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Frontiers in Microbiology 2022and species oxidize methanol pyrroloquinoline quinone-methanol dehydrogenases (MDHs). MDHs can be classified into two major groups, Ca-dependent MDH (MxaF) and...
and species oxidize methanol pyrroloquinoline quinone-methanol dehydrogenases (MDHs). MDHs can be classified into two major groups, Ca-dependent MDH (MxaF) and lanthanide (Ln)-dependent MDH (XoxF), whose expression is regulated by the availability of Ln. A set of a siderophore, TonB-dependent receptor, and an ABC transporter that resembles the machinery for iron uptake is involved in the solubilization and transport of Ln. The transport of Ln into the cytosol enhances XoxF expression. A unique protein named lanmodulin from strain AM1 was identified as a specific Ln-binding protein, and its biological function was implicated to be an Ln shuttle in the periplasm. In contrast, it remains unclear how Ln levels in the cells are maintained, because Ln is potentially deleterious to cellular systems due to its strong affinity to phosphate ions. In this study, we investigated the function of a lanmodulin homolog in strain 22A. The expression of a gene encoding lanmodulin () was induced in response to the presence of La. A recombinant LanM underwent conformational change upon La binding. Phenotypic analyses on deletion mutant and overexpressing strains showed that LanM is not necessary for the wild-type and XoxF-dependent mutant's methylotrophic growth. We found that expression was regulated by MxcQE (a two-component regulator for MxaF) and TonB_Ln (a TonB-dependent receptor for Ln). The expression level of was altered to be negatively dependent on Ln concentration in ∆ whereas it was constant in the wild type. Furthermore, when exposed to La, ∆ showed an aggregating phenotype, cell membrane impairment, La deposition in the periplasm evidenced by electron microscopy, differential expression of proteins involved in membrane integrity and phosphate starvation, and possibly lower La content in the membrane vesicle (MV) fractions. Taken together, we concluded that lanmodulin is involved in the complex regulation mechanism of MDHs and homeostasis of cellular Ln levels by facilitating transport and MV-mediated excretion.
PubMed: 35814700
DOI: 10.3389/fmicb.2022.921636 -
Nature Communications Nov 2022Mechanosensitive channels of small conductance, found in many living organisms, open under elevated membrane tension and thus play crucial roles in biological response...
Mechanosensitive channels of small conductance, found in many living organisms, open under elevated membrane tension and thus play crucial roles in biological response to mechanical stress. Amongst these channels, MscK is unique in that its activation also requires external potassium ions. To better understand this dual gating mechanism by force and ligand, we elucidate distinct structures of MscK along the gating cycle using cryo-electron microscopy. The heptameric channel comprises three layers: a cytoplasmic domain, a periplasmic gating ring, and a markedly curved transmembrane domain that flattens and expands upon channel opening, which is accompanied by dilation of the periplasmic ring. Furthermore, our results support a potentially unifying mechanotransduction mechanism in ion channels depicted as flattening and expansion of the transmembrane domain.
Topics: Potassium Channels; Ion Channel Gating; Mechanotransduction, Cellular; Cryoelectron Microscopy; Models, Molecular; Ion Channels; Potassium
PubMed: 36371466
DOI: 10.1038/s41467-022-34737-0 -
MBio Aug 2021Antimicrobial resistance in Neisseria gonorrhoeae has reached an alarming level, severely impacting the effective treatment of gonorrhea. Belonging to the...
Antimicrobial resistance in Neisseria gonorrhoeae has reached an alarming level, severely impacting the effective treatment of gonorrhea. Belonging to the resistance-nodulation-cell division (RND) superfamily of efflux transporters, the MtrD membrane protein of N. gonorrhoeae provides resistance to a broad range of antimicrobial compounds. A unique feature of MtrD is an 11-residue sequence (from N917 to P927 [N917-P927]) that connects transmembrane helices (TMS) 9 and 10; this sequence is not present in homologous RND proteins. This study explores the structural and functional roles of the N917-P927 region by means of mutant analysis and molecular dynamics simulations. We show that N917-P927 plays a key role in modulating substrate access to the binding cleft and influences the overall orientation of the protein within the inner membrane necessary for optimal functioning. Removal of N917-P927 significantly reduced MtrD-mediated resistance to a range of antimicrobials and mutations of three single amino acids impacted MtrD-mediated multidrug resistance. Furthermore, molecular dynamics simulations showed deletion of N917-P927 in MtrD may dysregulate access of the substrate to the binding cleft and closure of the substrate-binding pocket during the transport cycle. These findings indicate that N917-P927 is a key region for interacting with the inner membrane, conceivably influencing substrate capture from the membrane-periplasm interface and thus is essential for full multidrug resistance capacity of MtrD. The historical sexually transmitted infection gonorrhea continues to be a major public health concern with an estimated global annual incidence of 86.9 million cases. N. gonorrhoeae has been identified by the World Health Organization as one of the 12 antimicrobial-resistant bacterial species that poses the greatest risk to human health. As the major efflux pump in gonococci, the MtrD transporter contributes to the cell envelope barrier in this organism and pumps antimicrobials from the periplasm and inner membrane, resulting in resistance. This study demonstrates that a unique region of the MtrD protein that connects TMS 9 and TMS 10 forms a structure that may interact with the inner membrane positioning TMS 9 and stabilizing the protein facilitating substrate capture from the inner membrane-periplasm interface. Analysis of mutants of this region identified that it was essential for MtrD-mediated multidrug resistance. Characterization of the structure and function of this unique local region of MtrD has implications for drug efflux mechanisms used by related proteins and is important knowledge for development of antibiotics that bypass efflux.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Base Sequence; Biological Transport; Drug Resistance, Multiple, Bacterial; Humans; Membrane Transport Proteins; Molecular Dynamics Simulation; Neisseria gonorrhoeae
PubMed: 34465021
DOI: 10.1128/mBio.01675-21 -
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 -
Nature Chemical Biology Feb 2023Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic...
Colibactin, a DNA cross-linking agent produced by gut bacteria, is implicated in colorectal cancer. Its biosynthesis uses a prodrug resistance mechanism: a non-toxic precursor assembled in the cytoplasm is activated after export to the periplasm. This activation is mediated by ClbP, an inner-membrane peptidase with an N-terminal periplasmic catalytic domain and a C-terminal three-helix transmembrane domain. Although the transmembrane domain is required for colibactin activation, its role in catalysis is unclear. Our structure of full-length ClbP bound to a product analog reveals an interdomain interface important for substrate binding and enzyme stability and interactions that explain the selectivity of ClbP for the N-acyl-D-asparagine prodrug motif. Based on structural and biochemical evidence, we propose that ClbP dimerizes to form an extended substrate-binding site that can accommodate a pseudodimeric precolibactin with its two terminal prodrug motifs in the two ClbP active sites, thus enabling the coordinated activation of both electrophilic warheads.
Topics: Peptide Hydrolases; Escherichia coli; Prodrugs; Peptides; Escherichia coli Proteins
PubMed: 36253550
DOI: 10.1038/s41589-022-01142-z -
Protein Science : a Publication of the... Nov 2023Investigating the evolution of structural features in modern multidomain proteins helps to understand their immense diversity and functional versatility. The class of...
Investigating the evolution of structural features in modern multidomain proteins helps to understand their immense diversity and functional versatility. The class of periplasmic binding proteins (PBPs) offers an opportunity to interrogate one of the main processes driving diversification: the duplication and fusion of protein sequences to generate new architectures. The symmetry of their two-lobed topology, their mechanism of binding, and the organization of their operon structure led to the hypothesis that PBPs arose through a duplication and fusion event of a single common ancestor. To investigate this claim, we set out to reverse the evolutionary process and recreate the structural equivalent of a single-lobed progenitor using ribose-binding protein (RBP) as our model. We found that this modern PBP can be deconstructed into its lobes, producing two proteins that represent possible progenitor halves. The isolated halves of RBP are well folded and monomeric proteins, albeit with a lower thermostability, and do not retain the original binding function. However, the two entities readily form a heterodimer in vitro and in-cell. The x-ray structure of the heterodimer closely resembles the parental protein. Moreover, the binding function is fully regained upon formation of the heterodimer with a ligand affinity similar to that observed in the modern RBP. This highlights how a duplication event could have given rise to a stable and functional PBP-like fold and provides insights into how more complex functional structures can evolve from simpler molecular components.
Topics: Periplasmic Binding Proteins; Carrier Proteins; Amino Acid Sequence; Ligands; Protein Binding; Evolution, Molecular
PubMed: 37788980
DOI: 10.1002/pro.4793 -
Frontiers in Microbiology 2021In the bacterial flagellar motor, the cell-wall-anchored stator uses an electrochemical gradient across the cytoplasmic membrane to generate a turning force that is... (Review)
Review
In the bacterial flagellar motor, the cell-wall-anchored stator uses an electrochemical gradient across the cytoplasmic membrane to generate a turning force that is applied to the rotor connected to the flagellar filament. Existing theoretical concepts for the stator function are based on the assumption that it anchors around the rotor perimeter by binding to peptidoglycan (P). The existence of another anchoring region on the motor itself has been speculated upon, but is yet to be supported by binding studies. Due to the recent advances in electron cryotomography, evidence has emerged that polar flagellar motors contain substantial proteinaceous periplasmic structures next to the stator, without which the stator does not assemble and the motor does not function. These structures have a morphology of disks, as is the case with spp., or a round cage, as is the case with . It is now recognized that such additional periplasmic components are a common feature of polar flagellar motors, which sustain higher torque and greater swimming speeds compared to peritrichous bacteria such as and . This review summarizes the data available on the structure, composition, and role of the periplasmic scaffold in polar bacterial flagellar motors and discusses the new paradigm for how such motors assemble and function.
PubMed: 33776972
DOI: 10.3389/fmicb.2021.639490 -
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 -
Frontiers in Bioengineering and... 2023Despite the long history of use and the knowledge of the genetics and biochemistry of , problems are still possible in obtaining a soluble form of recombinant proteins...
Despite the long history of use and the knowledge of the genetics and biochemistry of , problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of . We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02 µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003 µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists.
PubMed: 37456729
DOI: 10.3389/fbioe.2023.1187761