Applied Microbiology and Biotechnology最新文献

Environmental stresses due to climate changes, such as high temperatures and land degradation, significantly impact crop yield, making innovative strategies necessary to increase plant stress tolerance. This study investigates the potential of plant growth-promoting rhizobacteria (PGPR) to enhance wheat resilience under multiple environmental stresses, such as high salinity and temperature. For this, 15 bacterial strains were isolated from the rhizosphere and roots of Pancratium maritimum and screened for their ability to withstand high salinity (50–600 mM NaCl) and elevated temperatures (up to 42 °C). The isolates were identified by 16S rRNA sequencing and tested for their PGP traits under combined abiotic stresses. Most of the strains exhibited PGP features, such as biofilm formation, phosphate solubilization, and phytohormone production. To enhance the growth of wheat plants, used as a model crop of commercial interest, three different consortia were designed and tested in vitro. The consortium (CONSIII), composed of Serratia marcescens ERA6, Enterobacter cloacae ERA9, and Bacillus proteolyticus ESOB2, provided synergistic effects that led to an enhancement in plant growth and stress resilience in vitro. This positive effect was confirmed in pot trials under double abiotic stress (37 °C, 132 mM NaCl), where CONSIII was able to boost the root and shoot growth, increase chlorophyll and carotenoid content, and enhance antioxidant activity, mitigating reactive oxygen species accumulation. These findings underscore the potential of PGPR consortia as bioinoculants for sustainable agriculture, demonstrating their effectiveness in the simultaneous presence of salinity and heat stresses—a challenging and under-investigated environmental scenario.

• PGPR strains isolated from Pancratium maritimum rhizosphere are able to grow and exhibit PGP traits under combined salinity and heat conditions

• The formulated consortium of PGPR strains (CONSIII) significantly enhances wheat growth and stress resilience under a multi-stress environment

• CONSIII increases plant biomass, pigment content, and antioxidant activity, proving its value as a sustainable bioinoculant

Thioredoxins (TRXs) are small, conserved redox-active proteins that play central roles in oxidative stress responses. Here, we identified and functionally characterized a novel thioredoxin, MpTRX1, from the newly isolated yeast Metschnikowia persimmonesis. The full-length MpTRX1 gene was cloned and expressed in Escherichia coli and Saccharomyces cerevisiae to analyze its biochemical and physiological functions. MpTRX1 encodes a 103-amino-acid protein containing a canonical CXXC redox motif, and structural modeling confirmed a conserved thioredoxin fold. Recombinant MpTRX1 exhibited clear disulfide reductase activity in both DTNB (5,5′-dithiobis-(2-nitrobenzoic acid)) and insulin reduction assays. Mutation of either catalytic cysteine residue abolished activity, confirming their essential roles. Moreover, heterologous expression of MpTRX1 in S. cerevisiae enhanced tolerance to hydrogen-peroxide-induced oxidative stress. Although the functional assays were conducted in a heterologous system, these findings demonstrate that MpTRX1 is a bona fide thioredoxin that may contribute to oxidative stress protection in M. persimmonesis. This work provides the first molecular characterization of a protein from M. persimmonesis and establishes a foundation for future studies on its potential ecological and biotechnological applications.

• Identification of MpTRX1, a novel thioredoxin from M. persimmonesis.

• Recombinant MpTRX1 reduces both chemical and protein substrates.

• Overexpression of MpTRX1 enhances oxidative stress tolerance in S. cerevisiae.

In nature, remarkable geological structures, such as stromatolites, thrombolites, and beachrocks, are formed through biocementation, a process involving the successive dissolution and reprecipitation of CaCO₃. This research demonstrates mimicking natural cement via bio-sintering of limestone, a process that involves successive dissolution and reprecipitation of limestone facilitated by bacteria under ambient environmental conditions. When the bacterium Acetobacter aceti (ATCC 15973) was introduced into a mixture of ethanol and limestone powder, the pH dropped rapidly, leading to the dissolution of limestone into calcium acetate. After ethanol was fully consumed, the pH gradually increased due to acetate oxidation, causing biocement crystals to precipitate. All reaction rates were measured, and the products characterized through Fourier transform infrared spectroscopy, scanning electron microscopy, quantitative X-ray diffraction, and a particle size analyzer. The detailed characterization indicates that the precipitate is mainly calcite and that the dissolved calcium carbonate sinters microbially. This pathway offers new opportunities in biocementation research by using limestone as a direct calcium source, reducing the overall carbon footprint, and eliminating the need for urea or other chemical additives. The biocement produced shows potential for practical applications in soil stabilization, eco-concrete, and other sustainable construction solutions, providing a foundation for environmentally conscious alternatives in next-generation construction. 

• Novel bio-sintering emulates natural CaCO₃ cementation via microbial action.

• Acetobacter aceti mediates limestone dissolution and calcite precipitation.

• The process forms calcite without urea or synthetic additives.

Porcine deltacoronavirus (PDCoV) is an emerging enteric coronavirus causing high mortality in neonatal piglets and posing a significant threat to the swine industry. Evidence indicates that PDCoV has cross-species transmission potential and may pose a zoonotic risk, emphasizing the need for reliable serological tools for epidemiological surveillance and vaccine evaluation. Here, we developed a double-antigen sandwich enzyme-linked immunosorbent assay (DAgS-ELISA) based on the PDCoV S1 protein, expressed in CHO cells and used as both coating and HRP-conjugated antigen. The assay reliably detected PDCoV-specific antibodies in sera from pigs, chickens, rabbits, and mice, showing high sensitivity (92.86%) and specificity (99.11%) as determined by receiver operating characteristic curve analysis, with excellent reproducibility. No cross-reactivity was observed with antibodies against other common swine pathogens. Concordance with indirect immunofluorescence assay was 96.18% (kappa = 0.923), and assay results correlated strongly with neutralizing antibody titers (Pearson r = 0.865). Overall, this S1-based DAgS-ELISA provides a sensitive, specific, and cross-species applicable method for PDCoV serological detection, supporting its use for epidemiological surveillance and evaluation of vaccine-induced neutralizing antibodies.

• A novel S1-based double-antigen sandwich ELISA was established for PDCoV detection.

• The assay shows high sensitivity, strong specificity, and broad cross-species applicability.

• ELISA results strongly correlate with neutralizing antibody titers, aiding vaccine evaluation.

The increasing spread of antimicrobial resistance has prompted the search for innovative alternatives to conventional antibiotics. Chitosan, a biopolymer derived from chitin, is known for its broad-spectrum antimicrobial activity. This study evaluated both direct and indirect antimicrobial activity of chitosan obtained from Hermetia illucens, a novel and sustainable source compared to the traditionally crustacean-derived biopolymer. Chitosan produced from H. illucens larvae, pupal exuviae and adults, through heterogeneous and homogeneous deacetylation, was tested for both its indirect and direct antimicrobial effects. The indirect effect was evaluated by measuring the induction of Human Beta-Defensin-2 (HBD-2) expression in HaCaT keratinocytes stimulated with lipopolysaccharide of Salmonella typhimurium, a Gram-negative bacterium. The direct antimicrobial activity was assessed against Gram-positive pathogens (Enterococcus faecalis, Staphylococcus epidermidis, and Streptococcus agalactiae), using a microdilution assay and plate colony count. Results demonstrated significant bacteriostatic effects at 0.5 mg/mL, with some samples, particularly the homogeneous unbleached pupal exuviae chitosan and the heterogeneous unbleached larvae chitosan, comparable to or even superior to commercial chitosan in terms of biological activity. Furthermore, insect-chitosan significantly up-regulated HBD-2 expression, suggesting immunomodulatory activity. These findings validated H. illucens as a promising alternative source of chitosan with dual antimicrobial activity, and supported its potential use in clinical, pharmaceutical and biomedical applications.

• Insect-chitosan activates innate immunity via strong HBD-2 induction

• Chitosan samples showed notable growth-inhibition toward key Gram-positive strains

• Hermetia illucens chitosans provide efficacy comparable or superior to the commercial biopolymer

The escalating challenge of antibiotic resistance in aquaculture critically threatens global fish health and food security, underscoring an urgent need for novel antimicrobial strategies. This study explored the bioactive potential of metabolites from the marine actinomycete Streptomyces zaomyceticus, isolated from a deep-sea sediment sample off Sharm El-Sheikh, Egypt. Bioactivity-guided fractionation led to the isolation and structural elucidation of SPIROST-8-EN-11-ONE, 3-HYDROXY- (SEOH), identified as a novel spirostenoid. SEOH exhibited significant broad-spectrum in vitro growth inhibition against a diverse panel of aquaculture-relevant pathogens, including Gram-positive and Gram-negative bacteria, and opportunistic fungi. It demonstrated potent minimum inhibitory concentrations (MICs) ranging from 0.25 to 1.0 µg/mL, notably effective against multidrug-resistant (MDR) Klebsiella pneumoniae (0.25 µg/mL) and Enterococcus faecalis (0.5 µg/mL). Scanning electron microscopy (SEM) revealed that SEOH treatment (2× MIC) induced significant morphological alterations, including visible cell surface modifications and reduced cell numbers, in both bacterial (E. faecalis, K. pneumoniae, P. aeruginosa) and fungal (C. albicans) pathogens. Preliminary cytotoxicity assessment using the MTT assay on HepG2 cells yielded a promising IC₅₀ value of 71.76 ± 0.62 µg/ml, indicating a favorable in vitro safety profile. The novel structure of SEOH coupled with its potent, broad-spectrum in vitro antimicrobial activity against crucial aquaculture pathogens positions it as a highly promising candidate. These compelling in vitro findings strongly warrant comprehensive in vivo efficacy and safety studies to fully establish SEOH’s potential as a novel therapeutic agent or feed additive for advancing aquaculture sustainability and animal health.

• Novel Spirostenoid Discovery: SEOH, a new spirostenoid from Streptomyces zaomyceticus, was identified

• Potent Broad-Spectrum Activity: It shows strong inhibition against MDR aquaculture pathogens (MICs = 1.0 µg/mL)

• Warrants Further Study: Its promising safety profile and potency merit in vivo testing for aquaculture use

Porins govern nutrient uptake and antibiotic influx in Gram-negative bacteria, making their characterization critical for understanding permeability, resistance mechanisms, and structure-function relationships. From a biotechnological point of view, they are effective tools for modulating the transport of substances across the outer bacterial membrane or for building catalytically active nanoreactors and biosensors. Quantitative data on mass transport through membranes is of great interest, but not trivial to obtain, as in vivo analyses are confounded by cellular complexity and variability. Here, we present a synthetic bottom-up approach, based on polymersomes containing reconstituted purified porins, enabling direct, quantitative measurement of substrate translocation, while minimizing interferences from native processes. Encapsulation of Gaussia luciferase allowed real-time monitoring of coelenterazine (CLZ) translocation across the polymeric membrane in the absence and presence of porins. The typically flash-type luciferase kinetics adapts a glow-type light emission profile, whose signal increases over time. This allows conclusions to be drawn about the substrate concentration accessible to the enzyme, enabling quantitative calculations of the transport rates. The novel approach was exemplarily used to compare the transport characteristics of three Escherichia coli porins: Outer membrane protein F (OmpF), a deletion variant selected for larger pore size OmpF∆, and Phosphoporin E (PhoE). OmpF∆ exhibited the highest transport rate of 78 molecules s⁻¹ per porin trimer, exceeding OmpF (10.8 molecules s⁻¹) more than sevenfold, whereas PhoE showed a lower rate of 2.8 molecules s⁻¹ for the neutral CLZ substrate. Analysis of two CLZ derivatives of slightly higher molecular mass and notably greater hydrophobicity revealed that transport through OmpF and OmpF∆ was reduced by half, whereas PhoE exhibited lower selectivity for the selected substrates.

• Synthetic polymersomes enable direct, quantitative analysis of porin transport

• OmpF∆ exhibits a sevenfold higher molecular flux than wildtype OmpF porins

• The assay provides a versatile platform to study porin selectivity and permeability

Solventogenic Clostridium species can efficiently produce n-butanol and other valuable chemicals via acetone–butanol–ethanol (ABE) fermentation from plant-based feedstocks. For economic and ecological sustainability, cheap and abundant substrates such as lignocellulosic and hemicellulosic residues from agricultural or forestry side streams are preferable. Cereal brans, rich in hemicellulose, represent a promising substrate. However, for direct fermentation of this material, only low product titers are reported. In this study, we characterized the utilization of arabinoxylan, the main polysaccharide component of cereal bran, by the industrial ABE producer Clostridium saccharobutylicum DSM 13864^T and report inefficient degradation of the substrate. Supplementation with hemicellulolytic enzyme mixtures derived from the thermophilic organism Thermoclostridium stercorarium subsp. stercorarium DSM 8532^T significantly enhanced substrate utilization. The best improvement was achieved by the addition of the arabinofuranosidase Axh43A, which reduced the residual sugar content in the fermentation broth from 48.2 to 17.8%. Analysis of the remaining oligosaccharides after growth on arabinoxylan showed that C. saccharobutylicum cannot remove O-2 and O-3 α-L-arabinofuranosyl groups from double-substituted xyloses, creating a key bottleneck in arabinoxylan degradation that is overcome by Axh43A addition. Plasmid-based expression of Axh43A in C. saccharobutylicum DSM 13864^T replicated the enzymatic supplementation effects, confirming the enzyme’s role in overcoming this limitation. This underscores the potential of genetic engineering to enhance the valorization of lignocellulosic biomass in biotechnological fermentation processes.

Benzyl alcohol (BA) and two BA esters were examined for their ability to support growth of Burkholderia vietnamiensis G4 and expression of toluene-2-monooxygenase (T2MO) activity. Resting cell assays and an activity-based labeling (ABL) approach were used to quantify T2MO activity and levels of T2MO hydroxylase subunit in cells grown either on substrates already known to support T2MO expression (toluene, phenol); novel aromatic substrates (BA, benzyl acetate (BAc); benzyl butyrate (BBu)); and non-aromatics (lactate, acetate, and butyrate). Specific rates of TCE oxidation and levels of activity-based fluorescent labeling of T2MO hydroxylase α-subunits in cells grown on BA or BA esters were comparable to or greater than those of cells grown on toluene or phenol. In contrast, levels of activity and protein labeling of cells grown on acetate or butyrate were like those of lactate-grown cells. Cells grown on BA also degraded 1,2-cis-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC). While toluene-grown cells generally exhibited higher specific rates of chloroethene transformation, BA-grown cells had consistently higher transformation capacities for these compounds. Stable accumulation of BA in batch cultures grown on BBu in the presence of propyne and the inhibitory effects of BA on TCE transformation both suggest BA is a T2MO substrate. Substrate-specific O₂ uptake studies also suggest 2-hydroxybenzyl alcohol is a likely product of T2MO-dependent BA oxidation. Our results suggest that BA and benzyl esters could be useful for promoting cometabolic transformations, because unlike toluene or phenol, these are “generally regarded as safe” (GRAS) compounds with little or no human toxicity.

• Burkholderia vietnamiensis G4 cometabolizes TCE when grown on benzyl alcohol (BA).

• Activity-based labeling showed BA and BA producing esters support T2MO expression.

• BA and BA producing esters are safe compounds to promote aerobic TCE transformation.