-
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 -
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 -
Nanotechnology Mar 2020Combining abiotic photosensitisers such as quantum dots (QDs) with non-photosynthetic bacteria presents an intriguing concept into the design of artificial...
Combining abiotic photosensitisers such as quantum dots (QDs) with non-photosynthetic bacteria presents an intriguing concept into the design of artificial photosynthetic organisms and solar-driven fuel production. Shewanella oneidensis MR-1 (MR-1) is a versatile bacterium concerning respiration, metabolism and biocatalysis, and is a promising organism for artificial photosynthesis as the bacterium's synthetic and catalytic ability provides a potential system for bacterial biohydrogen production. MR-1's hydrogenases are present in the periplasmatic space. It follows that for photoenergised electrons to reach these enzymes, QDs will need to be able to enter the periplasm, or electrons need to enter the periplasm via the Mtr pathway that is responsible for MR-1's extracellular electron transfer ability. As a step towards this goal, various QDs were tested for their photo-reducing potential, nanotoxicology and further for their interaction with MR-1. CdTe/CdS/TGA, CdTe/CdS/Cysteamine, a commercial, negatively charged CdTe and CuInS/ZnS/PMAL QDs were examined. The photoreduction potential of the QDs was confirmed by measuring their ability to photoreduce methyl viologen with different sacrificial electron donors. The commercial CdTe and CuInS/ZnS/PMAL QDs showed no toxicity towards MR-1 as evaluated by a colony-forming units method and a fluorescence viability assay. Only the commercial negatively charged CdTe QDs showed good interaction with MR-1. With transmission electron microscopy, QDs were observed both in the cytoplasm and periplasm. These results inform on the possibilities and bottlenecks when developing bionanotechnological systems for the photosynthetic production of biohydrogen by MR-1.
Topics: Anti-Bacterial Agents; Bacterial Proteins; Cadmium Compounds; Hydrogenase; Microbial Viability; Microscopy, Electron, Transmission; Periplasm; Photosynthesis; Quantum Dots; Shewanella; Tellurium; Zinc Compounds
PubMed: 31810073
DOI: 10.1088/1361-6528/ab5f78 -
Proceedings of the National Academy of... Oct 2023A large number of small membrane proteins have been uncovered in bacteria, but their mechanism of action has remained mostly elusive. Here, we investigate the mechanism...
A large number of small membrane proteins have been uncovered in bacteria, but their mechanism of action has remained mostly elusive. Here, we investigate the mechanism of a physiologically important small protein, MgrB, which represses the activity of the sensor kinase PhoQ and is widely distributed among enterobacteria. The PhoQ/PhoP two-component system is a master regulator of the bacterial virulence program and interacts with MgrB to modulate bacterial virulence, fitness, and drug resistance. A combination of cross-linking approaches with functional assays and protein dynamic simulations revealed structural rearrangements due to interactions between MgrB and PhoQ near the membrane/periplasm interface and along the transmembrane helices. These interactions induce the movement of the PhoQ catalytic domain and the repression of its activity. Without MgrB, PhoQ appears to be much less sensitive to antimicrobial peptides, including the commonly used C18G. In the presence of MgrB, C18G promotes MgrB to dissociate from PhoQ, thus activating PhoQ via derepression. Our findings reveal the inhibitory mechanism of the small protein MgrB and uncover its importance in antimicrobial peptide sensing.
Topics: Bacterial Proteins; Antimicrobial Peptides; Membrane Proteins; Periplasm; Gene Expression Regulation, Bacterial
PubMed: 37792514
DOI: 10.1073/pnas.2309607120 -
Protein Expression and Purification May 2022Suppressor of copper sensitivity (Scs) proteins play a role in the bacterial response to copper stress in many Gram-negative bacteria, including in the human pathogen...
Suppressor of copper sensitivity (Scs) proteins play a role in the bacterial response to copper stress in many Gram-negative bacteria, including in the human pathogen Proteus mirabilis. Recently, the ScsC protein from P. mirabilis (PmScsC) was characterized as a trimeric protein with isomerase activity that contributes to the ability of the bacterium to swarm in the presence of copper. The CXXC motif catalytic cysteines of PmScsC are maintained in their active reduced state by the action of its membrane-bound partner protein, the Proteus mirabilis ScsB (PmScsB). Thus, PmScsC and PmScsB form a redox relay in vivo. The predicted domain arrangement of PmScsB comprises a central transmembrane β-domain and two soluble, periplasmic domains, the N-terminal α-domain and C-terminal γ-domain. Here, we provide a procedure for the recombinant expression and purification of the full-length PmScsB protein. Using Lemo21 (DE3) cells we expressed PmScsB and, after extraction and purification, we were able to achieve a yield of 3 mg of purified protein per 8 L of bacterial culture. Furthermore, using two orthogonal methods - AMS labelling of free thiols and a scrambled RNase A activity assay - PmScsB is shown to catalyze the reduction of PmScsC. Our results demonstrate that the PmScsC and PmScsB redox relay can be reconstituted in vitro using recombinant full-length PmScsB membrane protein. This finding provides a promising starting point for the in vitro biochemical and structural characterization of the P. mirabilis ScsC and ScsB interaction.
Topics: Bacterial Proteins; Copper; Humans; Membrane Proteins; Periplasm; Proteus mirabilis
PubMed: 35026386
DOI: 10.1016/j.pep.2022.106047 -
Journal of Bacteriology Nov 2023Survival during starvation hinges on the ability to manage intracellular energy reserves and to initiate appropriate metabolic responses to perturbations of such...
Survival during starvation hinges on the ability to manage intracellular energy reserves and to initiate appropriate metabolic responses to perturbations of such reserves. How manage carbon storage systems under starvation stress, as well as transpose changes in intracellular metabolite levels into regulatory signals, is not well understood. Endogenous trehalose metabolism may be at the center of these processes, coupling carbon storage with carbon starvation responses. The coupled transport to the periplasm and subsequent hydrolysis of trehalose back to glucose for transport to the cytoplasm may function as a crucial metabolic signaling pathway. Although trehalose has been characterized as a stress protectant in , the disaccharide also functions as both an energy storage compound and a regulator of carbohydrate metabolism in fungi, plants, and other bacteria. Our research explores the metabolic regulatory properties of trehalose in and a potential mechanism by which the intracellular carbon pool is interconnected with regulatory circuits, enabling long-term survival.
Topics: Escherichia coli; Trehalose; Periplasm; Signal Transduction; Carbon
PubMed: 37916804
DOI: 10.1128/jb.00292-23 -
Proceedings of the National Academy of... Mar 2022SignificanceHow flagella sense complex environments and control bacterial motility remain fascinating questions. Here, we deploy cryo-electron tomography to determine in...
SignificanceHow flagella sense complex environments and control bacterial motility remain fascinating questions. Here, we deploy cryo-electron tomography to determine in situ structures of the flagellar motor in wild-type and mutant cells of , revealing that three flagellar proteins (FliL, MotA, and MotB) form a unique supramolecular complex in situ. Importantly, FliL not only enhances motor function by forming a ring around the stator complex MotA/MotB in its extended, active conformation but also facilitates assembly of the stator complex around the motor. Our in situ data provide insights into how cooperative remodeling of the FliL-stator supramolecular complex helps regulate the collective ion flux and establishes the optimal function of the flagellar motor to guide bacterial motility in various environments.
Topics: Bacterial Physiological Phenomena; Bacterial Proteins; Borrelia burgdorferi; Flagella; Gene Expression Regulation, Bacterial; Membrane Proteins; Models, Biological; Models, Molecular; Molecular Motor Proteins; Periplasm
PubMed: 35254893
DOI: 10.1073/pnas.2117245119 -
STAR Protocols Dec 2021Bacteriophages of the family densely package their genomes into precursor capsids alongside internal virion proteins called ejection proteins. In phage T7 these...
Bacteriophages of the family densely package their genomes into precursor capsids alongside internal virion proteins called ejection proteins. In phage T7 these proteins (gp14, gp15, and gp16) are ejected into the host envelope forming a DNA-ejectosome for genome delivery. Here, we describe the purification and characterization of recombinant gp14, gp15, and gp16. This protocol was used for high-resolution cryo-EM structure analysis of the T7 periplasmic tunnel and can be adapted to study ejection proteins from other phages. For complete details on the use and execution of this protocol, please refer to Swanson et al. (2021).
Topics: Bacteriophage T7; Cryoelectron Microscopy; Escherichia coli; Periplasm; Recombinant Proteins; Viral Proteins
PubMed: 34825220
DOI: 10.1016/j.xpro.2021.100960 -
Biochimica Et Biophysica Acta Nov 2004Disulfide bond formation is a catalyzed process in vivo. In prokaryotes, the oxidation of cysteine pairs is achieved by the transfer of disulfides from the highly... (Review)
Review
Disulfide bond formation is a catalyzed process in vivo. In prokaryotes, the oxidation of cysteine pairs is achieved by the transfer of disulfides from the highly oxidizing DsbA/DsbB catalytic machinery to substrate proteins. The oxidizing power utilized by this system comes from the membrane-embedded electron transport system, which utilizes molecular oxygen as a final oxidant. Proofreading of disulfide bond formation is performed by the DsbC/DsbD system, which has the ability to rearrange non-native disulfides to their native configuration. These disulfide isomerization reactions are sustained by a constant supply of reducing power provided by the cytoplasmic thioredoxin system, utilizing NADPH as the ultimate electron source.
Topics: Catalysis; Cysteine; Disulfides; Escherichia coli; Periplasm; Protein Disulfide-Isomerases; Protein Engineering; Recombinant Proteins
PubMed: 15546661
DOI: 10.1016/j.bbamcr.2004.02.012 -
Nature Communications Nov 2019Horizontal gene transfer through natural transformation is a major driver of antibiotic resistance spreading in many pathogenic bacterial species. In the case of...
Horizontal gene transfer through natural transformation is a major driver of antibiotic resistance spreading in many pathogenic bacterial species. In the case of Gram-negative bacteria, and in particular of Helicobacter pylori, the mechanisms underlying the handling of the incoming DNA within the periplasm are poorly understood. Here we identify the protein ComH as the periplasmic receptor for the transforming DNA during natural transformation in H. pylori. ComH is a DNA-binding protein required for the import of DNA into the periplasm. Its C-terminal domain displays strong affinity for double-stranded DNA and is sufficient for the accumulation of DNA in the periplasm, but not for DNA internalisation into the cytoplasm. The N-terminal region of the protein allows the interaction of ComH with a periplasmic domain of the inner-membrane channel ComEC, which is known to mediate the translocation of DNA into the cytoplasm. Our results indicate that ComH is involved in the import of DNA into the periplasm and its delivery to the inner membrane translocator ComEC.
Topics: Bacterial Proteins; Biological Transport; DNA; DNA, Bacterial; Gene Transfer, Horizontal; Helicobacter pylori; Periplasm; Receptors, Cell Surface; Transformation, Bacterial
PubMed: 31767852
DOI: 10.1038/s41467-019-13352-6