Periplasmic bacterial biomineralization of copper sulfide nanoparticles

Metal sulfides are a common group of extracellular bacterial biominerals. Only few cases of intracellular biomineralization have been reported in this group, mostly limited to greigite (Fe3S4) in magnetotactic bacteria. Here, we report the intracellular but periplasmic biomineralization of copper sulfide by the magnetotactic bacterium Desulfamplus magnetovallimortis (strain BW-1) that is known to mineralize greigite and magnetite (Fe3O4) in the cytoplasm. BW-1 produces hundreds of spherical nanoparticles, composed of 1-2 nm substructures of a poorly crystalline hexagonal copper sulfide that remains in a thermodynamically unstable state. Differential proteomics suggests that periplasmic proteins, such as a DegP-like protein and a heavy metal-binding protein, could be involved in this process. The unexpected periplasmic formation of copper sulfide nanoparticles in BW-1 reveals previously unknown possibilities for intracellular biomineralization.

Membrane Vesicles of Pectobacterium as an Effective Protein Secretion System

Bacteria of genus Pectobacterium are Gram-negative rods of the family Pectobacteriaceae. They are the causative agent of soft rot diseases of crops and ornamental plants. However, their virulence mechanisms are not yet fully elucidated. Membrane vesicles (MVs) are universally released by bacteria and are believed to play an important role in the pathogenicity and survival of bacteria in the environment. Our study investigates the role of MVs in the virulence of Pectobacterium. The results indicate that the morphology and MVs production depend on growth medium composition. In polygalacturonic acid (PGA) supplemented media, Pectobacterium produces large MVs (100–300 nm) and small vesicles below 100 nm. Proteomic analyses revealed the presence of pectate degrading enzymes in the MVs. The pectate plate test and enzymatic assay proved that those enzymes are active and able to degrade pectates. What is more, the pathogenicity test indicated that the MVs derived from Pectobacterium were able to induce maceration of Zantedeschia sp. leaves. We also show that the MVs of β-lactamase producing strains were able to suppress ampicillin activity and permit the growth of susceptible bacteria. Those findings indicate that the MVs of Pectobacterium play an important role in host-pathogen interactions and niche competition with other bacteria. Our research also sheds some light on the mechanism of MVs production. We demonstrate that the MVs production in Pectobacterium strains, which overexpress a green fluorescence protein (GFP), is higher than in wild-type strains. Moreover, proteomic analysis revealed that the GFP was present in the MVs. Therefore, it is possible that protein sequestration into MVs might not be strictly limited to periplasmic proteins. Our research highlights the importance of MVs production as a mechanism of cargo delivery in Pectobacterium and an effective secretion system.

Secretin channel-interactors prevent antibiotic influx during type IV pili assembly in P. aeruginosa

Type IV pili (T4P) are important virulence factors involved in host attachment and other aspects of bacterial pathogenesis. In Gram-negative bacteria, the T4P filament is polymerized from pilin subunits at the platform complex in the inner membrane (IM) and exits the outer membrane (OM) through the OM secretin channel. Although it is essential for T4P assembly and function, the OM secretin complexes can potentially impair the permeability barrier function of the OM and allow the entry of antibiotics and other toxic molecules. The mechanism by which Gram-negative bacteria prevent secretin-mediated OM leakage is currently not well understood. Here, we report a discovery of SlkA and SlkB (PA5122 and PA5123) that prevent permeation of several classes of antibiotics through the secretin channel of Pseudomonas aeruginosa type IV pili. We found these periplasmic proteins interact with the OM secretin complex and prevent toxic molecules from entering through the channel when there is a problem in the assembly of the T4P IM subcomplexes or when docking between the OM and IM complexes is defective.

Responsive Quaternized PDMAEMA Copolymers with Antimicrobial Action

In this work, the antimicrobial action of partially quaternized poly(2-(dimethylamino)ethyl methacrylate) (PQDMAEMA) copolymers using different alkyl halides is presented. The poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) homopolymer was synthesized by group transfer polymerization, followed by the modification of its tertiary amine groups, using bromoethane, iodoethane, bromohexane and bromoethanol, to introduce permanent cationic, quaternary ammonium salt moieties, randomly distributed along the polymer chains. In all cases, the degree of quaternization was low, at ~10 mol%, as verified by proton nuclear magnetic resonance spectroscopy to preserve the thermo-responsive character of the PDMAEMA precursor polymer. The biocidal activity of the lightly quaternized PQDMAEMA copolymers against Escherichia coli and Staphylococcus aureus was evaluated by calculating the minimum inhibitory concentration (MIC) as well as the minimum bactericidal concentration (MBC) of the polymers and by comparing them to the respective values of the precursor non-quaternized PDMAEMA homopolymer. The antibacterial mechanism of action in the solution was studied by zeta potential measurements, scanning electron microscopy and protein leakage tests signifying the disruption of the outer membrane of the bacterial cells to release their periplasmic proteins.

Stress-Responsive Periplasmic Chaperones in Bacteria

Periplasmic proteins are involved in a wide range of bacterial functions, including motility, biofilm formation, sensing environmental cues, and small-molecule transport. In addition, a wide range of outer membrane proteins and proteins that are secreted into the media must travel through the periplasm to reach their final destinations. Since the porous outer membrane allows for the free diffusion of small molecules, periplasmic proteins and those that travel through this compartment are more vulnerable to external environmental changes, including those that result in protein unfolding, than cytoplasmic proteins are. To enable bacterial survival under various stress conditions, a robust protein quality control system is required in the periplasm. In this review, we focus on several periplasmic chaperones that are stress responsive, including Spy, which responds to envelope-stress, DegP, which responds to temperature to modulate chaperone/protease activity, HdeA and HdeB, which respond to acid stress, and UgpB, which functions as a bile-responsive chaperone.

Computational Structure Prediction Provides a Plausible Mechanism for Electron Transfer by the Outer Membrane Protein Cyc2 from Acidithiobacillus ferrooxidans

Cyc2 is the key protein in the outer membrane of Acidithiobacillus ferrooxidans that mediates electron transfer between extracellular inorganic iron and the intracellular central metabolism. This cytochrome c is specific for iron and interacts with periplasmic proteins to complete a reversible electron transport chain. A structure of Cyc2 has not yet been characterized experimentally. Here we describe a structural model of Cyc2, and associated proteins, to highlight a plausible mechanism for the ferrous iron electron transfer chain. A comparative modeling protocol specific for trans membrane beta barrel (TMBB) proteins in acidophilic conditions (pH ~2) was applied to the primary sequence of Cyc2. The proposed structure has three main regimes: extracellular loops exposed to low-pH conditions, a TMBB, and a N-terminal cytochrome-like region within the periplasmic space. The Cyc2 model was further refined by identifying likely iron and heme docking sites. This represents the first computational model of Cyc2 that accounts for the membrane microenvironment and the acidity in the extracellular matrix. This approach can be used to model other TMBBs which can be critical for chemolithotrophic microbial growth.

Removal of disulfide from acid stress chaperone HdeA does not wholly eliminate structure or function at low pH

HdeA is an acid-stress chaperone that operates in the periplasm of various strains of pathogenic gramnegative bacteria. Its primary function is to prevent irreversible aggregation of other periplasmic proteins when the bacteria enter the acidic environment of the stomach after contaminated food is ingested; its role is therefore to help the bacteria survive long enough to enter and infect the intestines. The mechanism of operation of HdeA is unusual in that this helical homodimer is inactive when folded at neutral pH but becomes activated at low pH after the dimer dissociates and becomes partially unfolded. Studies with chemical reducing agents have previously suggested that the intramolecular disulfide bond of HdeA aids in the maintenance of residual structure at low pH; it is believed that this residual structure is important for clustering exposed hydrophobic residues together for the purpose of binding unfolded client proteins. In order to explore its role in HdeA structure and chaperone function we performed a conservative mutation of the disulfide, swapping cysteines with serines. We found that, although residual structure is greatly diminished at low pH without the disulfide, it is not completely lost; conversely, the mutant is almost completely random coil at pH 6. Aggregation assays showed that mutated HdeA, although less successful as a chaperone than wild type, still maintains a surprising level of function, and is better at maintaining the solubility of malate dehydrogenase than chemically reduced wild type. These studies highlight that we still have much to learn about the factors that stabilize residual structure at low pH and the role of disulfide bonds.CRediT author statementImex Aguirre-Cardenas: Investigation, Visualization, Formal analysis, Writing – Review and Editing. Dane Geddes-Buehre: Investigation, Formal analysis, Writing – Review and Editing. Karin Crowhurst: Conceptualization, Funding acquisition, Supervision, Project administration, Investigation, Formal analysis, Visualization, Writing – Original Draft, Writing – Review and Editing.

Critical role of the periplasm in copper homeostasis in Gram-negative bacteria

Copper is essential for life, but is toxic in excess; that is, cells must keep an optimal internal copper concentration. Under aerobic conditions, less toxic Cu(II) taken up by bacterial cells is reduced to more toxic Cu(I) in the cytoplasm. Copper homeostasis is achieved in the cytoplasm and the periplasm as a unique feature of Gram-negative bacteria. The copper efflux pumps, CopA and CusCBA export Cu(I) from the cytoplasm or the periplasm to outside of the cells in Escherichia coli. In addition, the periplasmic proteins, such as a multi-copper oxidase CueO, play a role in the periplasmic detoxification. While the efflux pumps are highly conserved in Gram-negative bacteria, the periplasmic proteins are diversified, indicating that copper homeostasis in the periplasm could contribute to adaptation to various living environments. However, the role of the periplasm and periplasmic proteins in regard to whole-cell copper homeostasis remains unknown. In this study, we addressed the role of the periplasm and periplasmic proteins in copper homeostasis to adapt to various ecological niches. We have used a systems approach, alternating rounds of experiments and models, to further elucidate the dynamics of copper efflux system. We measured the response to copper of the main specific copper export systems in the wild type E. coli strain, and a series of deletion mutant strains. We interpreted these data using a detailed mathematical model and Bayesian model fitting routines, and verified copper homeostasis. Compared with the simulation and the growth in response to copper, we found that the growth was associated with copper abundance in the periplasm. In particular, CueO unique to Gram-negative bacteria contributes both to protection against Cu(I) toxicity and to incorporating copper into the periplasmic components/proteins, resulting in maximizing the growth. These results suggest that Gram-negative bacteria have evolved to utilize the periplasm as a sensor and store for copper, in order to enable Gram-negative bacteria to adapt to a wide range of environmental copper concentrations.