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Chemosphere Jul 2024Titanate nanotubes (TNs) functionalized with CuS nanoparticles using the microwave-assisted hydrothermal method were characterized via XRD, Raman spectroscopy, UV-Vis...
Titanate nanotubes (TNs) functionalized with CuS nanoparticles using the microwave-assisted hydrothermal method were characterized via XRD, Raman spectroscopy, UV-Vis spectrophotometry, and N physisorption. The as-synthesized CuS/TNs had anatase as the main crystalline phase and the band-gap energy was in the visible region, 2.9 eV. The TNs were recrystallized on titania and functionalized with CuS, forming spherical bundles. SEM showed agglomerates of cauliflower-like semispherical particles. The antimicrobial photoactive assets were evaluated against the bacteria Staphylococcus aureus and Escherichia coli. Inhibition was clearly visible in S. aureus after the first 20 min of exposure to a 6-W LED irradiation lamp. The visible-light catalyzed completely and irreversibly the inactivation of S. aureus after 60 min, however, in the case of E. coli, this material only slightly disturbed its growth, which was recovered after 60 min. The successful result obtained with S. aureus can be explained by the fact that it lacks periplasmic superoxide dismutase (SOD) but has staphyloxanthin for external protection against ROS. However, the CuS/TN particles could release Cu ions, which got attached to bacterium structures or entered the cytoplasm; these events together with the generation of ROS under visible LED light helped inactivate quickly staphyloxanthin, thus inflicting permanent damage to the periplasmic membrane.
Topics: Staphylococcus aureus; Titanium; Escherichia coli; Copper; Light; Anti-Infective Agents; Nanotubes; Anti-Bacterial Agents
PubMed: 38735492
DOI: 10.1016/j.chemosphere.2024.142317 -
ACS Infectious Diseases Jun 2024The design of siderophore-antibiotic conjugates is a promising strategy to overcome drug resistance in negative bacteria. However, accumulating studies have shown that...
The design of siderophore-antibiotic conjugates is a promising strategy to overcome drug resistance in negative bacteria. However, accumulating studies have shown that only those antibiotics acting on the cell wall or cell membrane multiply their antibacterial effects when coupled with siderophores, while antibiotics acting on targets in the cytoplasm of bacteria do not show an obvious enhancement of their antibacterial effects when coupled with siderophores. To explore the causes of this phenomenon, we synthesized several conjugate probes using 3-hydroxypyridin-4(1)-ones as siderophores and replacing the antibiotic cargo with 5-carboxyfluorescein (5-FAM) or malachite green (MG) cargo. By monitoring changes in the fluorescence intensity of FAM conjugate in bacteria, the translocation of the conjugate across the outer membranes of Gram-negative pathogens was confirmed. Further, the use of the fluorogen activating protein(FAP)/MG system revealed that 3-hydroxypyridin-4(1)-one-MG conjugate was ultimately distributed mainly in the periplasm rather than being translocated into the cytosol of and PAO1. Additional mechanistic studies suggested that the uptake of the conjugate involved the siderophore-dependent iron transport pathway and the 3-hydroxypyridin-4(1)-ones siderophore receptor-dependent mechanism. Meanwhile, we demonstrated that the conjugation of 3-hydroxypyridin-4(1)-ones to the fluorescein 5-FAM can reduce the possibility of the conjugates crossing the membrane layers of mammalian Vero cells by passive diffusion, and the advantages of the mono-3-hydroxypyridin-4(1)-ones as a delivery vehicle in the design of conjugates compared to the tri-3-hydroxypyridin-4(1)-ones. Overall, this work reveals the localization rules of 3-hydroxypyridin-4(1)-ones as siderophores to deliver the cargo into Gram-negative bacteria. It provides a theoretical basis for the subsequent design of siderophore-antibiotic conjugates, especially based on 3-hydroxypyridin-4(1)-ones as siderophores.
Topics: Siderophores; Anti-Bacterial Agents; Pseudomonas aeruginosa; Gram-Negative Bacteria; Fluorescent Dyes; Escherichia coli; Pyridones; Pyridines; Animals; Fluorescein; Biological Transport; Microbial Sensitivity Tests
PubMed: 38725130
DOI: 10.1021/acsinfecdis.4c00287 -
Current Opinion in Microbiology Jun 2024Bacteria surround themselves with complex cell envelopes to maintain their integrity and protect against external insults. The envelope of Gram-negative organisms is... (Review)
Review
Bacteria surround themselves with complex cell envelopes to maintain their integrity and protect against external insults. The envelope of Gram-negative organisms is multilayered, with two membranes sandwiching the periplasmic space that contains the peptidoglycan cell wall. Understanding how this complicated surface architecture is assembled during cell growth and division is a major fundamental problem in microbiology. Additionally, because the envelope is an important antibiotic target and determinant of intrinsic antibiotic resistance, understanding the mechanisms governing its assembly is relevant to therapeutic development. In the last several decades, most of the factors required to build the Gram-negative envelope have been identified. However, surprisingly, little is known about how the biogenesis of the different cell surface layers is co-ordinated. Here, we provide an overview of recent work that is beginning to uncover the links connecting the different envelope biosynthetic pathways and assembly machines to ensure uniform envelope growth.
Topics: Gram-Negative Bacteria; Cell Wall; Peptidoglycan; Cell Membrane; Bacterial Proteins
PubMed: 38718542
DOI: 10.1016/j.mib.2024.102479 -
Biochemical and Biophysical Research... Jul 2024Sugar phosphates are potential sources of carbon and phosphate for bacteria. Despite that the process of internalization of Glucose-6-Phosphate (G6P) through plasma...
Sugar phosphates are potential sources of carbon and phosphate for bacteria. Despite that the process of internalization of Glucose-6-Phosphate (G6P) through plasma membrane remained elusive in several bacteria. VCA0625-27, made of periplasmic ligand binding protein (PLBP) VCA0625, an atypical monomeric permease VCA0626, and a cytosolic ATPase VCA0627, recently emerged as hexose-6-phosphate uptake system of Vibrio cholerae. Here we report high resolution crystal structure of VCA0625 in G6P bound state that largely resembles AfuA of Actinobacillus pleuropneumoniae. MD simulations on VCA0625 in apo and G6P bound states unraveled an 'open to close' and swinging bi-lobal motions, which are diminished upon G6P binding. Mutagenesis followed by biochemical assays on VCA0625 underscored that R34 works as gateway to bind G6P. Although VCA0627 binds ATP, it is ATPase deficient in the absence of VCA0625 and VCA0626, which is a signature phenomenon of type-I ABC importer. Further, modeling, docking and systematic sequence analysis allowed us to envisage the existence of similar atypical type-I G6P importer with fused monomeric permease in 27 other gram-negative bacteria.
Topics: Vibrio cholerae; Bacterial Proteins; Crystallography, X-Ray; Glucose-6-Phosphate; ATP-Binding Cassette Transporters; Molecular Dynamics Simulation; Protein Conformation; Models, Molecular; Protein Binding; Binding Sites
PubMed: 38704889
DOI: 10.1016/j.bbrc.2024.150030 -
Marine Pollution Bulletin Jun 2024The impact of heavy metal ions on the biodenitrification process remains unknown, which is the key to understand the nitrogen cycle in estuarine areas. Here,...
The impact of heavy metal ions on the biodenitrification process remains unknown, which is the key to understand the nitrogen cycle in estuarine areas. Here, denitrification rate and the abundance of five denitrifying enzyme genes (narG, nirK, napA, norB and nosZ) in Liaohe Estuary sediments were examined, and the community structure of nirK denitrifying bacteria was also analyzed. The results demonstrate a significant positive correlation between heavy metal content (Cu, Zn, and Cr) and the denitrification rate, and the abundance of napA/norB (periplasmic nitrate reductase and nitric-oxide reductase) in sediments. The dominant narG denitrifiers were Pseudomonas, Hydrogenophaga, and Serratia known to be tolerant to heavy metal pollution. Sediment particle size, NO, NO, Zn, and Cd were the key factors influencing the denitrifying community structure. These findings suggest that heavy metals may enhance the aerobic denitrification process in sediments and mitigate the adverse effects of high dissolved oxygen levels.
Topics: Denitrification; Geologic Sediments; Metals, Heavy; Estuaries; Water Pollutants, Chemical; Bacteria; China; Nitrate Reductase
PubMed: 38696947
DOI: 10.1016/j.marpolbul.2024.116408 -
Biochemistry May 2024Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import...
Periplasmic solute-binding proteins (SBPs) are key ligand recognition components of bacterial ATP-binding cassette (ABC) transporters that allow bacteria to import nutrients and metabolic precursors from the environment. Periplasmic SBPs comprise a large and diverse family of proteins, of which only a small number have been empirically characterized. In this work, we identify a set of 610 unique uncharacterized proteins within the SBP_bac_5 family that are found in conserved operons comprising genes encoding (i) ABC transport systems and (ii) putative amidases from the FmdA_AmdA family. From these uncharacterized SBP_bac_5 proteins, we characterize a representative periplasmic SBP from sp. A09 (Ami_SBP) and show that Ami_SBP binds l-amino acid amides but not the corresponding l-amino acids. An X-ray crystal structure of Ami_SBP bound to l-serinamide highlights the residues that impart distinct specificity for l-amino acid amides and reveals a structural Ca binding site within one of the lobes of the protein. We show that the residues involved in ligand and Ca binding are conserved among the 610 SBPs from experimentally uncharacterized FmdA_AmdA amidase-associated ABC transporter systems, suggesting these homologous systems are also likely to be involved in the sensing, uptake, and metabolism of l-amino acid amides across many Gram-negative nitrogen-fixing soil bacteria. We propose that Ami_SBP is involved in the uptake of such solutes to supplement pathways such as the citric acid cycle and the glutamine synthetase-glutamate synthase pathway. This work expands our currently limited understanding of microbial interactions with l-amino acid amides and bacterial nitrogen utilization.
Topics: Amides; Crystallography, X-Ray; Periplasmic Binding Proteins; ATP-Binding Cassette Transporters; Amino Acids; Mesorhizobium; Bacterial Proteins; Binding Sites; Models, Molecular; Amidohydrolases; Calcium; Protein Binding
PubMed: 38696389
DOI: 10.1021/acs.biochem.4c00096 -
Protein Expression and Purification Aug 2024Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of...
Phytate (inositol hexaphosphate) is the major storage form of phosphorus (P) in nature, and phytases catalyze the hydrolysis of P from phytate and the formation of inositol phosphate isomers. In this study, a bacterium that produces phytase was isolated in a phytase screening medium. The bacterium was identified as Klebsiella sp. using phenotypic and molecular techniques. The PhyK phytase gene was successfully amplified from the genome, inserted into the pET-21a (+) vector, and expressed as a recombinant protein in E. Coli BL21. The efficiency of a laboratory phytase (Lab-Ph, PhyK phytase) was determined and compared with a commercial phytase (Com-Ph, Quantum Blue 40P phytase, AB Vista) under an in vitro digestion assay. The native signal peptide effectively facilitated the translocation of the protein to the periplasmic space of E. Coli BL21, resulting in the proper folding of the protein and the manifestation of desirable enzyme activity. The Lab-Ph displayed the temperature and pH optima at 50 °C and 5 respectively. In addition, the Lab-Ph was inactivated at 80 °C. Under an in vitro digestion assay condition, Lab-Ph improved the P solubility coefficient in broiler diets. In comparison, the Com-Ph significantly increased the P solubility coefficient even when compared with the Lab-Ph. In summary, this study has shown that Lab-Ph possesses the necessary biochemical properties to be used in various industrial applications. However, Lab-Ph is extremely sensitive to heat treatment. The Lab-Ph and Com-Ph under an in vitro digestion assay improved the solubility coefficient of P in the broiler diet.
Topics: Animals; Chickens; Recombinant Proteins; 6-Phytase; Solubility; Klebsiella; Escherichia coli; Animal Feed; Bacterial Proteins; Hydrogen-Ion Concentration; Minerals; Phytic Acid
PubMed: 38685535
DOI: 10.1016/j.pep.2024.106489 -
Frontiers in Chemistry 2024Andrographolide is one of the main biologically active molecules isolated from , which is a traditional Chinese herb used extensively throughout Eastern Asia, India, and...
Andrographolide is one of the main biologically active molecules isolated from , which is a traditional Chinese herb used extensively throughout Eastern Asia, India, and China. often known as is a common clinical opportunistic pathogen with remarkable adaptability to harsh settings and resistance to antibiotics. possesses a wide array of virulence traits, one of which is biofilm formation, which contributes to its pathogenicity. One of the main modulators of the -controlled intramembrane proteolysis pathway is AlgW, a membrane-bound periplasmic serine protease. In this work, we have used a set of density functional theory (DFT) calculations to understand the variety of chemical parameters in detail between andrographolide and levofloxacin, which show strong bactericidal activity against . Additionally, the stability and interaction of andrographolide and levofloxacin with the protein AlgW have been investigated by molecular docking and molecular dynamics (MD) simulations . Moreover, the growth and inhibition of biofilm production by experiments were also investigated, providing insight that andrographolide could be a potential natural product to inhibit
PubMed: 38680458
DOI: 10.3389/fchem.2024.1388545 -
Trends in Biochemical Sciences Apr 2024The survival and virulence of Gram-negative bacteria require proper biogenesis and maintenance of the outer membrane (OM), which is densely packed with β-barrel OM... (Review)
Review
The survival and virulence of Gram-negative bacteria require proper biogenesis and maintenance of the outer membrane (OM), which is densely packed with β-barrel OM proteins (OMPs). Before reaching the OM, precursor unfolded OMPs (uOMPs) must cross the whole cell envelope. A network of periplasmic chaperones and proteases maintains unfolded but folding-competent conformations of these membrane proteins in the aqueous periplasm while simultaneously preventing off-pathway aggregation. These periplasmic proteins utilize different strategies, including conformational heterogeneity, oligomerization, multivalency, and kinetic partitioning, to perform and regulate their functions. Redundant and unique characteristics of the individual periplasmic players synergize to create a protein quality control team capable responding to changing environmental stresses.
PubMed: 38677921
DOI: 10.1016/j.tibs.2024.03.015 -
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