Mixed-species biofilms formed by carbapenem-resistant Klebsiella pneumoniae and carbapenem-resistant Acinetobacter baumannii can complicate the clinical management of device-associated infections; however, phage-based interventions targeting these communities remain insufficiently characterized. We assessed the antibiofilm activity of a lytic HZJ31 + HZY2308 phage cocktail against KPZ2-AB48 mixed-species biofilms. Compared with the corresponding single-species cultures, cocultures showed greater crystal violet-stained biomass, higher metabolic activity as measured by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay, and microscopic surface coverage. Phage-cocktail exposure during biofilm development or after biofilm formation reduced biomass, XTT reduction, culturable bacterial counts, and scanning electron microscopy-derived surface coverage, with similar changes observed on central venous catheter surfaces. In Galleria mellonella infection models, cocktail treatment increased survival and was associated with less extensive histopathological lesions and lower Kirschner wire-associated bacterial burdens. RNA sequencing and quantitative reverse transcription polymerase chain reaction identified treatment-associated transcriptional changes involving metabolic pathways, autoinducer-2 transport, cell-envelope functions, efflux systems, and stress responses. These findings support further preclinical investigation of phage cocktails targeting multidrug-resistant mixed-species biofilms.
Controlling phytopathogenic bacteria is essential for safeguarding crops and minimizing economic losses in agriculture. Although chemical pesticides are effective, they have adverse effects on ecosystems, beneficial organisms such as pollinators, and natural pathogen predators, creating a need for sustainable alternatives. Bacteriophages ɸEF1 and ɸEF2 (infecting Erwinia amylovora, the fire blight pathogen), and ɸXF1 (targeting Xanthomonas campestris, the black rot pathogen) have shown promise as biocontrol agents in vitro and in vivo. However, to be effective in the field these phages must maintain infectivity during storage and environmental exposure. We evaluated their stability under varying pH, temperature, UV and sunlight conditions and assessed various photoprotective additives. The phages remained infectious for up to 3 months at 4°C and 20°C and pH 7-9, but were inactivated at pH 3 and 37°C. UV rapidly inactivated the phages, but their stability was significantly enhanced by adding photoprotectants like Amino 22% or CaCO3, particularly CaCO3 at 24%, which preserved infectivity for over 8 h. These photoprotectants also improved phage recovery from plant surfaces. We conclude that effective biocontrol formulations should contain ≥ 106 pfu/mL, maintain neutral pH, include 24% CaCO3, and allow room temperature storage. This study is among the first to demonstrate the long-term stability of phage-based formulations at room temperature, together with improved field persistence, supporting their potential use as biopesticides.
Methylotrophic microbes are attractive alternatives to traditional heterotrophic production platforms, yet their efficiency is constrained by carbon loss through pyruvate decarboxylation and the oxidative branch of the RuMP cycle. The phosphoketolase (PKT) pathway provides a carbon-conserving alternative by cleaving fructose-6-phosphate and/or xylulose-5-phosphate into acetyl-phosphate, which can subsequently be converted to acetyl-coA without pyruvate decarboxylation. The remaining carbon intermediates are recycled through central metabolism to regenerate RuMP cycle intermediates without direct CO2 release. Here, we engineered this strategy in Bacillus methanolicus, a thermophilic methylotroph with strong industrial potential. We first established a versatile expression toolkit comprising inducible and constitutive promoters, benchmarked using an sfGFP reporter. Leveraging this system, we heterologously expressed the phosphoketolase B (pktB) gene from Methylotuvimicrobium buryatense 5GB1C which increased methanol-to-biomass yields by 18%-24% relative to controls and reduced biogenic CO2 production by 9%-12%. Chromosomal integration of pktB preserved these gains, demonstrating stability without reliance on plasmid-based expression. Together, these results show that PKT-driven metabolic rewiring enhances substrate yields in B. methanolicus and provides a scalable strategy to improve methylotrophic bioprocesses. This work expands the metabolic engineering toolbox for methylotrophs and highlights carbon-conserving pathway design as a key lever for advancing single carbon (C1) biomanufacturing.
ABSTRACT The secretory (Sec) pathway is the major pathway of Bacillus subtilis for protein export from the cytoplasm to the extracellular milieu. An amino‐terminal signal peptide is required to initiate protein translocation across the cytoplasmic membrane via Sec. Generally, Sec pathway signal peptides of B. subtilis are around 20–30 amino acids long and tend to be relatively hydrophobic compared to signal peptides from other organisms. In view of the importance of signal peptides for high‐level secretory protein production, understanding how signal peptide structure and length influence the efficiency of protein secretion is crucial. Accordingly, the present study was aimed at investigating the effects of signal peptide size and, in particular the subdomain sizes of a signal peptide, in relation to protein secretion efficiency. To this end, a rational engineering approach was employed for the design of shortened signal peptides that can still drive effective protein secretion. This involved in silico interaction studies with the signal recognition particle, which is involved in protein targeting from the ribosome to the membrane. Our results show that the hydrophobic H‐domain of a B. subtilis signal peptide can be shortened from seventeen to seven hydrophobic amino acid residues without a detectable decrease in protein secretion.
ABSTRACT Limited treatment options exist for refractory non‐Clostridioides difficile infection (non‐CDI) of the gut in critically ill patients; whether washed microbiota transplantation (WMT) is feasible and safe in this heterogeneous population is unknown. We retrospectively analysed prospectively registered consecutive cases (NCT03895593) between September 2015 and August 2022, in which severe non‐CDI gut pathogens were identified, and rescue WMT was administered after standard treatment failure. Primary outcomes were the clinical outcome and adverse events graded by CTCAE v5.0. Secondary descriptive outcomes were the change in total abdominal symptom score (TASS) on day 7, microbiological clearance, and 12‐week survival. Ten patients underwent 29 WMT infusions. Fifty percentage of patients (5/10) obtained clinical cure after WMT. Twenty percentage of patients (2/10) achieved clinical improvement and 30% (3/10) showed no response 7 days after WMT. One transient WMT‐related fever occurred (1/29, 3.45%). No serious WMT‐attributed events were observed. TASS decreased significantly (p = 0.007). Microbiological clearance was documented in 3 of 4 patients with available post‐WMT stool bacteriological test results. This case series provides preliminary evidence supporting the feasibility and potential efficacy of WMT for refractory non‐CDI gut pathogens.
ABSTRACT Bacterial volatile organic compounds (BVOCs) are increasingly recognised as effective signalling molecules that stimulate plant growth and stress responses. Although BVOC production may impose metabolic costs on bacteria, the benefits gained by VOC‐emitting bacteria through BVOC‐mediated communication with plants remain unclear. In this study, we established Pb‐stressed conditions for Bacillus megaterium, Arabidopsis thaliana or both organisms to investigate their reciprocal interactions. Pb‐stressed bacteria emitted a distinct VOC profile and exhibited significant reprogramming of sugar and nitrogen‐containing carbon metabolism compared with unstressed bacteria. Plant growth inhibition caused by Pb was alleviated by VOCs emitted from unstressed bacteria; however, this alleviation was enhanced by 36% when the bacteria were exposed to Pb stress, indicating that Pb‐induced changes in bacterial VOC metabolism trigger distinct plant responses. Specifically, Pb accumulation increased by up to 95% in plant shoot, whereas oxidative damage markers, including malondialdehyde and H2O2, decreased by up to 25% in plants exposed to VOCs from Pb‐stressed bacteria compared with those exposed to VOCs from Pb‐unstressed bacteria. These effects were associated with increased expression of antioxidant enzyme‐related genes in plants exposed to VOCs from Pb‐stressed bacteria. Finally, we confirmed that the Pb‐stressed bacteria‐mediated increases in plant growth and Pb uptake were accompanied by alleviation of the Pb stress‐induced reduction in bacterial population. Overall, our results demonstrate that VOC‐emitting bacteria can benefit from modulating plant physiology and suggest that a cooperative relationship between bacteria and plants can be established through BVOC‐mediated communication.
ABSTRACT The emerging zoonotic Getah virus (GETV) poses an increasing threat to both animal and human health, underscoring the need for rapid, sensitive and field‐deployable diagnostic tools. In this study, we developed and optimized a rapid, one‐step, visual detection (ROSVD) platform for GETV by integrating reverse transcription recombinase‐aided amplification (RT‐RAA) with CRISPR‐EsCas13d‐mediated collateral RNA cleavage. Notably, the ROSVD assay uses a simplified sample‐preparation strategy based on rapid nucleic acid release, eliminating the need for conventional nucleic acid extraction and purification. The entire workflow, including amplification and detection, is completed within 30 min at 37°C or ambient temperature (25°C) without specialized instrumentation. Detection results can be visualized directly under ultraviolet light or with a lateral flow assay. At 37°C, the ROSVD assay achieved sensitivity comparable to RT‐qPCR, and evaluation of clinical specimens showed 100% concordance with RT‐qPCR results. Collectively, these findings demonstrate that ROSVD is a rapid, sensitive, cost‐effective and instrument‐independent diagnostic platform, providing a practical solution for on‐site surveillance of GETV and a versatile framework for the detection of other emerging RNA pathogens.
ABSTRACT Co‐culturing fungi offers a promising strategy for generating hybrid fungal biomass with structural and functional properties for developing fungal‐based alternative leather. In this study, two filamentous fungi, Aspergillus oryzae and Rhizopus delemar, were co‐cultured to valorise food waste through production of fungal materials with enhanced mechanical properties owing to chitin‐rich biomass of A. oryzae and chitin‐chitosan‐rich biomass of R. delemar. After confirming symbiotic growth on solid media and in submerged semi‐synthetic media, the system was applied to a complex medium prepared from bread and lemon waste. Cultivation was scaled up to a 4.5 L bubble‐column bioreactor. The harvested biomass was tanned with chestnut tannin and processed into materials using wet‐laid method. Symbiotic growth was verified by polymerase chain reaction (PCR) amplification and visually via optical and scanning electron microscopy (SEM). Unlike the pelletised morphology typical of A. oryzae, the co‐culture produced dispersed mycelium favouring material formation. Co‐cultures yielded higher ethanol concentrations (12–14 g/L), with biomass yields exceeding those of R. delemar (~0.2 g/g) and comparable to A. oryzae (~0.3 g/g) monocultures. The highest tensile strength and elongation achieved were 11.7 MPa and 8% respectively. Overall, this work establishes fungal‐fungal co‐culture as a transformative approach for producing hybrid biomass for fungal‐based leather alternatives.
ABSTRACT Emerging research has revealed a diverse microbiota in the high myopia (HM) conjunctival sac, highlighting the therapeutic potential of modulating this community to influence disease outcomes. However, current research on targeted modulation of this microbiota for myopia intervention lacks in‐depth investigation and definitive conclusions regarding its efficacy and underlying mechanisms. In this study, high‐throughput sequencing and culturomics were first used to screen the conjunctival sac microbiota of healthy people, identifying Lactobacillus mucosae MM‐1 as a potential probiotic for myopia control. Then, animal studies further confirmed that L. mucosae MM‐1 significantly attenuated lens‐induced myopia, improved COL1A1 expression and scleral integrity and elevated Lactobacillus abundance. Furthermore, untargeted metabolomics analysis suggested that indole‐3‐lactic acid (ILA) may be a key substance for L. mucosae MM‐1 to exert its effects for myopia control. Mechanistically, pharmacological ILA supplementation supports the involvement of AHR in TGF‐β/Smad pathway inhibition, accompanied by altered COL1A1 and α‐SMA expression and attenuation of lens‐induced myopia in mice. Therefore, these findings suggest that L. mucosae MM‐1 may attenuate lens‐induced myopia in mice, at least in part, through ILA‐mediated regulation of the TGF‐β/Smad pathway, and offer a theoretical foundation for future therapeutic strategies based on conjunctival sac microbiome manipulation.
ABSTRACT Fungi can oxidise pyrite, a process which has potential implications for metal bioleaching and acid mine drainage formation. However, this capacity has been demonstrated primarily using only a few model fungal strains. Diverse fungal communities are known to inhabit pyrite‐rich mine tailings but how these indigenous fungi may mediate pyrite oxidation and by what mechanisms remain largely unexplored. Here, we isolated 51 pyrite‐oxidizing fungal strains spanning 28 genera from three mine tailings areas in South China. From this native library, we selected three potent pyrite oxidisers, Pithomyces cynodontis DC37, Penicillium janthinellum ZJS52, and Aspergillus niger DBS124, for in‐depth mechanistic characterisation. These indigenous strains solubilised 1.2%–1.8% of the total iron from pyrite in fungus‐pyrite co‐culture systems, exceeding values reported for model fungi under comparable conditions. They also acidified the medium to a pH as low as 2.15, providing dual lines of evidence for their robust oxidative capacity. Further analyses revealed distinct surface erosion features and changes in iron (Fe) and sulfur (S) speciation, confirming fungal‐mediated pyrite oxidation. All three strains secreted organic acids (e.g., oxalic and citric acids) and showed peroxidase activity, but they exhibited divergent patterns. By combining hybrid genome sequencing (Illumina and Nanopore) with time‐course transcriptomics, we uncovered the genetic basis for these divergent patterns. Genes coding for lignin and manganese peroxidases in P. cynodontis DC37 were co‐induced by reactive oxygen species and metal ions released during pyrite oxidation. Furthermore, A. niger DBS124 employed an oxalic‐acid‐based mechanism upon pyrite exposure, driven by coordinated upregulation of central carbon metabolism and oxalate biosynthesis. Collectively, our findings highlight the underexplored biotechnological potential of mine‐tailings fungi and establish a molecular foundation for understanding their roles in Fe‐ and S‐cycling within mining environments and for developing fungal‐mediated bioleaching technologies.
ABSTRACT Deep learning models based on both distant protein sequence homology and genetic neighbourhood context searches predicted that 1.5% of bacterial genes or 30 genes per E. coli genome represent anti‐phage systems (APS); many are colocalized on defence islands or are found on mobile DNA elements. Thousands of APS thus remain to be defined molecularly. Selected recent examples highlighting an astonishing molecular diversity of these defence systems are described in this editorial. The mechanisms include allosterically regulated dGTPase responding to competing nucleotide signals (Clover); bacterial defence systems activated by phage anti‐defence manoeuvres (Panoptes); a prophage encoded tRNA nuclease activated by a phage tail tip protein (HepS); a helicase‐nuclease complex that scans for ssDNA 3′ overhangs created by phage DNA transaction (Hachiman); and systems that cleave free ends of linear DNA (Shedu). Systems were described that synthesize template‐free poly‐dA chains which are degraded by a phage exonuclease thereby activating an ion channel (Hailong). Several systems interfere with phage DNA injection into the cell, for example, a cell membrane associated protein complex inhibiting injection (KIWA); or destroying phage DNA at injection (SNIPE); or a complex membrane motor system that senses phage DNA injection and activates nuclease effectors (Zorya). Other systems consist of a single pore building protein that combines sensor and effector functions (Rip1) or degrade NAD+ (Cat1). Defence‐associated reverse transcriptase (DRT) preceded by non‐coding RNA (ncRNA) come in different forms: in DRT2 a rolling circle reverse transcription leads to an endless protein inducing cell dormancy; in DRT3 a mixed templated and untemplated repeat DNA synthesis becomes cytotoxic in presence of a phage protein; DRT9 synthesizes a polyA strand that might sequester a phage protein needed for DNA replication. Striking are bacterial APS that resemble innate immune reaction directed against viruses in animals (gasdermin, Argonaute, RAZR, Schlafen, Thoeris 2, ubiquitin‐like proteins) pointing to an ancient origin.
ABSTRACT Mixed‐linkage β‐glucans (MLGs) are emerging as promising biopolymers with significant biotechnological potential due to their unique structural and rheological properties. In rhizobia, MLG biosynthesis is controlled by the second messenger cyclic di‐GMP (c‐di‐GMP) and mediated by the bicistronic operon bgsBA. However, the full composition of the biosynthetic machinery and strategies for enhanced production remain incompletely understood. In this study, we demonstrate that the outer membrane protein TolC is essential for MLG production in Sinorhizobium meliloti. Genetic disruption of tolC abolished MLG synthesis, while its complementation restored production. We propose that TolC functions as part of a tripartite complex with BgsA and BgsB, enabling efficient polymer synthesis and/or export. Furthermore, co‐overexpression of tolC, bgsBA, and a constitutively active diguanylate cyclase (pleD*) yielded an 8‐fold increase of MLG over a control plasmid without tolC, reaching up to ~10 g/L under bioreactor conditions. Additionally, this genetic module enabled de novo MLG production in otherwise non‐producer rhizobial hosts (e.g., Mesorhizobium japonicum), allowing bacterial chassis exchanges and highlighting its portability and potential for synthetic biology applications. Overall, our findings identify TolC as a key component of the MLG biosynthetic machinery and provide a robust platform for the scalable production of this valuable biopolymer.
ABSTRACT The active foraging behaviour of honey bees frequently exposes them to various xenobiotics. Honey bees rely primarily on endogenous enzymatic detoxification systems to metabolise these compounds; however, this capacity is constrained by limitations in their genomic detoxification repertoire. The gut microbiota may partially compensate for this deficiency through two complementary mechanisms: directly transforming or sequestering xenobiotics, and modulating host detoxification pathways. We therefore propose that the gut microbiota should be regarded as an extended detoxification organ in honey bees. This perspective also points to a microbial biotechnology agenda for pollinator protection, including precision probiotics, microbiome‐informed breeding and engineered symbionts. Viewing detoxification as a holobiont trait provides a more comprehensive framework for understanding bee resilience and for developing microbiome‐based interventions under real‐world chemical stress.
ABSTRACT N‐Linked glycosylation can have a significant impact on the yield of heterologously expressed proteins secreted by Saccharomyces cerevisiae. The yeast is a widely used host for the expression of unspecific peroxygenases (UPOs), a subclass of peroxide‐dependent oxidoreductases with high potential for industrial biocatalytic applications. However, the effects of N‐glycosylation on the expression of recombinant UPOs have not yet been investigated. Here, we studied respective protein modification on the expression of a UPO from Marasmius rotula, belonging to the protein subfamily of short peroxygenases, in S. cerevisiae. Two of the three N‐glycosylation sites that are actually occupied in rMroUPO were eliminated by substituting asparagine at positions N43 and N151 with serine. The single substitutions led to reduced amounts of secreted rMroUPO, with N43S having the highest impact (almost four times lower protein amount compared to the native enzyme) and N151S having a moderate effect. In the next step, the glycosylation probability at position 43 was enhanced by replacing the serine in the corresponding sequon with threonine, which increased the expression of the rMroUPO variant. The concentration of active UPO in the concentrated culture supernatant of the glycosylation‐optimised variant S45T was twice as high as that of the native rMroUPO. The results suggest that N‐linked glycosylation is an important factor for successful heterologous UPO expression in S. cerevisiae.
ABSTRACT Retirement need not mark the end of a scientific career. This opinion article describes how the MicroMundo citizen science programme transformed a secondary‐school laboratory into a place where students search for new antibiotics while experiencing authentic scientific research. The experience highlights the educational value of citizen science, promotes microbial literacy and STEM vocations, and illustrates how retired scientists can continue to contribute meaningfully by connecting universities, schools and society.
ABSTRACT Antifungal proteins from fungal origin (AFPs) are small, secreted, cationic, cysteine‐rich proteins with potential as novel biofungicides. However, their application requires safe and cost‐effective production platforms. PeAfpA is a highly active AFP naturally produced in large quantities by the phytopathogenic fungus Penicillium expansum and biotechnologically produced by the Generally Recognized As Safe fungus Penicillium chrysogenum although at lower yields . Here, the production of PeAfpA using the P. chrysogenum strain PCMG11552 was optimised in 0.5 L and scaled up to 5 L in stirred‐tank bioreactors. A competitive enzyme‐linked immunosorbent assay (cELISA) was developed to evaluate the influence of process parameters on PeAfpA production, identifying medium pH and agitation rate as key factors, while dissolved oxygen proved critical during scale‐up. The PeAfpA yields obtained were tenfold higher than those achieved previously in flask cultures, demonstrating the feasibility of scaling AFP production from laboratory to pilot scale for diverse applications. Finally, the P. chrysogenum marker‐free PeAfpA‐producing strain obtained via recyclable CRISPR/Cas9 system guarantees regulatory compliance and biosafety, making it suitable for its use in any commercial environment.
ABSTRACT The construction and performance of plants capable of detoxifying the organochlorine pesticide (lindane, γ‐hexachlorocyclohexane or γ‐HCH) is reported. To this end, the bacterial linA gene from Sphingobium japonicum strain UT26, which encodes a dehydrochlorinase initiating γ‐HCH degradation, was engineered into Arabidopsis thaliana. The resulting lines expressing linA exhibited markedly enhanced tolerance to lindane compared to wild‐type controls, both in synthetic media and in contaminated soils. Furthermore, thereby engineered plants also removed more than 90% of the pollutant from the medium within 4 weeks. Chemical analyses revealed not only the formation of 1,2,4‐trichlorobenzene (TCB), the expected γ‐HCH degradation intermediates, but also detected 1,4‐dichlorobenzene (DCB), suggesting that native plant activities could further push the degradation process beyond the canonical microbial pathway. This points to a potentially synergistic interaction between the introduced bacterial enzyme and the endogenous plant detoxification systems. By combining bacterial catabolic activity with plant resilience and root‐mediated soil interactions, our data advocate a new strategy for the remediation of γ‐HCH contaminated environments and demonstrate the feasibility of designing plants for enhanced degradation of persistent organic pollutants.
ABSTRACT Porins serve as the primary transport channels for substrate molecules across the outer membrane of Gram‐negative bacteria. Despite their potential to influence substrate uptake in microbial cell factories, porins are often overlooked in metabolic engineering approaches. In this study, we investigate the impact of modulation of sugar porin expression using laboratory and industrial workhorse Pseudomonas putida. We first examined the P. putida porin repertoire through bioinformatic analysis. Among the two selected porin sets, only the one comprising OprB‐I, OprB‐II and OprB‐III was found to be relevant for glucose catabolism in two biotechnologically important P. putida strains. Functional studies involving gene knockouts, complementation and overexpression revealed that the substrate specificity of P. putida OprB porins extends beyond glucose and includes the non‐native substrate xylose. Overexpression of oprB‐I alone was sufficient to restore sugar utilization in strains with all three oprB genes knocked out. Notably, when Gcd was active in P. putida, oprB‐I overexpression accelerated the utilization of glucose and xylose in mixed sugar conditions through altered sugar uptake and oxidation dynamics. This work exposes the relevance of porins in shaping the uptake of major lignocellulosic sugars and highlights the importance of incorporating outer membrane transport considerations into metabolic engineering strategies for Gram‐negative bacteria.
Komagataella pastoris is extensively used as a microbial cell factory for the production of recombinant proteins and high‐value compounds. However, tightly controlled promoter systems responsive to safe and economical inducers are required for precise metabolic and pathway engineering in this yeast species. Cumate‐inducible promoters are an ideal choice due to the safety and low cost of cumate. In this study, we systematically optimised the insertion sites of the CuO operator sequence within the strong promoter P GCW14 to isolate a high‐activity variant that we designated as P GCWCuO03 . To fine‐tune the expression of the repressor protein CymR, we developed a truncated promoter of P GAP , designated as P GAP200 . Based on the optimal promoter P GCWCuO03 and the CymR expression unit, we constructed a robust CymR/CuO‐mediated cumate‐inducible promoter, designated as P gc , in K. pastoris . P gc demonstrated outstanding induction properties, resulting in an approximately 11‐fold increase in target protein production following induction. Promoter substitution assays validated the effectiveness of P gc in temporal gene expression control, highlighting the significant potential of this promoter for both basic research and industrial bioprocessing applications in synthetic biology and biotechnology in K. pastoris .
