Probing the interaction between biomolecules under sub-zero temperature conditions by electrophoresis in ice grain boundaries.

BACKGROUND: Psychrophiles can survive under cryogenic conditions because of various biomolecules. These molecules interact with cells, ice crystals, and lipid bilayers to enhance their functionality. Previous studies typically measured these interactions by thawing frozen samples and conducting biological assays at room temperature; however, studying these interactions under cryogenic conditions is crucial. This is because these biomolecules can function at lower temperatures. Therefore, a platform for measuring chemical interactions under sub-zero temperature conditions must be established. RESULTS: The chemical interactions between biomolecules under sub-zero temperature conditions were evaluated within ice grain boundaries with a channel-like structure, which circumvents the need for thawing. An aqueous solution of sucrose was frozen within a microfluidic channel, facilitating the formation of freeze-concentrated solutions (FCSs) that functioned as size-tunable electrophoretic fields. Avidin proteins or single-stranded DNA (ssDNA) were introduced into the FCS in advance. Probe micro/nanospheres whose surfaces were modified with molecules complementary to the target analytes were introduced into the FCS. If the targets have functionalities under sub-zero temperature conditions, they interact with complementary molecules. The chemical interactions between the target molecules and nanospheres led to the aggregation of the particles. The size tunability of the diameter of the FCS channels enabled the recognition of aggregation levels, which is indicative of interaction reactivity. The avidin-biotin interaction and ssDNA hybridization served as models for chemical interactions, demonstrating interactivity under sub-zero temperature conditions. The results presented herein suggest the potential for in situ measurement of biochemical assays in the frozen state, elucidating the functionality of bio-related macromolecules at or slightly below 0 °C. SIGNIFICANCE: This is the first methodology to evaluate chemical interactions under sub-zero temperature conditions without employing the freeze-and-thaw process. This method has the advantage of revealing the chemical interactions only at low temperatures. Therefore, it can be used to screen and evaluate the functionality of cryo-related biomolecules, including cold-shock and antifreeze proteins.

Eliminating extracellular autoinducing peptide signals inhibits the Staphylococcus aureus quorum sensing agr system.

An accessory gene regulator (agr) in the quorum sensing (QS) system in Staphylococcus aureus contributes to host infection, virulence factor production, and resistance to oxidative damage. Artificially maintaining the inactive state of agr QS impedes the host infection strategy of S. aureus and inhibits toxin production. The QS system performs intercellular signal transduction, which is activated by the mature autoinducer peptide (AIP). It is released from cells after AgrD peptide processing as an intercellular signal associated with increased bacterial cell density. This study evaluated the effectiveness of inhibiting agr QS wherein AIP trap carriers were made to coexist when culturing Staphylococcus aureus. Immersing a nitrocellulose (NC) membrane in Staphylococcus aureus ATCC 12600 culture inhibited QS-dependent α-hemolysin production, which significantly reduced the hemolysis ratio of sheep red blood cells by the culture supernatant. A quartz crystal microbalance analysis supported AIP adsorption onto the NC membrane. Adding the NC membrane during culture was found to maintain the expression levels of the agr QS gene agrA and α-hemolysin gene hla lower than that when it was not added. Eliminating extracellular AIP signals allowed agr QS to remain inactive and prevented QS-dependent α-hemolysin expression. Isolating intercellular signals secreted outside the cell is an effective strategy to suppress gene expression in bacterial cells that collaborate via intercellular signaling.

Phase Transfer of AMIET-functionalized Gold Nanoparticles from Aqueous to Organic Solvents.

This paper presents a feasible and reliable phase transfer protocol for polyoxyethylene alkyl amine surfactant (AMIET)-coated gold nanoparticles (AuNPs) in aqueous media to chloroform using a pH-triggered method, through the liquid-liquid interface. In the initial stage, the colloidal aqueous dispersion is destabilized by pH adjustment towards the isoelectric pH of the nanoparticle, which promotes the separation of the particles from water. We further explored a mechanistic view of this phase transfer phenomenon, considering the orientation of hydrophilic and hydrophobic moieties depending on the nature of the surrounding solvent. It was proposed that the AMIET molecules bound to the AuNPs undergo conformational changes through phase transfer. Ultraviolet visible absorption spectra before and after the phase transfer reveal that the original morphology and dispersion states of the particles were preserved.

Effective quorum quenching with a conformation-stable recombinant lactonase possessing a hydrophilic polymeric shell fabricated via electrospinning.

Quorum sensing (QS) in Gram-negative bacteria is frequently regulated by the diffusible signal N-acylhomoserine lactone (AHL) along with the production of virulence factors in pathogens. To inhibit QS, we fabricated heat-resistant, long-term-stable AHL-lactonase AiiM by electrospinning (ES) aqueous polyvinyl alcohol (PVA) solution containing genetically engineered AiiM with a maltose-binding protein (MBP) tag. MBP-AiiM was immobilized via its inclusion within a dense PVA shell formed during the drying process of ES, followed by cross-linking between hydroxyl groups on PVA. Secondary structure analysis via circular dichroism suggested no conformational change in the MBP-AiiM during ES. Even after pre-heating of MBP-AiiM/PVA fiber mats at 70 °C for 24 h, QS-dependent prodigiosin production in the model pathogen Serratia marcescens AS-1 was effectively inhibited to 0.13% that of the control. Additionally, relative prodigiosin production was reduced to ~20% that of the control after 5-month storage in buffer solution. These results suggest that a shear-thinning process using an entangled PVA aggregate during elongational changes to fibrous domains and a drying process during ES contributes not to enzymatic inactivation caused by conformational changes, but rather to the fabrication of a dense PVA shell around the MBP-AiiM molecules to protect them from disruptors including heating. The developed quorum-quenching enzyme has high potential to inhibit AHL-mediated QS frequently appearing in various Gram-negative bacteria.

Structuring of multiple parallel pectin gel filaments by applied shear.

The bundled structure of micron-sized pectin gel filaments was formed by quick shear-induced gelation of the filamentous domains of pectin-polyethylene glycol (PEG) assemblies. Highly concentrated pectin with PEG in a separated pectin-rich phase under aqueous two-phase separation in the pectin/PEG/NaCl system enabled the formation of the pectin-PEG assembly, which was elongated in the flow direction, resulting in the generation of filamentous domains using a microfluidic device. The pectin gel filaments were formed by crosslinking with Ca2+ in the presence of shear-responsive PEG assemblies formed in the PEG-rich phase, because the filamentous PEG assemblies prevented fusion of the pectin filaments to form the seamless cylindrical gel. The shear-dependent elongation applied to the pectin-PEG assembly under the aqueous two-phase separation condition enabled the formation of the biomimetic bundled filamentous structure using bio-safe PEG as a sacrificial polymer, without the requirement of a multi-hole nozzle. Potential applications for gel filaments possessing a bundled structure are matrices in the biomedical field, such as a biodegradable scaffold for cell engineering.

Novel quorum quenching enzymes identified from draft genome of Roseomonas sp. TAS13.

Roseomonas sp. strain TAS13 isolated from an activated sludge sample degrades N-acylhomoserine lactones (AHLs) that are widely utilized as a signal in bacterial quorum sensing systems. The draft genome of Roseomonas sp. TAS13 contains 816 contigs (total 5,078,941 bp) which carries 4760 protein-coding genes and 52 tRNA genes (DDBJ/EMBL/GenBank accession numbers BDLP01000001 through BDLP01000816).

Phylogenetically novel LuxI/LuxR-type quorum sensing systems isolated using a metagenomic approach.

A great deal of research has been done to understand bacterial cell-to-cell signaling systems, but there is still a large gap in our current knowledge because the majority of microorganisms in natural environments do not have cultivated representatives. Metagenomics is one approach to identify novel quorum sensing (QS) systems from uncultured bacteria in environmental samples. In this study, fosmid metagenomic libraries were constructed from a forest soil and an activated sludge from a coke plant, and the target genes were detected using a green fluorescent protein (GFP)-based Escherichia coli biosensor strain whose fluorescence was screened by spectrophotometry. DNA sequence analysis revealed two pairs of new LuxI family N-acyl-L-homoserine lactone (AHL) synthases and LuxR family transcriptional regulators (clones N16 and N52, designated AubI/AubR and AusI/AusR, respectively). AubI and AusI each produced an identical AHL, N-dodecanoyl-L-homoserine lactone (C(12)-HSL), as determined by nuclear magnetic resonance (NMR) and mass spectrometry. Phylogenetic analysis based on amino acid sequences suggested that AusI/AusR was from an uncultured member of the Betaproteobacteria and AubI/AubR was very deeply branched from previously described LuxI/LuxR homologues in isolates of the Proteobacteria. The phylogenetic position of AubI/AubR indicates that they represent a QS system not acquired recently from the Proteobacteria by horizontal gene transfer but share a more ancient ancestry. We demonstrated that metagenomic screening is useful to provide further insight into the phylogenetic diversity of bacterial QS systems by describing two new LuxI/LuxR-type QS systems from uncultured bacteria.