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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 -
Microbiology (Reading, England) Aug 2023The bacterial predator is a model for the wider phenomenon of bacteria:bacteria predation, and the specialization required to achieve a lifestyle dependent on prey...
The bacterial predator is a model for the wider phenomenon of bacteria:bacteria predation, and the specialization required to achieve a lifestyle dependent on prey consumption. is able to recognize, enter and ultimately consume fellow Gram-negative bacteria, killing these prey from within their periplasmic space, and lysing the host at the end of the cycle. The classic phenotype-driven characterization (and observation of predation) has benefitted from an increased focus on molecular mechanisms and fluorescence microscopy and tomography, revealing new features of several of the lifecycle stages. Herein we summarize a selection of these advances and describe likely areas for exploration that will push the field toward a more complete understanding of this fascinating 'two-cell' system.
Topics: Bdellovibrio bacteriovorus; Gram-Negative Bacteria
PubMed: 37535060
DOI: 10.1099/mic.0.001380 -
Yakugaku Zasshi : Journal of the... 2024Iron is necessary for all living organisms, and bacteria that cause infections in human hosts also need ferrous ions for their growth and proliferation. In the human... (Review)
Review
Iron is necessary for all living organisms, and bacteria that cause infections in human hosts also need ferrous ions for their growth and proliferation. In the human body, most ferric ions (Fe) are tightly bound to iron-binding proteins such as hemoglobin, transferrin, lactoferrin, and ferritin. Pathogenic bacteria express highly specific iron uptake systems, including siderophores and specific receptors. Most bacteria secrete siderophores, which are low-molecular weight metal-chelating agents, to capture Fe outside cell. Siderophores are mainly classified as either catecholate or hydroxamate. Vibrio vulnificus, a Gram-negative pathogenic bacterium, is responsible for serious infections in humans and requires iron for growth. A clinical isolate, V. vulnificus M2799, secretes a catecholate siderophore, vulnibactin, that captures ferric ions from the environment. In our study, we generated deletion mutants of the genes encoding proteins involved in the vulnibactin mediated iron-utilization system, such as ferric-vulnibactin receptor protein (VuuA), periplasmic ferric-vulnibactin binding protein (FatB), ferric-vulnibactin reductase (VuuB), and isochorismate synthase (ICS). ICS and VuuA are required under low-iron conditions for ferric-utilization in M2799, but the alternative proteins FatB and VuuB can function as a periplasmic binding protein and a ferric-chelate reductase, respectively. VatD, which functions as ferric-hydroxamate siderophores periplasmic binding protein, was shown to participate in the ferric-vulnibactin uptake system in the absence of FatB. Furthermore, the ferric-hydroxamate siderophore reductase IutB was observed to participate in ferric-vulnibactin reduction in the absence of VuuB. We propose that ferric-siderophore periplasmic binding proteins and ferric-chelate reductases represent potential targets for drug discovery in the context of infectious diseases.
Topics: Iron; Siderophores; Humans; Drug Discovery; Bacterial Infections; Molecular Targeted Therapy; Hydroxamic Acids; Iron-Binding Proteins
PubMed: 38825472
DOI: 10.1248/yakushi.23-00197-2 -
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 -
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 -
BioRxiv : the Preprint Server For... Aug 2023Bacterial growth and division rely on intricate regulation of morphogenetic complexes to remodel the cell envelope without compromising envelope integrity. Significant...
Bacterial growth and division rely on intricate regulation of morphogenetic complexes to remodel the cell envelope without compromising envelope integrity. Significant progress has been made in recent years towards understanding the regulation of cell wall metabolic enzymes. However, other cell envelope components play a role in morphogenesis as well. Components required to maintain osmotic homeostasis are among these understudied envelope-associated enzymes that may contribute to cell morphology. A primary factor required to protect envelope integrity in low osmolarity environments is OpgH, the synthase of osmoregulated periplasmic glucans (OPGs). Here, we demonstrate that OpgH is essential in the α-proteobacterium . Unexpectedly, depletion of OpgH results in striking asymmetric bulging and cell lysis, accompanied by misregulation of cell wall insertion and mislocalization of morphogenetic complexes. The enzymatic activity of OpgH is required for normal cell morphology as production of an OpgH mutant that disrupts a conserved glycosyltransferase motif phenocopies the depletion. Our data establish a surprising function for an OpgH homolog in morphogenesis and reveal an essential role of OpgH in maintaining proper cell morphology during normal growth and division in .
PubMed: 37693447
DOI: 10.1101/2023.08.28.555136 -
Biophysical Reviews Oct 2023For the last decades, significant progress has been made in studying the biological functions of H-bond networks in membrane proteins, proton transporters, receptors,... (Review)
Review
For the last decades, significant progress has been made in studying the biological functions of H-bond networks in membrane proteins, proton transporters, receptors, and photosynthetic reaction centers. Increasing availability of the X-ray crystal and cryo-electron microscopy structures of photosynthetic complexes resolved with high atomic resolution provides a platform for their comparative analysis. It allows identifying structural factors that are ensuring the high quantum yield of the photochemical reactions and are responsible for the stability of the membrane complexes. The H-bond networks are known to be responsible for proton transport associated with electron transfer from the primary to the secondary quinone as well as in the processes of water oxidation in photosystem II. Participation of such networks in reactions proceeding on the periplasmic side of bacterial photosynthetic reaction centers is less studied. This review summarizes the current understanding of the role of H-bond networks on the donor side of photosynthetic reaction centers from purple bacteria. It is discussed that the networks may be involved in providing close association with mobile electron carriers, in light-induced proton transport, in regulation of the redox properties of bacteriochlorophyll cofactors, and in stabilization of the membrane protein structure at the interface of membrane and soluble phases.
PubMed: 37974998
DOI: 10.1007/s12551-023-01109-x -
Plants (Basel, Switzerland) Jan 2024There are limited studies on the cytology of bamboo leaf development from primordium to maturity. This study delves into the leaf morphological characteristics and...
There are limited studies on the cytology of bamboo leaf development from primordium to maturity. This study delves into the leaf morphological characteristics and growth patterns of 'Aureostriatus' and provides a three-dimensional anatomical analysis of cell division, expansion, and degradation. Leaves on the same branch develop bottom-up, while individual leaves develop the other way around. Like bamboo shoots and culms, the leaves follow a "slow-fast-slow" growth pattern, with longitudinal growth being predominant during their development. The growth zones of individual leaves included division, elongation, and maturation zones based on the distribution of growth space. By measuring 13,303 epidermal long cells and 3293 mesophyll cells in longitudinal sections of rapidly elongating leaves, we observed that in the rapid elongation phase (S4-S5), the division zone was located in the 1-2 cm segment at the bottom of the leaf blade and maintained a constant size, continuously providing new cells for leaf elongation, whereas in the late rapid elongation phase (S6), when the length of the leaf blade was approaching that of a mature leaf, its cells at the bottom of the blade no longer divided and were replaced by the ability to elongate. Furthermore, to gain an insight into the dynamic changes in the growth of the 'Aureostriatus' leaves in the lateral and periclinal directions, the width and thickness of 1459 epidermal and 2719 mesophyll cells were counted in the mid-cross section of leaves at different developmental stages. The results showed that during the early stages of development (S1-S3), young leaves maintained vigorous division in the lateral direction, while periplasmic division gradually expanded from the bottom to the top of the leaf blade and the number of cell layers stabilized at S4. The meristematic tissues on both sides of the leaf were still able to divide at S4 but the frequency of the division gradually decreased, while cell division and expansion occurred simultaneously between the veins. At S6, the cells at the leaf margins and between the veins were completely differentiated and the width of the leaf blade no longer expanded. These findings revealed changes in cell growth anisotropically during the leaf development of 'Aureostriatus' and demonstrated that leaf elongation was closely related to the longitudinal expansion of epidermal cells and proliferative growth of mesophyll cells, whereas the cell division of meristematic tissues and expansion of post-divisional cells contributed to the increases in blade width and thickness. The presented framework will facilitate a further exploration of the molecular regulatory mechanisms of leaf development in 'Aureostriatus' and provide relevant information for developmental and taxonomic studies of bamboo plants.
PubMed: 38337866
DOI: 10.3390/plants13030332 -
MBio Oct 2023Exclusively in the Bacteroidetes phylum, most proteins exported across the inner membrane via the Sec system and released into the periplasm by type I signal peptidase...
Exclusively in the Bacteroidetes phylum, most proteins exported across the inner membrane via the Sec system and released into the periplasm by type I signal peptidase have N-terminal glutamine converted to pyroglutamate. The reaction is catalyzed by the periplasmic enzyme glutaminyl cyclase (QC), which is essential for the growth of and other periodontopathogens. Apparently, pyroglutamyl formation stabilizes extracytoplasmic proteins and/or protects them from proteolytic degradation in the periplasm. Given the role of as the keystone pathogen in periodontitis, QC is a promising target for the development of drugs to treat and/or prevent this highly prevalent chronic inflammatory disease leading to tooth loss and associated with severe systemic diseases.
Topics: Humans; Aminoacyltransferases; Periodontitis; Pyrrolidonecarboxylic Acid; Glutamine
PubMed: 37750700
DOI: 10.1128/mbio.00980-23 -
Scientific Reports Feb 2024Antimicrobial resistance has emerged as one of the leading public health threats of the twenty-first century. Gram-negative pathogens have been a major contributor to...
Antimicrobial resistance has emerged as one of the leading public health threats of the twenty-first century. Gram-negative pathogens have been a major contributor to the declining efficacy of antibiotics through both acquired resistance and tolerance. In this study, a pan-drug resistant (PDR), NDM-1 and CTX-M-15 co-producing isolate of K. pneumoniae, CDC Nevada, (Kp Nevada) was exposed to the clinical combination of aztreonam + ceftazidime/avibactam (ATM/CAZ/AVI) to overcome metallo-β-lactamases. Unexpectedly, the β-lactam combination resulted in long filamentous cell formation induced by PBP3 inhibition over 168 h in the hollow fiber infection model experiments with eventual reversion of the total population upon drug removal. However, the addition of imipenem to the two drug β-lactam combination was highly synergistic with suppression of all drug resistant subpopulations over 5 days. Scanning electron microscopy and fluorescence microscopy for all imipenem combinations in time kill studies suggested a role for imipenem in suppression of long filamentous persisters, via the formation of metabolically active spheroplasts. To complement the imaging studies, salient transcriptomic changes were quantified using RT-PCR and novel cassette assay evaluated β-lactam permeability. This showed significant upregulation of both spheroplast protein Y (SPY), a periplasmic chaperone protein that has been shown to be related to spheroplast formation, and penicillin binding proteins (PBP1, PBP2, PBP3) for all combinations involving imipenem. However, with aztreonam alone, pbp1, pbp3 and spy remained unchanged while pbp2 levels were downregulated by > 25%. Imipenem displayed 207-fold higher permeability as compared with aztreonam (mean permeability coefficient of 17,200 nm/s). Although the clinical combination of aztreonam/avibactam and ceftazidime has been proposed as an important treatment of MBL Gram-negatives, we report the first occurrence of long filamentous persister formation. To our knowledge, this is the first study that defines novel β-lactam combinations involving imipenem via maximal suppression of filamentous persisters to combat PDR CDC Nevada K. pneumoniae.
Topics: Ceftazidime; Klebsiella pneumoniae; Aztreonam; Anti-Bacterial Agents; Imipenem; beta-Lactamases; Drug Combinations; Microbial Sensitivity Tests; Azabicyclo Compounds
PubMed: 38326428
DOI: 10.1038/s41598-024-53130-z