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Journal of Bacteriology Aug 2021Two-component signaling systems (TCSs) are comprised of a sensory histidine kinase and a response regulator protein. In response to environmental changes, sensor kinases...
Two-component signaling systems (TCSs) are comprised of a sensory histidine kinase and a response regulator protein. In response to environmental changes, sensor kinases directly phosphorylate their cognate response regulator to affect gene expression. Bacteria typically express multiple TCSs that are insulated from one another and regulate distinct physiological processes. There are examples of cross-regulation between TCSs, but this phenomenon remains relatively unexplored. We have identified regulatory links between the ChvG-ChvI (ChvGI) and NtrY-NtrX (NtrYX) TCSs, which control important and often overlapping processes in alphaproteobacteria, including maintenance of the cell envelope. Deletion of and in Caulobacter crescentus limited growth in defined medium, and a selection for genetic suppressors of this growth phenotype uncovered interactions among , , and , which encodes a previously uncharacterized periplasmic protein. Significant overlap in the experimentally defined ChvI and NtrX transcriptional regulons provided support for the observed genetic connections between and . Moreover, we present evidence that the growth defect of strains lacking is influenced by the phosphorylation state of NtrX and, to some extent, by levels of the TonB-dependent receptor ChvT. Measurements of NtrX phosphorylation indicated that NtrZ is an upstream regulator of NtrY and that NtrY primarily functions as an NtrX phosphatase. We propose a model in which NtrZ functions in the periplasm to inhibit NtrY phosphatase activity; regulation of phosphorylated NtrX levels by NtrZ and NtrY provides a mechanism to modulate and balance expression of the NtrX and ChvI regulons under different growth conditions. TCSs enable bacteria to regulate gene expression in response to physiochemical changes in their environment. The ChvGI and NtrYX TCSs regulate diverse pathways associated with pathogenesis, growth, and cell envelope function in many alphaproteobacteria. We used Caulobacter crescentus as a model to investigate regulatory connections between ChvGI and NtrYX. Our work defined the ChvI transcriptional regulon in C. crescentus and revealed a genetic interaction between ChvGI and NtrYX, whereby modulation of NtrYX signaling affects the survival of cells lacking ChvGI. In addition, we identified NtrZ as a periplasmic inhibitor of NtrY phosphatase activity . Our work establishes C. crescentus as an excellent model to investigate multilevel regulatory connections between ChvGI and NtrYX in alphaproteobacteria.
Topics: Bacterial Proteins; Caulobacter crescentus; Gene Expression Regulation, Bacterial; Phosphorylation; Regulon; Signal Transduction
PubMed: 34124942
DOI: 10.1128/JB.00199-21 -
Molecules (Basel, Switzerland) Jun 2020Phosphodiesters of glucose-2-phosphate (G2P) are found only in few natural compounds such as agrocinopine D and agrocin 84. Agrocinopine D is a G2P phosphodiester...
Phosphodiesters of glucose-2-phosphate (G2P) are found only in few natural compounds such as agrocinopine D and agrocin 84. Agrocinopine D is a G2P phosphodiester produced by plants infected by C58 and recognized by the bacterial periplasmic binding protein AccA for being transported into the bacteria before cleavage by the phosphodiesterase AccF, releasing G2P, which promotes virulence by binding the repressor protein AccR. The G2P amide agrocin 84 is a natural antibiotic produced by the non-pathogenic K84 strain used as a biocontrol agent by competing with C58. G2P esters are also found in irregular glycogen structures. The rare glucopyranosyl-2-phophoryl moiety found in agrocin 84 is the key structural signature enabling its action as a natural antibiotic. Likewise, G2P and G2P esters can also dupe the agrocinopine catabolism cascade. Such observations illustrate the importance of G2P esters on which we have recently focused our interest. After a brief review of the reported phosphorylation coupling methods and the choice of carbohydrate building blocks used in G2P chemistry, a flexible access to glucose-2-phosphate esters using the phosphoramidite route is proposed.
Topics: Adenine Nucleotides; Agrobacterium; Esters; Glucosephosphates; Glycogen; Periplasmic Binding Proteins
PubMed: 32575421
DOI: 10.3390/molecules25122829 -
International Journal of Molecular... Dec 2022In the Gram-negative bacteria, many important virulence factors reach their destination via two-step export systems, and they must traverse the periplasmic space before... (Review)
Review
In the Gram-negative bacteria, many important virulence factors reach their destination via two-step export systems, and they must traverse the periplasmic space before reaching the outer membrane. Since these proteins must be maintained in a structure competent for transport into or across the membrane, they frequently require the assistance of chaperones. Based on the results obtained for the model bacterium and related species, it is assumed that in the biogenesis of the outer membrane proteins and the periplasmic transit of secretory proteins, the SurA peptidyl-prolyl isomerase/chaperone plays a leading role, while the Skp chaperone is rather of secondary importance. However, detailed studies carried out on several other Gram-negative pathogens indicate that the importance of individual chaperones in the folding and transport processes depends on the properties of client proteins and is species-specific. Taking into account the importance of SurA functions in bacterial virulence and severity of phenotypes due to mutations, this folding factor is considered as a putative therapeutic target to combat microbial infections. In this review, we present recent findings regarding SurA and Skp proteins: their mechanisms of action, involvement in processes related to virulence, and perspectives to use them as therapeutic targets.
Topics: Carrier Proteins; Escherichia coli Proteins; Virulence; Bacterial Outer Membrane Proteins; Molecular Chaperones; Peptidylprolyl Isomerase; Escherichia coli; Virulence Factors; Gram-Negative Bacteria; Protein Folding; DNA-Binding Proteins
PubMed: 36613738
DOI: 10.3390/ijms24010295 -
Advanced Science (Weinheim,... Oct 2022Metal sulfides are a common group of extracellular bacterial biominerals. However, only a few cases of intracellular biomineralization are reported in this group, mostly...
Metal sulfides are a common group of extracellular bacterial biominerals. However, only a few cases of intracellular biomineralization are reported in this group, mostly limited to greigite (Fe S ) in magnetotactic bacteria. Here, a previously unknown periplasmic biomineralization of copper sulfide produced by the magnetotactic bacterium Desulfamplus magnetovallimortis strain BW-1, a species known to mineralize greigite (Fe S ) and magnetite (Fe O ) in the cytoplasm is reported. BW-1 produces hundreds of spherical nanoparticles, composed of 1-2 nm substructures of a poorly crystalline hexagonal copper sulfide structure that remains in a thermodynamically unstable state. The particles appear to be surrounded by an organic matrix as found from staining and electron microscopy inspection. Differential proteomics suggests that periplasmic proteins, such as a DegP-like protein and a heavy metal-binding protein, could be involved in this biomineralization process. The unexpected periplasmic formation of copper sulfide nanoparticles in BW-1 reveals previously unknown possibilities for intracellular biomineralization that involves intriguing biological control and holds promise for biological metal recovery in times of copper shortage.
Topics: Bacteria; Biomineralization; Copper; Ferrosoferric Oxide; Iron; Magnetosomes; Nanoparticles; Periplasmic Proteins; Sulfides
PubMed: 35975419
DOI: 10.1002/advs.202203444 -
Protein Science : a Publication of the... Apr 2023Outer membrane protein (OMP) biogenesis in gram-negative bacteria is managed by a network of periplasmic chaperones that includes SurA, Skp, and FkpA. These chaperones...
Outer membrane protein (OMP) biogenesis in gram-negative bacteria is managed by a network of periplasmic chaperones that includes SurA, Skp, and FkpA. These chaperones bind unfolded OMPs (uOMPs) in dynamic conformational ensembles to suppress aggregation, facilitate diffusion across the periplasm, and enhance folding. FkpA primarily responds to heat-shock stress, but its mechanism is comparatively understudied. To determine FkpA chaperone function in the context of OMP folding, we monitored the folding of three OMPs and found that FkpA, unlike other periplasmic chaperones, increases the folded yield but decreases the folding rate of OMPs. The results indicate that FkpA behaves as a chaperone and not as a folding catalyst to influence the OMP folding trajectory. Consistent with the folding assay results, FkpA binds all three uOMPs as determined by sedimentation velocity (SV) and photo-crosslinking experiments. We determine the binding affinity between FkpA and uOmpA by globally fitting SV titrations and find it to be intermediate between the known affinities of Skp and SurA for uOMP clients. Notably, complex formation steeply depends on the urea concentration, suggesting an extensive binding interface. Initial characterizations of the complex using photo-crosslinking indicate that the binding interface spans the entire FkpA molecule. In contrast to prior findings, folding and binding experiments performed using subdomain constructs of FkpA demonstrate that the full-length chaperone is required for full activity. Together these results support that FkpA has a distinct and direct effect on OMP folding that it achieves by utilizing an extensive chaperone-client interface to tightly bind clients.
Topics: Humans; Carrier Proteins; Peptidylprolyl Isomerase; Escherichia coli Proteins; Escherichia coli; Bacterial Outer Membrane Proteins; Protein Folding; Molecular Chaperones; Periplasm
PubMed: 36775935
DOI: 10.1002/pro.4592 -
The EMBO Journal Dec 2022Secretory preproteins of the Sec pathway are targeted post-translationally and cross cellular membranes through translocases. During cytoplasmic transit, mature domains...
Secretory preproteins of the Sec pathway are targeted post-translationally and cross cellular membranes through translocases. During cytoplasmic transit, mature domains remain non-folded for translocase recognition/translocation. After translocation and signal peptide cleavage, mature domains fold to native states in the bacterial periplasm or traffic further. We sought the structural basis for delayed mature domain folding and how signal peptides regulate it. We compared how evolution diversified a periplasmic peptidyl-prolyl isomerase PpiA mature domain from its structural cytoplasmic PpiB twin. Global and local hydrogen-deuterium exchange mass spectrometry showed that PpiA is a slower folder. We defined at near-residue resolution hierarchical folding initiated by similar foldons in the twins, at different order and rates. PpiA folding is delayed by less hydrophobic native contacts, frustrated residues and a β-turn in the earliest foldon and by signal peptide-mediated disruption of foldon hierarchy. When selected PpiA residues and/or its signal peptide were grafted onto PpiB, they converted it into a slow folder with enhanced in vivo secretion. These structural adaptations in a secretory protein facilitate trafficking.
Topics: Protein Folding; Protein Sorting Signals; Proteins; Cell Membrane; Hydrophobic and Hydrophilic Interactions
PubMed: 36031863
DOI: 10.15252/embj.2022111344 -
Proceedings of the National Academy of... Sep 2021Many tailed bacteriophages assemble ejection proteins and a portal-tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel...
Many tailed bacteriophages assemble ejection proteins and a portal-tail complex at a unique vertex of the capsid. The ejection proteins form a transenvelope channel extending the portal-tail channel for the delivery of genomic DNA in cell infection. Here, we report the structure of the mature bacteriophage T7, including the ejection proteins, as well as the structures of the full and empty T7 particles in complex with their cell receptor lipopolysaccharide. Our near-atomic-resolution reconstruction shows that the ejection proteins in the mature T7 assemble into a core, which comprises a fourfold gene product 16 (gp16) ring, an eightfold gp15 ring, and a putative eightfold gp14 ring. The gp15 and gp16 are mainly composed of helix bundles, and gp16 harbors a lytic transglycosylase domain for degrading the bacterial peptidoglycan layer. When interacting with the lipopolysaccharide, the T7 tail nozzle opens. Six copies of gp14 anchor to the tail nozzle, extending the nozzle across the lipopolysaccharide lipid bilayer. The structures of gp15 and gp16 in the mature T7 suggest that they should undergo remarkable conformational changes to form the transenvelope channel. Hydrophobic α-helices were observed in gp16 but not in gp15, suggesting that gp15 forms the channel in the hydrophilic periplasm and gp16 forms the channel in the cytoplasmic membrane.
Topics: Bacteriophage T7; Capsid; Capsid Proteins; Cell Membrane; Cryoelectron Microscopy; DNA, Viral; Lipid Bilayers; Models, Molecular; Periplasm; Structure-Activity Relationship; Transduction, Genetic; Viral Proteins
PubMed: 34504014
DOI: 10.1073/pnas.2102003118 -
New Biotechnology Nov 2023The Gram-negative periplasm is a convenient location for the accumulation of many recombinant proteins including biopharmaceutical products. It is the site of disulphide...
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.
Topics: Periplasmic Proteins; Escherichia coli; Periplasm; Biological Products; Recombinant Proteins
PubMed: 37708933
DOI: 10.1016/j.nbt.2023.09.003 -
Cell Chemical Biology Jun 2021In this issue of Cell Chemical Biology, Hentrich et al. (2021) describe the application of the SpyCatcher technology to antibody discovery and validation. Fab-SpyTag...
In this issue of Cell Chemical Biology, Hentrich et al. (2021) describe the application of the SpyCatcher technology to antibody discovery and validation. Fab-SpyTag fusion proteins can be expressed in the periplasm of protease-deficient bacteria and coupled in a modular manner to a variety of SpyCatcher-tagged proteins for improved assay performance.
Topics: Immunoglobulin Fragments; Recombinant Proteins
PubMed: 34143957
DOI: 10.1016/j.chembiol.2021.05.017 -
The Biochemical Journal Feb 2023Gram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Together they form the envelope (or cell wall),...
Gram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Together they form the envelope (or cell wall), crucial for energy production, lipid biosynthesis, structural integrity, and for protection against physical and chemical environmental challenges. To achieve envelope biogenesis, periplasmic and outer-membrane proteins (OMPs) must be transported from the cytosol and through the inner-membrane, via the ubiquitous SecYEG protein-channel. Emergent proteins either fold in the periplasm or cross the peptidoglycan (PG) layer towards the outer-membrane for insertion through the β-barrel assembly machinery (BAM). Trafficking of hydrophobic proteins through the periplasm is particularly treacherous given the high protein density and the absence of energy (ATP or chemiosmotic potential). Numerous molecular chaperones assist in the prevention and recovery from aggregation, and of these SurA is known to interact with BAM, facilitating delivery to the outer-membrane. However, it is unclear how proteins emerging from the Sec-machinery are received and protected from aggregation and proteolysis prior to an interaction with SurA. Through biochemical analysis and electron microscopy we demonstrate the binding capabilities of the unoccupied and substrate-engaged SurA to the inner-membrane translocation machinery complex of SecYEG-SecDF-YidC - aka the holo-translocon (HTL). Supported by AlphaFold predictions, we suggest a role for periplasmic domains of SecDF in chaperone recruitment to the protein translocation exit site in SecYEG. We propose that this immediate interaction with the enlisted chaperone helps to prevent aggregation and degradation of nascent envelope proteins, facilitating their safe passage to the periplasm and outer-membrane.
Topics: Periplasm; Escherichia coli Proteins; Escherichia coli; Peptidoglycan; Molecular Chaperones; Membrane Proteins; Bacterial Outer Membrane Proteins; Membrane Transport Proteins; Carrier Proteins; Peptidylprolyl Isomerase
PubMed: 36701201
DOI: 10.1042/BCJ20220480