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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 -
Antioxidants & Redox Signaling May 2013The cell envelope of aerobic bacteria is an oxidizing environment in which most cysteine residues are involved in disulfide bonds. However, reducing redox pathways are... (Review)
Review
SIGNIFICANCE
The cell envelope of aerobic bacteria is an oxidizing environment in which most cysteine residues are involved in disulfide bonds. However, reducing redox pathways are also present in this cellular compartment where they provide electrons to a variety of cellular processes. The membrane protein DsbD plays a central role in these pathways by functioning as an electron hub that dispatches electrons received from the cytoplasmic thioredoxin system to periplasmic oxidoreductases.
RECENT ADVANCES
Recent data have revealed that DsbD provides reducing equivalents to a large array of periplasmic redox proteins. Those proteins use the reducing power received from DsbD to correct non-native disulfides, mature c-type cytochromes, protect cysteines on secreted proteins from irreversible oxidation, reduce methionine sulfoxides, and scavenge reactive oxygen species such as hydrogen peroxide.
CRITICAL ISSUES
Despite the prominent role played by DsbD, we have a poor understanding of how this protein transfers electrons across the inner membrane. Another critical issue will be to grasp the full physiological significance of the new reducing pathways that have been identified in the cell envelope such as the peroxide reduction pathway.
FUTURE DIRECTIONS
A detailed understanding of DsbD's mechanism will require solving the structure of this intriguing protein. Moreover, bioinformatic, biochemical, and genetic approaches need to be combined for a better comprehension of the broad spectrum of periplasmic reducing systems present in bacteria, which will likely lead to the discovery of novel pathways.
Topics: Bacterial Proteins; Electron Transport; Escherichia coli Proteins; Membrane Proteins; Metabolic Networks and Pathways; Oxidation-Reduction; Oxidoreductases; Periplasm
PubMed: 23025488
DOI: 10.1089/ars.2012.4962 -
MBio Oct 2014A defining characteristic of Chlamydia spp. is their developmental cycle characterized by outer membrane transformations of cysteine bonds among cysteine-rich outer...
UNLABELLED
A defining characteristic of Chlamydia spp. is their developmental cycle characterized by outer membrane transformations of cysteine bonds among cysteine-rich outer membrane proteins. The reduction-oxidation states of host cell compartments were monitored during the developmental cycle using live fluorescence microscopy. Organelle redox states were studied using redox-sensitive green fluorescent protein (roGFP1) expressed in CF15 epithelial cells and targeted to the cytosol, mitochondria, and endoplasmic reticulum (ER). The redox properties of chlamydiae and the inclusion were monitored using roGFP expressed by Chlamydia trachomatis following transformation. Despite the large morphological changes associated with chlamydial infection, redox potentials of the cytosol (Ψ(cyto) [average, -320 mV]), mitochondria (Ψ(mito) [average, -345 mV]), and the ER (ΨER [average, -258 mV]) and their characteristic redox regulatory abilities remained unchanged until the cells died, at which point Ψ(cyto) and Ψ(mito) became more oxidized and Ψ(ER) became more reduced. The redox status of the chlamydial cytoplasm was measured following transformation and expression of the roGFP biosensor in C. trachomatis throughout the developmental cycle. The periplasmic and outer membrane redox states were assessed by the level of cysteine cross-linking of cysteine-rich envelope proteins. In both cases, the chlamydiae were highly reduced early in the developmental cycle and became oxidized late in the developmental cycle. The production of a late-developmental-stage oxidoreductase/isomerase, DsbJ, may play a key role in the regulation of the oxidoreductive developmental-stage-specific process.
IMPORTANCE
Infectious Chlamydia organisms have highly oxidized and cysteine cross-linked membrane proteins that confer environmental stability when outside their host cells. Once these organisms infect a new host cell, the proteins become reduced and remain reduced during the active growth stage. These proteins become oxidized at the end of their growth cycle, wherein infectious organisms are produced and released to the environment. How chlamydiae mediate and regulate this key step in their pathogenesis is unknown. Using biosensors specifically targeted to different compartments within the infected host cell and for the chlamydial organisms themselves, the oxidoreductive states of these compartments were measured during the course of infection. We found that the host cell redox states are not changed by infection with C. trachomatis, whereas the state of the chlamydial organisms remains reduced during infection until the late developmental stages, wherein the organisms' cytosol and periplasm become oxidized and they acquire environmental resistance and infectivity.
Topics: Cell Line; Cell Membrane; Chlamydia trachomatis; Cytoplasm; Epithelial Cells; Genes, Reporter; Humans; Organelles; Oxidation-Reduction; Periplasm
PubMed: 25352618
DOI: 10.1128/mBio.01924-14 -
PloS One 2015Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to...
Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.
Topics: Bacterial Adhesion; Bacterial Proteins; Cellulose; Fibrobacter; Fimbriae Proteins; Models, Biological; Periplasm; Proteomics; Transcriptome
PubMed: 26629814
DOI: 10.1371/journal.pone.0143809 -
MBio Nov 2019Photoferrotrophy is a form of anoxygenic photosynthesis whereby bacteria utilize soluble or insoluble forms of ferrous iron as an electron donor to fix carbon dioxide...
Photoferrotrophy is a form of anoxygenic photosynthesis whereby bacteria utilize soluble or insoluble forms of ferrous iron as an electron donor to fix carbon dioxide using light energy. They can also use poised electrodes as their electron donor via phototrophic extracellular electron uptake (phototrophic EEU). The electron uptake mechanisms underlying these processes are not well understood. Using TIE-1 as a model, we show that a single periplasmic decaheme cytochrome , PioA, and an outer membrane porin, PioB, form a complex allowing extracellular electron uptake across the outer membrane from both soluble iron and poised electrodes. We observe that PioA undergoes postsecretory proteolysis of its N terminus to produce a shorter heme-attached PioA (holo-PioA, where PioA represents the C terminus of PioA), which can exist both freely in the periplasm and in a complex with PioB. The extended N-terminal peptide controls heme attachment, and its processing is required to produce wild-type levels of holo-PioA and holo-PioAB complex. It is also conserved in PioA homologs from other phototrophs. The presence of PioAB in these organisms correlate with their ability to perform photoferrotrophy and phototrophic EEU. Some anoxygenic phototrophs use soluble iron, insoluble iron minerals (such as rust), or their proxies (poised electrodes) as electron donors for photosynthesis. However, the underlying electron uptake mechanisms are not well established. Here, we show that these phototrophs use a protein complex made of an outer membrane porin and a periplasmic decaheme cytochrome (electron transfer protein) to harvest electrons from both soluble iron and poised electrodes. This complex has two unique characteristics: (i) it lacks an extracellular cytochrome , and (ii) the periplasmic decaheme cytochrome undergoes proteolytic cleavage to produce a functional electron transfer protein. These characteristics are conserved in phototrophs harboring homologous proteins.
Topics: Bacterial Proteins; Biological Transport; Carbon Dioxide; Cytochromes c; Electrodes; Electron Transport; Electrons; Iron; Periplasm; Photosynthesis; Porins; Rhodopseudomonas
PubMed: 31690680
DOI: 10.1128/mBio.02668-19 -
Scientific Reports Apr 2017A main challenge in chemotherapy is to determine the in cellulo parameters modulating the drug concentration required for therapeutic action. It is absolutely urgent to...
A main challenge in chemotherapy is to determine the in cellulo parameters modulating the drug concentration required for therapeutic action. It is absolutely urgent to understand membrane permeation and intracellular concentration of antibiotics in clinical isolates: passing the membrane barrier to reach the threshold concentration inside the bacterial periplasm or cytoplasm is the pivotal step of antibacterial activity. Ceftazidime (CAZ) is a key molecule of the combination therapy for treating resistant bacteria. We designed and synthesized different fluorescent CAZ derivatives (CAZ*, CAZ**) to dissect the early step of translocation-accumulation across bacterial membrane. Their activities were determined on E. coli strains and on selected clinical isolates overexpressing ß-lactamases. The accumulation of CAZ* and CAZ** were determined by microspectrofluorimetry and epifluorimetry. The derivatives were properly translocated to the periplasmic space when we permeabilize the outer membrane barrier. The periplasmic location of CAZ** was related to a significant antibacterial activity and with the outer membrane permeability. This study demonstrated the correlation between periplasmic accumulation and antibiotic activity. We also validated the method for approaching ß-lactam permeation relative to membrane permeability and paved the way for an original matrix for determining "Structure Intracellular Accumulation Activity Relationship" for the development of new therapeutic candidates.
Topics: Anti-Bacterial Agents; Ceftazidime; Cell Membrane; Gram-Negative Bacteria; Microbial Sensitivity Tests; Microspectrophotometry; Molecular Structure; Periplasm; Permeability
PubMed: 28428543
DOI: 10.1038/s41598-017-00945-8 -
Cell Apr 2003DegS, the periplasmic stress sensor, becomes activated when its PDZ domain recognizes the improperly exposed C-terminal sequences of outer membrane porins. This... (Review)
Review
DegS, the periplasmic stress sensor, becomes activated when its PDZ domain recognizes the improperly exposed C-terminal sequences of outer membrane porins. This interaction relieves the inhibition of the neighboring protease domain of DegS, triggering a proteolysis cascade that leads to the sigma(E)-driven expression of periplasmic chaperones.
Topics: Bacteria; Bacterial Proteins; Periplasm; Protein Folding; Protein Structure, Tertiary; Signal Transduction; Transcription Factors
PubMed: 12679025
DOI: 10.1016/s0092-8674(03)00192-2 -
The Journal of Biological Chemistry Nov 2023Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas...
Enzymatic modifications of bacterial exopolysaccharides enhance immune evasion and persistence during infection. In the Gram-negative opportunistic pathogen Pseudomonas aeruginosa, acetylation of alginate reduces opsonic killing by phagocytes and improves reactive oxygen species scavenging. Although it is well known that alginate acetylation in P. aeruginosa requires AlgI, AlgJ, AlgF, and AlgX, how these proteins coordinate polymer modification at a molecular level remains unclear. Here, we describe the structural characterization of AlgF and its protein interaction network. We characterize direct interactions between AlgF and both AlgJ and AlgX in vitro and demonstrate an association between AlgF and AlgX, as well as AlgJ and AlgI, in P. aeruginosa. We determine that AlgF does not exhibit acetylesterase activity and is unable to bind to polymannuronate in vitro. Therefore, we propose that AlgF functions to mediate protein-protein interactions between alginate acetylation enzymes, forming the periplasmic AlgJFXK (AlgJ-AlgF-AlgX-AlgK) acetylation and export complex required for robust biofilm formation.
Topics: Acetylation; Alginates; Bacterial Proteins; Biofilms; Periplasm; Protein Processing, Post-Translational; Pseudomonas aeruginosa
PubMed: 37797696
DOI: 10.1016/j.jbc.2023.105314 -
Proceedings of the National Academy of... Jun 2017Multicomponent efflux complexes constitute a primary mechanism for Gram-negative bacteria to expel toxic molecules for survival. As these complexes traverse the...
Multicomponent efflux complexes constitute a primary mechanism for Gram-negative bacteria to expel toxic molecules for survival. As these complexes traverse the periplasm and link inner and outer membranes, it remains unclear how they operate efficiently without compromising periplasmic plasticity. Combining single-molecule superresolution imaging and genetic engineering, we study in living cells the tripartite efflux complex CusCBA of the resistance-nodulation-division family that is essential for bacterial resistance to drugs and toxic metals. We find that CusCBA complexes are dynamic structures and shift toward the assembled form in response to metal stress. Unexpectedly, the periplasmic adaptor protein CusB is a key metal-sensing element that drives the assembly of the efflux complex ahead of the transcription activation of the operon for defending against metals. This adaptor protein-mediated dynamic pump assembly allows the bacterial cell for efficient efflux upon cellular demand while still maintaining periplasmic plasticity; this could be broadly relevant to other multicomponent efflux systems.
Topics: Copper; Escherichia coli; Escherichia coli Proteins; Ion Transport; Membrane Proteins; Membrane Transport Proteins; Periplasm; Silver
PubMed: 28607072
DOI: 10.1073/pnas.1704729114 -
Chemistry & Biology Aug 2015Mycobacterium tuberculosis mycobacterial membrane protein large (MmpL) proteins are important in substrate transport across the inner membrane. Here, we show that MmpL...
Mycobacterium tuberculosis mycobacterial membrane protein large (MmpL) proteins are important in substrate transport across the inner membrane. Here, we show that MmpL proteins are classified into two phylogenetic clusters, where MmpL cluster II contains three soluble domains (D1, D2, and D3) and has two full-length members, MmpL3 and MmpL11. Significantly, MmpL3 is currently the most druggable M. tuberculosis target. We have solved the 2.4-Å MmpL11-D2 crystal structure, revealing structural homology to periplasmic porter subdomains of RND (multidrug) transporters. The resulting predicted cluster II MmpL membrane topology has D1 and D2 residing, and possibly interacting, within the periplasm. Crosslinking and biolayer interferometry experiments confirm that cluster II D1 and D2 bind with weak affinities, and guided D1-D2 heterodimeric model assemblies. The predicted full-length MmpL3 and MmpL11 structural models reveal key substrate binding and transport residues, and may serve as templates to set the stage for in silico anti-tuberculosis drug development.
Topics: Amino Acid Sequence; Bacterial Proteins; Membrane Transport Proteins; Models, Molecular; Molecular Sequence Data; Mycobacterium tuberculosis; Periplasm; Protein Structure, Secondary; Protein Structure, Tertiary; Structure-Activity Relationship
PubMed: 26278184
DOI: 10.1016/j.chembiol.2015.07.013