Search Results (273)

Glyphosate is the most widely used herbicide worldwide, but many current detection methods rely on lab-based chromatography, requiring costly equipment and expert users. Here, we describe a low-cost, field-deployable fluorescence biosensing platform for glyphosate detection in water and soil. An engineered variant of the Escherichia coli periplasmic binding protein PhnD was optimized through strategic fluorophore placement to produce a robust fluorescence signal increase upon glyphosate binding. The biosensor was integrated into a self-contained, 3D-printed device that functions as a miniature fluorometer, providing a simple yes-or-no output for non-expert users while retaining access to raw fluorescence data. The device exhibits nanomolar fluorescence sensitivity with results comparable to a benchtop fluorometer. Using this platform, glyphosate was reliably detected in buffered solutions, commercial herbicides, tap water, and soil extracts. To mitigate false positives arising from phosphate interference, we developed a dual-sensor strategy incorporating an independent phosphate biosensor and a second-generation device capable of multi-wavelength fluorescence detection. Together, these results demonstrate an affordable and versatile biosensing platform with strong potential for field-based environmental monitoring. Full article

The MarR (multiple antibiotic resistance regulator) family regulators, which are widely conserved across various organisms, play pivotal roles in metabolism, stress response mechanisms, and virulence factor production. However, the regulatory functions of these factors in the degradation of aromatic compounds within Corynebacterium glutamicum remain largely uncharacterized. In this study, we identified a MarR-type regulator, designated AcsR (encoded by ncgl2425), which directly represses the expression of the catechol 2,3-dioxygenase gene ncgl2007 (c23o) and the heavy metal (nickel) transport system permease gene ncgl2351, while activating the expression of ncgl2258 encoding an ABC-type C4-dicarboxylate-binding periplasmic protein. AcsR binds specifically as a dimer to a 6 bp inverted repeat sequence, and this binding is disrupted by catechol in vitro. Correspondingly, catechol induces the expression of c23o in vivo. Phenotypic analysis revealed that the ΔacsR mutant exhibited enhanced resistance to multiple aromatic compounds but increased sensitivity to antibiotics, heavy metals, and oxidants. Collectively, these findings demonstrate that AcsR is an important regulator of stress adaptation in C. glutamicum and provide new insights into the regulatory mechanisms of aromatic compound degradation in this industrially important bacterium. Full article

Glandular trichomes (GTs) are epidermal outgrowths that function as “natural cell factories” for the synthesis of specialized metabolites. Beyond their traditional understanding, GTs on cucumber fruits can form an undesirable trait known as bloom, which negatively affects market value. However, the secretory process, metabolite profiles, and genetic regulation underlying GT development in cucumber remain largely unclear. In this study, we employed scanning electron microscopy (SEM), transmission electron microscopy (TEM), histochemical staining, multi-omics analyses, and liquid chromatography–mass spectrometry (LC-MS) to systematically investigate GT development. The secretory process was classified into four distinct stages via SEM observations: morphogenesis, active metabolism, head sunken, and metabolite release. TEM revealed progressive ultrastructural changes, including increased organelle abundance and expansion of the periplasmic space, which facilitate metabolite transport and release. This process occurs through an autonomous mechanism involving osmiophilic substances and eventual cell rupture. LC-MS analysis identified 744 metabolites belonging to 11 classes, with phenylpropanoids/polyketides—particularly flavonoids—being the most abundant. While metabolite classes are conserved between European greenhouse and North China ecotypes, specific metabolite contents vary significantly. Multi-transcriptome analysis identified 60 candidate genes associated with GT development. Among these, CsaV4_3G003418 was functionally validated through virus-induced gene silencing (VIGS) to be involved in early GT development. Collectively, this work elucidates the secretory mechanism and metabolic characteristics of cucumber GTs, providing a foundation for future functional studies and biotechnological applications of secondary metabolites. Full article

Pseudomonas aeruginosa causes severe healthcare-associated infections, yet no vaccine has been licenced. To circumvent the antigenic variability of classical surface antigens, we evaluated LolB—an essential outer-membrane lipoprotein whose periplasmic orientation favours T-cell-dominant mechanisms with potential antibody access via outer-membrane vesicles (OMVs) or bacteriolysis. An integrative in silico pipeline combined multi-strain conservation (20 isolates), epitope discovery (B- and T-cell), safety filters, physicochemical profiling, de novo/refined 3D modelling, molecular dynamics (MD), and docking to TLR4/MD-2. LolB was highly conserved (95–100% identity) under strong purifying selection (dN/dS = 0.15). A conformational B-cell hotspot centred on Q72 mapped to a solvent-accessible flexible loop. Two class II epitopes—LAAQNSPLT and FLGSAAAVS—showed predicted high affinity (IC₅₀ < 10 nM), non-toxicity, and broad coverage, with the pooled set achieving 98.6% global HLA coverage in silico. The final 119-aa construct (N-terminal hBD-3 adjuvant; GPGPG linkers) was compact and tractable (MW = 12.7 kDa; instability index < 40; near-neutral GRAVY) and scored higher for antigenicity than native LolB (VaxiJen 0.82 vs. 0.41). MD supported thermal stability up to 350 K, linker RMSF < 1.5 Å, and a stable 18.2 ± 2.8 Å interdomain spacing. Docking predicted a 1420 Ų interface and ΔG = −10.2 kcal·mol⁻¹ (Kd = 28 nM) with reproducible polar contacts, suggesting productive TLR4/MD-2 engagement. A conservative R42A/K variant is proposed to temper IFN-γ bias. This work therefore suggests an essentiality-anchored LolB-derived multi-epitope construct as a computational vaccine candidate against multidrug-resistant P. aaeruginosa and defines specific experimentally testable hypotheses for future in vitro/in vivo assessment. Essentiality-anchored epitope selection plus adjuvant-surface engineering yielded a structurally coherent, immunologically rational LolB-derived multi-epitope vaccine warranting experimental validation. Full article

Polyethylene terephthalate (PET) waste accumulation requires sustainable recycling alternatives. While Ideonella sakaiensis PETase offers a green solution, its industrial application is hindered by low solubility and poor thermostability. In this study, we systematically evaluated the synergistic effects of maltose-binding protein (MBP) fusion and periplasmic translocation strategies to optimize PETase production in Escherichia coli. Our results demonstrate that MBP acts as a potent solubilizing partner for PETase, with the cytosolic MBP–PETase variant achieving a high purification yield of 8.4 mg per gram of wet cell weight–a significant improvement over the PelB–PETase control (1.1 mg per gram of wet cell weight). Furthermore, the periplasmic MalE–MBP–PETase construct provided an optimal intermediate compromise between the yield, thermal stability, and catalytic activity by leveraging the oxidative environment of the periplasm for critical disulfide bond formation. Although PelB–PETase exhibited higher specific activity, its low yield limits industrial scalability. This study establishes a robust plug-and-play platform for high-throughput PET depolymerization, providing a foundational step toward a circular plastic economy. Full article

Carbapenemase-producing bacteria, particularly those expressing the KPC-3 variant, pose a critical global health threat due to their resistance to nearly all β-lactam antibiotics, including carbapenems. Rapid and reliable detection tools are urgently needed to improve infection control and guide patient management. Nanobodies (VHHs) present a promising alternative to conventional antibodies thanks to their high stability, small size, and capacity to access cryptic epitopes. Here, we report the generation and characterization of a nanobody specifically targeting KPC-3. An immune VHH phage display library was constructed, with over 90% of clones containing correctly sized inserts. After three rounds of biopanning, high-specificity binders were identified by ELISA screening. Sequencing identified a nanobody with hallmark VHH features, which was expressed and validated by ELISA and Western blot. Although kinetic assays showed no inhibition of KPC-3 enzymatic activity, interestingly, the nanobody demonstrated high-binding recognition of both KPC-2 and KPC-3 in periplasmic extracts from clinical strains. Structural modeling further supported these results, highlighting favorable interaction surfaces. This study provides the first evidence of a nanobody raised against KPC-3 that recognizes a conserved epitope shared by KPC-3 and KPC-2, underscoring its promising use as a molecular tool for detecting KPC variants and establishing a basis for future affinity maturation toward therapeutic applications. Full article

The TonB-dependent receptors (TBDRs) FepA and FhuA transport the siderophores ferric enterobactin (FeEnt) and ferrichrome (Fc), respectively, through the Gram-negative bacterial outer membrane. Their uptake mechanism involves conformational change in an ~150 residue N-terminal luminal domain (NTLD), located within their C-terminal β-barrel (CTβB) channels. We identified four internal sites (1–4) in TBDR that form a conserved network of ion pairs encircling the NTLD-CTβB interface. We tested the mechanistic importance of these electrostatic interactions by engineering systematic Ala substitutions in FepA and FhuA for the acidic or basic side chains that comprise them. Siderophore nutrition assays, colicin susceptibility tests and fluorescence spectroscopic uptake measurements of the mutants showed the importance of site-2, that adheres the base of NL1/Nβ3 and Nβ5 of the NTLD to β14 and β17 on the interior of the CTβB. Disruption of electrostatic bonds at site-2 reduced or eliminated ferric siderophore uptake and severely curtailed colicin susceptibility. Despite these reductions in ligand transport, fluorescent spectroscopic binding measurements showed that the site-2 mutations did not alter the affinity of FepA for FeEnt, nor FhuA for Fc. Elimination of ionic interactions at the three other locations in FepA (sites-1, -3, -4) did not reduce FeEnt uptake. Lastly, the disruption of ionic bonding at site-2 in FepA rendered it more susceptible to proteolysis, in part by OmpT, suggesting that ablation of ionic interactions in site-2 destabilized the NTLD within the CTβB. Overall, the experiments demonstrated that the ion pairs at site-2 in FepA and FhuA, that are evolutionarily conserved in the TBDR superfamily, are essential to the movement of ferric siderophores through the CTβB into the periplasm. Full article

The metal-binding periplasmic protein CusF has been proposed as a bifunctional tag that enhances the solubility of recombinant proteins and enables purification using Cu affinity chromatography. However, evidence for its performance remains limited to a few model proteins. Here, we evaluated CusF as a solubility tag for two heterologous proteins: a putative poly(A)-polymerase from Enterococcus faecalis (Efa PAP) and the red fluorescent protein mCherry. The proteins were fused to CusF, expressed in E. coli BL21 (DE3) pLysS and Rosetta 2 (DE3) strains, and assessed for solubility and IMAC binding. Native Efa PAP was completely insoluble under all tested conditions, and fusion to CusF did not improve its solubility. Similarly, CusF–mCherry accumulated predominantly in the insoluble fraction, with only trace amounts detectable in soluble lysates. Soluble CusF–mCherry did not bind Cu²⁺-charged IMAC resin, while moderate binding to Ni²⁺-charged resin was attributable to the vector-encoded His tag rather than CusF. These results indicate that CusF does not universally enhance protein solubility and may not consistently bind Cu-based IMAC resin. Our findings expand empirical knowledge of solubility tag performance and emphasize the necessity of testing multiple tags to identify optimal strategies for recombinant protein production. Full article

Galactomannans are a typical reserve polysaccharide in the endosperm of leguminous seeds; they turn the endosperm hard when dry and gelatinous and swollen when hydrated. Although galactomannans of several species have been biochemically characterized, little is known about their deposition within the endosperm. This study aimed to clarify how polysaccharides, galactomanans according to the literature, are produced and stored in the endosperm of Caesalpinia pulcherrima seeds by describing its structural and ultrastructural features throughout development. Samples of seeds at different developmental stages were collected and processed for study under light and electron microscopy. During development, the endosperm of C. pulcherrima undergoes substantial anatomical modifications associated with cellular cycles of polysaccharide release that gradually accumulates in the intercellular spaces. Endosperm cells exhibit an active Golgi apparatus with intense polysaccharide production, confirming their secretory function. In the mature endosperm, polysaccharides are stored in periplasmic and intercellular spaces rather than in thickened cell walls, as previously reported for other Leguminosae. By showing that galactomannans accumulate in periplasmic and intercellular spaces rather than in cell walls, our findings expand current understanding of endosperm diversity in Leguminosae and provide a foundation for future comparative studies on galactomannan synthesis and deposition across the family. Full article

Palladium, a non-toxic platinum-group metal, is widely used in catalysis, electronics, hydrogen storage, and chemical industries because of its excellent physical and chemical properties. However, given that the number of palladium-producing countries is limited, recycling is considered essential for ensuring a stable and sustainable global supply. Here, I describe a simple and efficient method for palladium recovery from electronic waste (e-waste) using Enterobacter oligotrophicus CCA6^T. To clarify biomineralization capacity, the role of electron donors in modulating biomineralization capacity was examined. Findings showed that formic acid was the most effective donor, enhancing the relative recovery rate to 44% compared to 23% without electron donors. Transmission electron microscopy analysis revealed palladium particles (1–10 nm) distributed across the cell wall, periplasmic space and cytoplasm, confirming active biomineralization rather than passive biosorption. Moreover, based on a comparison with the biomineralization mechanism of Escherichia coli, the biomineralization mechanism of E. oligotrophicus CCA6^T was estimated . Reaction parameters were then optimized by testing the effects of formic acid concentration, reaction temperature, and reaction pH. Under optimized conditions, the relative recovery rate exceeded 99% within 6 h using 40 mg/L palladium. When this method was applied to a metal dissolution solution prepared from e-waste , a recovery rate of 94% was achieved from trace concentrations (36 µg/L), and palladium loss from bacteria after the palladium recovery test was negligible (<0.01%). Taken together, these results demonstrate that biomineralization using E. oligotrophicus CCA6^T could potentially be applied to the recovery of palladium from e-waste, particularly for trace-level concentrations where conventional methods are ineffective. Full article

A novel heavy metal-resistant bacterium with significant bioremediation capabilities, Sinirhodobacter sp. 1C5-22 was isolated from moderately polluted Shenzhen Futian mangrove rhizosphere sediments. This strain showed exceptional tolerance (MIC ≥ 600 mg/L for Cu/Zn; > 500 mg/L for Ni). Analyses revealed distinct metal-specific distribution strategies: Cd and Ni were predominantly bound extracellularly (>80%); Cu was bound intracellularly (~60%); and Zn exhibited balanced partitioning. Integrated omics analysis identified a molecular defense mechanism coordinated by the CreB transcriptional regulator. This Adsorption–Sequestration–Efflux (ASE) system integrates extracellular polymer binding, periplasmic sequestration via stable metal-binding proteins, and efflux pump activity, resolving the apparent adsorption-tolerance paradox at elevated concentrations. For bioremediation applications, we developed a polyvinyl alcohol–sodium alginate immobilized consortium (PVA-SA 1C5-22). The engineered agent displayed significantly enhanced biosorption capacity compared to free cells and effectively mitigated heavy metal-induced oxidative damage, evidenced by stabilized malondialdehyde levels. It demonstrated robust reusability, maintaining high metal enrichment across five adsorption–desorption cycles in multi-metal wastewater with efficient HCl-driven desorption (55–70%). Critically, it achieved stable nickel removal performance (~20% adsorption, >50% desorption) from authentic electroplating wastewater (1850 mg/L Ni²⁺) through successive multiple cycles. Our integrated approach bridges microbial ecology and environmental biotechnology, establishing this immobilized system as a highly sustainable strategy for complex industrial effluent remediation. Full article

Mycobacterium tuberculosis, the causative agent of tuberculosis, utilizes biofilm formation as a key mechanism to withstand host-derived stresses. To identify novel factors involved in this process, we performed a CRISPRi screen in the model organism Mycobacterium smegmatis. This screen identified trypsin HtrA as a critical factor for growth and biofilm formation. Deletion of htrA led to a profound upregulation of the cell wall amidase Ami3. We demonstrated that Ami3 is a crucial negative regulator of biofilm formation, as overexpression of ami3 recapitulated the biofilm and growth defects of the ΔhtrA strain. Furthermore, we found that the essential role of periplasmic protease HtrA for normal growth could be suppressed by novel mutations in pmt, a gene encoding a phosphomyoinositol mannosyltransferase, at residues F53 and N55, distinct from the previously reported D68 site. Our findings establish a novel regulatory pathway in which HtrA modulates mycobacterial biofilm formation by controlling the levels of Ami3 and reveal new genetic interactions within this network. Full article

Various surfactants have been applied for the remediation of polycyclic aromatic hydrocarbon (PAH)-contaminated environments, but their roles in bioremediation remain controversial. This study focused on rhamnolipid (a typical surfactant) and Burkholderia sp. FM-2 (a high-efficiency phenanthrene-degrading bacterium), investigating its effects on phenanthrene solubilization and biodegradation by analyzing cell surface characteristics and gene expression differences. Results showed that low concentrations of rhamnolipid (20–120 mg/L) promoted phenanthrene degradation, while high concentration (400 mg/L) exerted an inhibitory effect. At 20–56 mg/L, rhamnolipid altered the bacterial surface morphology and functional groups, facilitated lipopolysaccharide release, enhanced cell surface hydrophobicity, and increased zeta potential. When the rhamnolipid concentration was 20 mg/L, the phenanthrene degradation rates of cytoplasmic enzymes, periplasmic enzymes, and extracellular enzymes produced by the bacterium reached over 98% after 15 days of enzyme system culture, demonstrating its role in promoting enzyme production and activity. Transcriptomic analysis revealed that 56 mg/L (1 CMC) rhamnolipid enhanced degradation through multi-pathway regulation of gene expression: upregulating the gene encoding protocatechuate 3,4-dioxygenase to strengthen benzene ring cleavage; increasing the expression of genes related to ABC transporters and protein transport to promote phenanthrene transmembrane transport; and activating genes involved in metabolic processes such as pyruvate metabolism and the tricarboxylic acid (TCA) cycle to enhance central carbon metabolic flux. This regulatory mode optimizes energy supply and redox balance, and indirectly improves phenanthrene bioavailability by modulating membrane structure and function. Full article

Antimicrobial peptides (AMPs), also referred to as host defense peptides, are promising molecules in the development of the next generation of antibiotics against drug-resistant bacterial pathogens. Thanatin comprises a family of naturally occurring cationic AMPs derived from several species of insects. The first thanatin, 21 residues long, was identified from the spined soldier bug, and more thanatin peptides have been discovered in recent studies. The 16-residue thanatin from Anasa tristis, or Ana-thanatin, represents the shortest sequence in the family. However, the antimicrobial activity and mechanistic process underpinning bacterial cell killing have yet to be reported for Ana-thanatin peptide. In this work, we examined the antibacterial activity, structures, and target interactions of Ana-thanatin. Our results demonstrated that Ana-thanatin exerts potent antibiotic activity against strains of Gram-negative and Gram-positive bacteria. Biophysical studies demonstrated that Ana-thanatin interacts with LPS outer membrane and can permeabilize the OM barrier in the process. Atomic-resolution structures of the peptide in free solution and in complex with lipopolysaccharide (LPS) micelle were solved by NMR, determining canonical β-sheet structures. Notably, in complex with LPS, the β-sheet structure of the peptide was better defined in terms of the packing of amino acid residues. Further, MD simulations demonstrated rapid binding of the Ana-thanatin peptide with the LPS molecules within the lipid bilayers. These studies have revealed structural features which could be responsible for LPS-OM disruption of the Gram-negative bacteria. In addition, NMR heteronuclear single quantum coherence (HSQC) studies have demonstrated that Ana-thanatin can strongly interact with the LPS transport periplasmic protein LptA_m, potentially inhibiting OM biogenesis. Taken together, we surmise that the Ana-thanatin peptide could serve as a template for the further development of novel antibiotics. Full article

The cellular envelope of Gram-negative bacteria is a space where processes that are extremely important for the proper functioning of bacteria and determining their virulence take place. The extracytoplasmic protein quality control system, which includes chaperones, protein-folding catalysts, and proteases, is responsible for maintaining homeostasis in this cellular compartment. This system has been well studied in the model bacterium Escherichia coli, but little is known about its function in other bacteria. In bacteria evolutionarily distant from Enterobacteriaceae, the protein quality control system appears to function differently. For example, in the phylum Campylobacterota, a number of homologs of folding factors and proteases, whose functions are important for maintaining homeostasis in the periplasm of E. coli, have not been identified. Instead, there are quality control components that have no similar counterparts in the Enterobacteriaceae. In this review, we present the current state of knowledge on the extracytoplasmic protein quality control system in the model Campylobacterota, C. jejuni and H. pylori. Full article