ABSTRACTThis study aimed to assess the potential of Lentilactobacillus hilgardii as a novel candidate for malolactic fermentation (MLF) in winemaking, through comparative genomics and experimental validation, in direct comparison with Oenococcus oeni. We performed a pangenome analysis on 16 L. hilgardii and 7 O. oeni strains to explore their genetic diversity, focusing on wine‐related traits. Functional predictions were generated using genome‐scale metabolic models (ModelSEED/KBase), including in silico co‐inoculation with Saccharomyces cerevisiae EC1118 and post‐alcoholic fermentation simulations. The reference strains L. hilgardii DSM 20176 and O. oeni DSM 20252 were experimentally tested for MLF performance in a synthetic wine‐like medium at 25°C and 10°C. Core‐genome comparison revealed that 67.9% of the malolactic enzyme sequence is conserved between the two species, with comparable docking affinity to L‐malic acid. L. hilgardii harboured unique enzymes with potential oenological interest (phenolic acid decarboxylase, mannitol dehydrogenase, glucosidase) and distinctive stress‐related proteins (YaaA, HrcA, ASP23), suggesting improved tolerance to oxidative, temperature, and alkaline stresses. Notably, L. hilgardii showed genomic potential to degrade putrescine, arginine, and ornithine, precursors of ethyl carbamate. Experimentally, L. hilgardii reduced L‐malic acid from 2.5 g/L to < 0.1 g/L within 12 days at 10°C, while O. oeni showed no MLF activity at this temperature. At 25°C, both strains completed MLF within 6–7 days. L. hilgardii also consumed > 80% of residual fructose at 10°C, whereas O. oeni showed minimal utilisation. Our results demonstrate that L. hilgardii combines a favourable genomic repertoire for wine adaptation with superior MLF performance at low temperature, suggesting its potential as an alternative to O. oeni in cool‐climate winemaking. This work provides the first genome‐scale comparative and functional evaluation of L. hilgardii in the winemaking context, highlighting its technological promise to improve fermentation reliability, reduce spoilage risk, and expand the biodiversity of malolactic starters.

ABSTRACTIn this study, we established a highly sensitive on‐site detection technology for Bacillus anthracis. Firstly, we integrated Multiple Enzyme Isothermal Rapid Amplification (MIRA) with the clustered regularly interspaced short palindromic repeats (CRISPR) /associated protein 13a (CRISPR/Cas13a) detection system to develop a highly sensitive CRISPR/Cas13a assay. After testing crRNA selection, MIRA primers, reaction temperature, and CRISPR detection conditions, the CRISPR/Cas13a detection system employing dual crRNAs achieved a detection limit of 1000 copies/mL for B. anthracis. Quantitative analysis was additionally attempted. Compared with other common respiratory pathogens, the assay demonstrated high specificity. In clinically simulated samples, all 20 positive specimens were correctly identified, and all 13 negatives were unambiguously classified as negative. Based on these findings, we established a CRISPR point‐of‐care testing technology. By developing a CRISPR point‐of‐care testing device together with a tested lyophilised reagent system, the device achieved a detection limit of 250 copies/mL and delivered results within 30 min. All positive samples were accurately identified, and every negative sample was classified as negative. Consequently, this study presents a highly sensitive and portable technology for on‐site detection of B. anthracis. It holds significant value for on‐site detection of emerging infectious diseases.

Despite the availability of vaccines, foot-and-mouth disease (FMD) remains a significant concern in many developing countries, causing severe economic losses and affecting local farming communities. Virus-like particle (VLP) vaccines are highly regarded for their safety and efficacy. N-glycosylation for stabilisation and recognition by antigen-presenting cells has been a widely adopted strategy, particularly in enveloped viruses. Here, FMD virus (FMDV) VLPs were employed as a model for artificial glycosylation. N-glycosylation was introduced by mutating the potential glycosylation site of VP1 and then N-glycosylated FMDV VLPs were successfully produced in Pichia pastoris. Glycan profiling revealed that the majority of associated glycans (72.93%) were of the high-mannose type, with additional hybrid type (4.16%) and complex type (22.92%) detected. Functional analyses demonstrated that glycosylation significantly enhanced the stability of VLPs and facilitated the uptake by antigen-presenting cells. Animal experiments further revealed that glycosylation could induce a higher cellular immune response compared to WT VLPs, offering a reference for the glycosylation design of VLP vaccines.

ABSTRACTSmall‐conductance mechanosensitive channels (MscS) are established l‐glutamate exporters in industrially relevant bacteria, yet their role in the methylotrophic bacterium Bacillus methanolicus, a promising platform for sustainable methanol‐based l‐glutamate production, remains unexplored. Our research on B. methanolicus MGA3 identifies its MscS as the sole mechanosensitive channel in this organism and a key exporter of the amino acid l‐glutamate, providing valuable insights into its potential for industrial applications. Transcriptomic analysis of B. methanolicus wild type cultured on an l‐glutamate production medium revealed downregulation of the fatty acid biosynthesis genes fadR, fadF, mutB2, and acdA, suggesting that fatty acid metabolism is influenced by l‐glutamate overproduction, with consequent changes in membrane fluidity likely driving mechanosensitive channel‐mediated l‐glutamate efflux. The MscS‐like channel in B. methanolicus shares structural and functional similarities with MscS in Escherichia coli and with MscCG in Corynebacterium glutamicum. In silico structural predictions show that MGA3 MscS forms a homoheptameric structure with a transmembrane TM‐barrel, resembling that of E. coli MscS. The opening mechanism of the channel, driven by membrane dynamics, involves coordinated rotation and flipping of its transmembrane helices, with variations in lipid composition potentially influencing the channel's activity. Additionally, under biotin‐replete conditions, where this essential coenzyme supports carboxylases involved in fatty acid biosynthesis, l‐glutamate overproduction was suppressed in MGA3. Finally, metabolic engineering experiments inducing MscS gain‐ and loss‐of‐function further confirmed the channel's critical role in B. methanolicus amino acid production, proportionally enhancing and reducing l‐glutamate efflux, respectively. These findings open doors to novel strategies for engineering B. methanolicus and related methylotrophic organisms for sustainable amino acid production.

Rapid and reliable analytical techniques play important roles in various research fields and are particularly crucial for diagnosing infectious diseases in clinical settings. African swine fever (ASF) is a devastating viral pig disease for which no effective vaccine is available. The ongoing ASF pandemic has highlighted the importance of rapid and accurate diagnosis, which enables the timely implementation of control and eradication measures. In this study, a ready-to-use bioluminescence immunosensor based on a split-nanoluciferase (NanoLuc) reporter system was proposed for the one-step sensitive detection of ASF virus (ASFV) antibodies. Specifically, the NanoLuc subunits SmBiT/LgBiT were each genetically fused to the ASFV p30 protein and protein G and used as probes. The simultaneous binding of the probes to ASFV IgGs induced the reconstitution of functional NanoLuc, which can generate a strong bioluminescent signal output by catalysing the substrate furimazine. This immunosensor allows the rapid and homogeneous detection of ASFV antibodies in solution, requiring only one incubation step of 10 min. This immunosensor also has high sensitivity, high specificity, and a wide dynamic range and is particularly promising for point-of-care testing. Comparative analysis of clinical samples validated the reliability and robustness of this approach and demonstrated high consistency with enzyme-linked immunosorbent assay (ELISA) results (concordance rate: 98.71%). These results suggest that the proposed immunosensor provides an attractive alternative to conventional immunoassays and could be easily repurposed by generating specific probes for antibody detection in other diseases.

ABSTRACTPseudomonas syringae is a plant pathogen complex responsible for bacterial canker in cherry. In the absence of any control measures, bacteriophages (phages) have the potential for biocontrol. However, it is crucial to first evaluate the role of bacterial antiviral defence systems (ADS) in phage infection dynamics for careful design of a phage cocktail (mixture). Investigating 250 Pseudomonas strains revealed the Ps complex possessed diverse ADS with defence profiles being influenced by phylogeny. Phage host range assays revealed five MR phages with distinct genotypes possessed strong lytic activity against several bacterial canker‐causing Ps pathovars, including syringae and morsprunorum race 1 and 2. Phage susceptibility and resistance appeared to be associated with individual ADS rather than defence profiles as a whole. Multisequence alignment of lipopolysaccharide biosynthesis genes glucose‐1‐phosphate thymidylyltransferase (gpt), glycosyltransferase family 1 (gst1) and lipopolysaccharide kinase (lpk) found these potential receptor genes to be highly conserved within Ps phylogroups. However, gpt alone appeared to influence phage infectivity. Our findings indicate that the gpt gene is a potential primary predictor of MR phage susceptibility, hypothesised to influence phage absorption, while individual ADS only have a secondary role in phage resistance. This study highlights that understanding the genetic mechanisms underlying phage‐bacterial interactions is crucial for designing more effective phage cocktails capable of targeting a broader range of pathogenic strains, but phage screening still is a powerful tool to select phages for biocontrol treatments.

This paper explores the potential of microbiology education at the secondary school level as a catalyst for enhancing both science, technology, engineering and mathematics (STEM) literacy and essential 21st-century competencies. As an inherently interdisciplinary field, microbiology offers an effective platform to develop scientific and technological knowledge while cultivating broader competencies, such as emotional intelligence, creativity and critical thinking, as outlined by UNESCO (UNESCO, 2016). Scientific, societal and educational (SSE) challenges for the next decade are interlinked, and many of these trans-dimensional issues require a solid foundation in biology and microbiology. Thus, research and education must evolve in step with these trends. Despite growing advocacy for microbiology in high school curricula, a significant gap remains in understanding how its teaching aligns with these broader developmental goals. This disconnection is particularly evident in Europe, where STEM engagement among youth remains low. As an experimental and accessible science, microbiology offers unique pedagogical strategies that address not only the cognitive dimensions of STEM but also the skills and attitudes needed for a complex, digital and interdependent world. By integrating microbiology into secondary education, teachers can bridge the gap between scientific literacy and future readiness, empowering students to build a generation capable of shaping a sustainable and innovative future. In this discourse, we describe examples of topics and strategies that could be implemented at the secondary education level, as well as at other educational levels, including university.

ABSTRACTGas‐fermenting acetogens, such as Clostridium autoethanogenum, have emerged as promising biocatalysts capable of converting CO and CO2‐containing gases into fuels and chemicals relevant for a circular economy. However, the functionalities of the majority of genes in acetogens remain uncharacterised, hindering the development of acetogen cell factories through targeted genetic engineering. We previously identified gene targets through adaptive laboratory evolution (ALE) that potentially realise enhanced autotrophic phenotypes in C. autoethanogenum. In this study, we deleted one of the targets—CLAU_0471 (proposed amino acid permease)—with high mutation occurrence in ALE isolates and extensively characterised the autotrophic growth of strain RE3 in batch bottle and bioreactor continuous cultures. In addition, we characterised two previously reverse‐engineered strains RE1 (deletion of CLAU_3129; putative sporulation transcriptional activator Spo0A) and RE2 (SNP in CLAU_1957; proposed two‐component transcriptional regulator winged helix family). Strikingly, the strains recovered the superior phenotypes of ALE isolates, including faster autotrophic growth, no need for yeast extract, and robustness in bioreactor operation (e.g., low sensitivity to gas ramping, high biomass, and dilution rates). Notably, RE3 exhibited elevated 2,3‐butanediol production, while RE1 performed similarly to the best‐performing previously characterised ALE isolate LAbrini. The three reverse‐engineered strains showed similarities in proteome expression, and bioinformatic analyses suggest that the targeted genes may be involved in overlapping regulatory networks. Our work provides insights into genotype–phenotype relationships for a better understanding of the metabolism of an industrially relevant acetogen.

Polymicrobial biofilms are a conglomeration of diverse microbial consortia encased in a self-produced exopolysaccharide layer that forms on any biotic or abiotic surface. They are more resilient and persistent due to their enhanced drug resistance compared to monospecies biofilms, making it more difficult to eliminate using standard antimicrobial therapies. The present review discusses various inter- and intra-kingdom interactions taking place in polymicrobial biofilms and accounts for the various underlying drug resistance mechanisms in this complex and heterogeneous niche. In addition, this review provides insights into developing new diagnostic approaches by exploiting metabolites and byproducts produced by drug-resistant pathogens and other microorganisms in polymicrobial biofilms. As drug resistance is an ever-evolving mechanism in polymicrobial biofilms, synergistic combinations of natural products and antibiotics alone are not a panacea for eradicating these drug-resistant polymicrobial biofilms. Therefore, this review summarises both chemical and physical measures undertaken to combat these drug-resistant biofilms and stresses the need to employ 'omics' approaches, gene editing technologies and the integration of artificial intelligence/machine learning tools as future perspectives to eradicate these complex biofilms.

Many environmental pollutants have a fluorine or chlorine atom on a carbon atom adjacent to a carboxylic acid. These α-halocarboxylic acids include heavily regulated compounds such as per- and polyfluorinated substances (PFAS). Due to PFAS persistence in the environment, there is intense interest in characterising the biodegradation of α-halocarboxylic acids. Their initial biodegradation often proceeds via defluorinase enzymes that catalyse hydrolytic removal of alpha fluorine or chlorine atoms. These enzymes can dehalogenate both mono-halocarboxylate and dihalocarboxylate substrates, generating α-hydroxy and α-ketocarboxylic acid products, respectively. To enable continuous monitoring of defluorinase activity, we identified, purified and optimised dehydrogenases from Limosilactobacillus fermentum JN248 and Enterococcus faecium IAM10071 that reacted with the specific α-hydroxy and α-ketocarboxylic acid products of the defluorinases. The dehydrogenases make or consume NADH, measured by absorbance readings at 340 nm, thus allowing continuous measurement of defluorinase activity using a spectrophotometer. Using the coupled assay, purified defluorinases from a Delftia sp. and a Dechloromonas sp. were compared with respect to substrate specificity. The Delftia defluorinase demonstrated superior activity with most substrates, including difluoroacetate. To our knowledge, this is the first report of a coupled-enzyme continuous assay method for enzymes that catalyse the hydrolysis of α-halocarboxylic acids.