, fusion to a small well-folded and stable periplasmic protein is more promising for periplasmic production of disulfide bonded proteins. In the past decades, several full-length proteins or domains were used for enhancing translocation and solubility. Here, protein fusion tags that significantly increase the yields of target proteins in the periplasmic space are reviewed.
PubMed: 28330113
DOI: 10.1007/s13205-016-0397-7 -
Environmental Microbiology Reports Oct 2015This review emphasizes the biological roles of the osmoregulated periplasmic glucans (OPGs). Osmoregulated periplasmic glucans occur in almost all α-, β- and... (Review)
Review
This review emphasizes the biological roles of the osmoregulated periplasmic glucans (OPGs). Osmoregulated periplasmic glucans occur in almost all α-, β- and γ-Proteobacteria. This polymer of glucose is required for full virulence. The roles of the OPGs are complex and vary depending on the species. Here, we outline the four major roles of the OPGs through four different pathogenic and one symbiotic bacterial models (Dickeya dadantii, Salmonella enterica, Pseudomonas aeruginosa, Brucella abortus and Sinorhizobium meliloti). When periplasmic, the OPGs are a part of the signal transduction pathway and indirectly regulate genes involved in virulence. The OPGs can also be secreted. When outside of the cell, they interact directly with antibiotics to protect the bacterial cell or interact with the host cell to facilitate the invasion process. When OPGs are not found, as in the ε-Proteobacteria, OPG-like oligosaccharides are present. Their presence strengthens the evidence that OPGs play an important role in virulence.
Topics: Alphaproteobacteria; Anti-Bacterial Agents; Gammaproteobacteria; Glucans; Osmoregulation; Periplasm; Signal Transduction; Virulence
PubMed: 26265506
DOI: 10.1111/1758-2229.12325 -
Molecular Cell Aug 2021Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our...
Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.
Topics: Bacteriophage T7; Computational Biology; Cryoelectron Microscopy; Cytoplasm; DNA, Viral; Lipid Bilayers; Periplasm; Podoviridae; Viral Core Proteins
PubMed: 34214465
DOI: 10.1016/j.molcel.2021.06.001 -
ACS Synthetic Biology May 2024is 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...
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 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.
Topics: Biosensing Techniques; Escherichia coli; Periplasmic Proteins; Recombinant Proteins; Periplasm; Stress, Physiological; Escherichia coli Proteins
PubMed: 38676700
DOI: 10.1021/acssynbio.3c00720 -
Journal of Molecular Biology Feb 2024The 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....
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.
Topics: Diffusion; Escherichia coli; Escherichia coli Proteins; Osmotic Pressure; Periplasm; Single Molecule Imaging
PubMed: 38143021
DOI: 10.1016/j.jmb.2023.168420 -
Cell Reports Jun 2020Type VI secretion systems (T6SSs) are nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain...
Type VI secretion systems (T6SSs) are nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized a Salmonella T6SS antibacterial effector called Tlde1 that is toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tldi1). Microscopy analysis reveals that cells expressing Tlde1 stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovers similarities between Tlde1 and the catalytic domain of l,d-transpeptidases. Point mutations on conserved catalytic residues abrogate toxicity. Biochemical assays reveal that Tlde1 displays both l,d-carboxypeptidase activity by cleaving peptidoglycan tetrapeptides between meso-diaminopimelic acid and d-alanine and l,d-transpeptidase exchange activity by replacing d-alanine by a non-canonical d-amino acid. Phylogenetic analysis shows that Tlde1 homologs constitute a family of T6SS-associated effectors broadly distributed among Proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition and reveals a different mechanism of bacterial antagonism.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Cell Division; Escherichia coli; Evolution, Molecular; Peptidoglycan; Peptidyl Transferases; Periplasm; Proteobacteria; Salmonella typhimurium; Type VI Secretion Systems
PubMed: 32579939
DOI: 10.1016/j.celrep.2020.107813 -
ACS Infectious Diseases Jun 2018The study of the bacterial periplasm requires techniques with sufficient spatial resolution and sensitivity to resolve the components and processes within this...
The study of the bacterial periplasm requires techniques with sufficient spatial resolution and sensitivity to resolve the components and processes within this subcellular compartment. Peroxidase-mediated biotinylation has enabled targeted labeling of proteins within subcellular compartments of mammalian cells. We investigated whether this methodology could be applied to the bacterial periplasm. In this study, we demonstrated that peroxidase-mediated biotinylation can be performed in mycobacteria and Escherichia coli. To eliminate detection artifacts from natively biotinylated mycobacterial proteins, we validated two alternative labeling substrates, tyramide azide and tyramide alkyne, which enable biotin-independent detection of labeled proteins. We also targeted peroxidase expression to the periplasm, resulting in compartment-specific labeling of periplasmic versus cytoplasmic proteins in mycobacteria. Finally, we showed that this method can be used to validate protein relocalization to the cytoplasm upon removal of a secretion signal. This novel application of peroxidase-mediated protein labeling will advance efforts to characterize the role of the periplasm in bacterial physiology and pathogenesis.
Topics: Bacterial Proteins; Biotin; Biotinylation; Click Chemistry; Cytoplasm; Escherichia coli; Periplasmic Proteins; Peroxidase; Recombinant Fusion Proteins; Staining and Labeling
PubMed: 29708735
DOI: 10.1021/acsinfecdis.8b00044 -
BioTechniques Apr 2019Fractionation in Gram-negative bacteria is used to identify the subcellular localization of proteins, in particular the localization of exported recombinant proteins....
Fractionation in Gram-negative bacteria is used to identify the subcellular localization of proteins, in particular the localization of exported recombinant proteins. The process of cell fractionation can be fraught with cross-contamination issues and often lacks supporting data for fraction purity. Here, we compare three periplasm extraction and two cell disruption techniques in different combinations to investigate which process gives uncontaminated compartments from Escherichia coli. From these data, a robust method named PureFrac was compiled that gives pure periplasmic fractions and a superior recovery of soluble cytoplasmic proteins. The process extracts periplasm using cold osmotic shock with magnesium, prior to sonication and ultracentrifugation to separate the cytoplasm from insoluble material. This method handles cells cultivated in various conditions and allows preparation of active proteins in their respective compartments.
Topics: Blotting, Western; Cell Fractionation; Cold Temperature; Escherichia coli; Escherichia coli Proteins; Osmotic Pressure; Periplasm; Recombinant Proteins
PubMed: 30987443
DOI: 10.2144/btn-2018-0135