Advances in molecular biology tools are essential for streamlining and accelerating genetic engineering of cells across industrial and academic applications. While CRISPR-Cas improves genome editing efficiency, current systems have limitations and are often host specific, which restricts their versatility. This study describes a versatile CRISPR-Cas9 system for genome editing in industrially relevant Bacillus species. By adapting the well-established pJOE8999 vector-based CRISPR-Cas9 genome editing system, we constructed an inducer-independent, broad-host-range genome editing system. It maintains the benefits of low toxicity to the target cell and the cloning host as well as the ease to use of a single-plasmid CRISPR-Cas9 system. We utilized the constitutive Sigma70-type promoter from the conserved veg gene of Bacillus, to develop and test the suitability of promoter variants of different strengths for Cas9 expression. Successful gene deletions in three different Bacillus species demonstrated the versatility of the modified system for this industrially important genus. This was further confirmed by the integration of a reporter gene fusion and the introduction of a single point mutation in the genome of Bacillus licheniformis. This one-step CRISPR-based transformation protocol developed in this study enables fast genome editing workflows with minimal hands-on time. KEY POINTS: • Editing and screening of promoter variants for balanced Cas9 expression in Bacillus. • Development of a versatile inducer-independent, single-plasmid CRISPR-Cas-based system. • Verification of the modified CRISPR-based system for genome editing in different Bacilli.

Heavy metals (HMs) are naturally occurring elements which can be essential, such as zinc, copper, and iron, or non-essential, including cadmium, mercury, and lead. While essential metals serve as cofactors in critical enzymatic processes, elevated concentrations of both essential and non-essential HMs pose severe toxicity risks, primarily through oxidative stress, disruption of metal homeostasis, and biomolecular damage. Microorganisms have evolved diverse mechanisms to cope with metal-induced stress, including metal sequestration, enzymatic transformation, efflux systems, and surface immobilization. Among these, metallothioneins (Mts) are small, cysteine-rich proteins capable of high-affinity metal binding, contributing to cellular detoxification. Although Mts have been extensively studied in eukaryotes, knowledge of bacterial Mts remains limited, with characterized examples largely confined to cyanobacteria and a few other bacterial species. In this study, we identified a novel hybrid protein, TrxA, from Runella aurantiaca, containing a thioredoxin (Trx) domain fused to a Mt domain. The presence of the Trx domain may confer improved stability and solubility, supporting potential recombinant applications. In fact, the recombinant protein, named TrxMt, was heterologously expressed in Escherichia coli, displaying both disulfide-reducing activity and heavy metal-binding capability. Notably, TrxMt expression enhanced bacterial tolerance to multiple HMs, demonstrating its functional relevance in vivo. These findings expand the understanding of bacterial Mt diversity and suggest that TrxMt is a promising candidate for the bioremediation of heavy metal-contaminated environments, combining metal detoxification with favorable biochemical properties for industrial and environmental applications. KEY POINTS: • Identification of TrxA, a novel hybrid thioredoxin-metallothionein in R. aurantiaca • Recombinant protein TrxMt shows reductase activity and binds HMs • Overexpression of TrxMt enhances tolerance to different HMs in E. coli.

The incidence of Candida albicans infections has risen markedly, particularly among immunocompromised individuals and cancer patients undergoing chemotherapy. Fluconazole is commonly used as a first-line treatment for C. albicans infections; however, increasing clinical resistance poses a significant therapeutic challenge. As a result, developing new strategies to enhance the management of C. albicans infections has become critical. This study aims to assess the prevalence of azole's resistance among clinical C. albicans isolates in Egypt and to evaluate disulfiram/fluconazole combination against fluconazole resistant isolates. A total of 64 C. albicans clinical isolates were tested for fluconazole susceptibility using disc diffusion method and broth microdilution method (alone and in combination with disulfiram). The combination of fluconazole and disulfiram was further assessed using checkerboard method. The expression levels of resistance determinants were analyzed using RT-PCR. Molecular docking was used for further analysis. Sixteen isolates (25%) were resistant to fluconazole. Combination with subinhibitory concentration of disulfiram reduced the minimum inhibitory concentration (MIC) of fluconazole in resistant isolates (2-128 fold). Checkerboard assay revealed that fluconazole/disulfiram combination was mostly synergistic. In fluconazole-resistant isolates, Cdr1 was overexpressed, whereas disulfiram (at subinhibitory concentration) has triggered its downregulation. Docking studies showed that disulfiram may compete with ATP for binding and may disrupt fluconazole's interaction with Cdr1. Additionally, disulfiram exhibited a potent anti-biofilm activity against the tested C. albicans isolates. Fluconazole/disulfiram combination appears to be a promising approach for overcoming fluconazole resistance in C. albicans. Further studies are needed to validate this finding and explore its clinical potential. KEY POINTS: • Fluconazole/disulfiram combination is a promising approach to overcome fluconazole resistance in C. albicans • Disulfiram triggered downregulation of Cdr1 expression in fluconazole-resistant C. albicans. • Disulfiram competes with ATP for Cdr1 binding, disrupting fluconazole-Cdr1 interaction.

Synthetic consortia is an emerging field in biotechnology, offering advantages such as division of labor between the individual members and reduced risk of contamination. Consortia combining phototrophic and heterotrophic bacteria are attractive, as phototrophs can utilize CO2 and light energy to sustain growth. Several studies have explored such systems where sucrose is most commonly used as the provided carbon source. An alternative is acetate, a smaller two-carbon molecule produced as a by-product by many microorganisms, including the model cyanobacterium Synechocystis PCC 6803 (thereafter Synechocystis). Although acetate production during phototrophic growth is typically low, previous studies have demonstrated that metabolic engineering-specifically the introduction of phosphoketolase (PK) and overexpression of phosphotransacetylase (Pta)-enabled the development of a high-producing strain (WT_PKPa_RBS_BsPta_Δacs) capable of secreting significant levels of acetate into the medium (Roussou and Lindblad 2026). In this study, Escherichia coli and Pseudomonas taiwanensis were engineered to produce 1-butanol, an industrially relevant bulk chemical, and cultivated using acetate as the sole carbon source. These strains were then individually co-cultivated with the previously engineered Synechocystis strain, WT_PKPa_RBS_BsPta_Δacs, forming two distinct consortia that were maintained for 42 days. Growth dynamics were successfully monitored, and acetate concentrations in the consortia were lower than in a corresponding axenic Synechocystis culture. At the same time, 1-butanol production was detected in the two co-cultures, demonstrating the feasibility of coupling photosynthetically derived acetate to heterotrophic production of a value-added bulk chemical. KEY POINTS: • Photosynthetic/heterotrophic synthetic consortia for 1-butanol production. • Engineered Synechocystis PCC 6803 cells produce acetate from CO2. • Modified Escherichia coli and Pseudomonas taiwanensis cells grow on acetate and produce 1-butanol.

The WalRK two-component system monitors cell wall biosynthesis, and its activity decreases when biosynthesis is inhibited. As WalRK is essential for many Gram-positive bacteria, including Staphylococcus aureus and Bacillus subtilis, directly inhibiting WalK would disrupt cell wall biosynthesis and result in cell death. This study presents the construction of new reporter strains with two WalRK-dependent promoters: PiseA and PssaA. These reporter strains enable indirect in vivo measurement of WalRK activity based on luminescence. We evaluated these reporters in combination with the established PliaI reporter system, which is regulated by LiaRS, in order to improve the identification of cell wall inhibitors during the screening of natural products. The WalRK-dependent PiseA reporter yielded specific responses to almost all known cell wall biosynthesis inhibitors, including β-lactams, tunicamycin, glycopeptides, daptomycin, and fosfomycin, with no discernible response to other antibiotic classes. Furthermore, combining the PliaI reporter system with the PiseA reporter enhanced the range of potential cell wall inhibitors that could be detected. Deleting the gene of the eukaryotic-like Ser/Thr kinase PrkC increased the PiseA signal obtained with certain antibiotics, as well as after a longer incubation period. Additionally, deletion of prkC lowered PliaI expression in the presence of lipid II-targeting antibiotics. Screening natural products with an unknown mode of action identified several compounds that elicited vancomycin-like patterns with the reporter systems, indicating potential cell wall inhibitory activity. Overall, combining WalRK and LiaRS reporters enables a rapid, complementary evaluation of cell wall stress responses, providing a powerful method for discovering new cell wall-targeting antibiotics. KEY POINTS: • The PiseA/PssaA reporters enable continuous in vivo observation of WalRK activity • Cell envelope stress responses are differentially sensed by WalRK and LiaRS • The PiseA reporter strain detects nearly all cell wall biosynthesis inhibitors.

The efficient recovery of plant polyphenolic bioactives is facing technological challenges of low concentrations and complex mixtures starting from plant tissue. In the search of alternatives, we developed a high-yield biotechnological production process for hydroxycinnamic acid-tyramine conjugates in Escherichia coli by converting soluble substrates into insoluble products. A recombinant plant-enzyme cascade was established by co-expression of Arabidopsis thaliana 4-coumarate:CoA ligase and Solanum tuberosum hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase. We overcame initial enzymatic limitations by developing a temperature adapted and sequentially phased bioprocess on a 1 L reactor scale. With 5 mM/h substrate supply rate we achieved 31.9 g/L (102 mM) of feruloyltyramine (FerT), 14.4 g/L (50.7 mM) coumaroyltyramine, and 14.6 g/L (46.5 mM) isoferuloyltyramine with a space time yield of 1.75 g *L[-1] *h[-1] FerT. The vast majority of the product was obtained as filterable precipitate, simplifying the downstream process compared to classical procedures. Washed filter cake from a FerT production after filtration via 16-40 µm glass filter, showed 99% mass content of FerT. Product identity was confirmed by NMR after recrystallization. The newly developed process is robust, cost-effective and scalable, paving the way for commercial applications of biotechnologically produced hydroxycinnamic acid-tyramines. KEY POINTS: • Plant derived enzymes expressed at low temperature enable high productivity • In situ precipitated product is readily filtered from fermentation broth • Carbon transfer rate mirrors toxicity of phenolics and guide process optimization.

Selenium nanoparticles (SeNPs) have attracted significant attention owing to their unique physicochemical properties and promising biomedical applications. In the present study, a newly isolated halotolerant marine Bacillus sonorensis 2MNHR strain was employed for the extracellular biosynthesis of SeNPs. Bacterial isolates capable of reducing selenium oxyanions were obtained from seawater samples collected at Alexandria Harbor, Mediterranean Sea, Egypt. The ability of ten morphologically distinct bacterial isolates to reduce sodium selenite and sodium selenate under aerobic conditions was evaluated. Among them, only one isolate consistently reduced sodium selenite, as evidenced by the development of a characteristic red coloration in both liquid and solid media within 72 h, whereas no reduction was observed with sodium selenate. Extracellular reduction was confirmed using a cell-free supernatant assay. UV-Vis and FTIR analyses of the filtrate confirmed the involvement of biomolecules in selenite biotransformation, while GC-MS analysis revealed diverse organic compounds potentially responsible for reduction and stabilization. The biosynthesized SeNPs were comprehensively characterized using UV-visible spectroscopy, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), zeta potential analysis, scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX), and transmission electron microscopy (TEM). These analyses confirmed the formation of spherical, amorphous nanoparticles with sizes ranging from 80 to 200 nm (average ~ 160 nm) and a characteristic UV-Vis peak at 226 nm. A zeta potential of -38 mV indicated high colloidal stability. The SeNPs exhibited strong antimicrobial activity, with inhibition zones ranging from 23 to 32 mm and MIC values from 19.5 to 156.25 µg/mL. MBC/MIC ratios (≤ 2) confirmed bactericidal activity against most tested bacteria, while a ratio of 4 indicated borderline activity against Pseudomonas aeruginosa and fungistatic behavior against Candida albicans. These findings highlight the potential of halotolerant marine bacteria as sustainable platforms for the production of stable and biologically active SeNPs. KEY POINTS: • Isolation of marine Bacillus sonorensis 2MNHR with high selenite-reducing potential • Efficient extracellular biosynthesis of stable selenium nanoparticles (SeNPs) • Integrated physicochemical characterization and antimicrobial evaluation of SeNPs.

The search for antibacterial agents remains a global priority, particularly against multidrug-resistant pathogens. In this study, microbial transformation of 1,8-dihydroxyanthraquinone (chrysazin) was performed to obtain derivatives with enhanced antibacterial activity. Biotransformation using Absidia corymbifera AUMC 7104 and Beauveria bassiana AUMC 5133 yielded two metabolites: 1,2,5,8-tetrahydroxyanthraquinone (1, quinalizarin) and 1,8-dihydroxy-3-methyl-anthraquinone (2, chrysophanol), respectively. Their structures were elucidated by HRESIMS, 1D/2D NMR analyses. To the best of our knowledge, this is the first report of quinalizarin production through microbial biotransformation. The antibacterial activities of both derivatives (1 and 2), along with their parent compound, were evaluated against multidrug-resistant Acinetobacter baumannii through in vitro assays. Compound 1 exhibited the least MIC values of 512-1024 µg/mL against A. baumannii isolates and was selected for further in vivo and in silico studies. In the murine infection model, the compound 1-treated group (group IV) exhibited a significant reduction (p < 0.05) in bacterial burden compared to the positive control group (group II). Immunohistochemical analysis of kidney, liver, and spleen tissues revealed modulation of pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α, indicating anti-inflammatory properties alongside antibacterial activity. Molecular docking suggests a potential multi-target mechanism for compound 1, with favorable interactions with five critical bacterial proteins: MetRS, MurB, LptG/F, DXR, and MacB efflux transporter. These findings highlight quinalizarin as a potential lead compound for further antibacterial development. KEY POINTS: • Fungal biotransformation of chrysazin yielded the antibacterial agent quinalizarin. • Quinalizarin revealed in vitro and in vivo actions against Acinetobacter baumannii. • Docking studies suggested favorable interactions with multiple bacterial targets.

Magnetosomes are membrane-bound iron-rich organelles synthesized by magnetotactic bacteria as sensors for navigation in the Earth's magnetic field. In the well-studied model Magnetospirillum gryphiswaldense, their multistep biosynthesis is tightly controlled by > 30 specific genes clustered within a genomic magnetosome island. The lack of versatile genetic tools for strong, tunable, and orthogonal transcriptional control has impeded the analysis, engineering, and transplantation of this complex biosynthetic pathway. In addition, recent studies revealed an intricate transcriptional architecture of magnetosome gene clusters, but its significance has still remained unclear. Here, by using a bioluminescence reporter assay, we identify several novel constitutive promoters and establish the cumate-inducible repressor/promoter system CymR/Pcym as a robust and tunable regulatory module for rapid, dose-dependent transcriptional control in M. gryphiswaldense. We demonstrate its successful application for genetic tuning of magnetosome biosynthesis and inducible overproduction by controlling expression of a single essential magnetosome gene in a duplicated hybrid magnetosome gene cluster background. Additionally, we show its versatility by creating a hybrid promoter within the essential long mamAB operon, which experimentally confirms the significance of the intragenic P(mamH) promoter for the generation of long polycistronic transcripts. In summary, our data underlines the importance of balanced magnetosome gene expression. We also provide a versatile framework for dynamic transcriptional control and genetic engineering strategies in M. gryphiswaldense and other bacteria.

Breast cancer is the most frequently diagnosed cancer in women and remains a leading cause of cancer-related mortality worldwide. Recently, bacteria-based delivery systems have emerged as promising strategies for targeted cancer therapy due to their biocompatibility, low systemic toxicity, and ability to overcome drug resistance. In this study, bacterial ghosts (BGs) derived from Escherichia coli DH5-α were successfully prepared using a modified sponge-like reduced protocol, achieving approximately 92% reduction in optical density and releasing 246.3 ± 5.46 µg/mL DNA and 2.5 ± 0.054 mg/mL proteins, confirming efficient cytoplasmic evacuation. Heat-killed E. coli (HKEc) was prepared via thermal inactivation. Ethanolic extract of fenugreek seeds (Trigonella foenum-graecum) was obtained with a yield of 14% and characterized by GC-MS, identifying 26 bioactive compounds, including linoleic acid ethyl ester (19.39%) and β-sitosterol (14.14%). LC-MS/MS analysis further confirmed the presence of key metabolites such as trigonelline, diosgenin, orientin, and vitexin. The extract was successfully loaded into BGs and HKEc with loading capacities of 14 µg/mg and 11.6 µg/mg, and entrapment efficiencies of 21% and 13.4%, respectively. In vitro release studies demonstrated sustained release behavior from BGs, with only 14% released after 4 h at pH 7.4 compared to 90% for free extract, indicating effective controlled delivery. Cytotoxicity analysis using the WST-1 assay revealed enhanced anticancer activity of FEE-loaded BGs, with IC50 values of 1.2 mg/mL in MDA-MB-231 and 0.87 mg/mL in ZR-75-1 cells, compared to 2.2 and 1.74 mg/mL for free extract, respectively. Mechanistically, treatment resulted in significant downregulation of STAT3, mTOR, and β-catenin, alongside upregulation of STAT5, and a twofold increase in caspase-3 activity, confirming apoptosis induction. Overall, these findings highlight E. coli-derived bacterial ghosts as an effective and low-cost delivery platform for plant-derived anticancer compounds, offering enhanced therapeutic efficacy and controlled release for breast cancer treatment. KEY POINTS: • Escherichia coli ghosts are safe, versatile carriers with high drug-loading capacity. • E. coli ghosts enhance natural extract delivery, improving anticancer efficacy and targeting. • Fenugreek extract-loaded E. coli ghosts modulate key cancer pathways.

Washing machines are known to contain significant amounts of microorganisms with negative implications for laundry and machine hygiene. In particular, the formation of machine and laundry odour and the potential role that microorganisms might play are still far from being understood. Here, we used Oxford Nanopore-based sequencing of full-length 16S rRNA gene amplicons and quantitative PCR to qualitatively and quantitatively investigate the bacterial community composition of different parts (detergent drawer, door seal, sump) of machines categorized as odorous (n = 8) and non-odorous (n = 8) by their users. DNA was extracted from swab samples taken in a standardized manner from each machine. In total, full-length 16S rRNA gene sequences (~ 1.5 kb) were obtained from the investigated machines and assigned to 1492 distinct species. The observed community compositions corroborated previous investigations using shorter gene sequences for identification. For instance, odour-related Moraxella osloensis showed the highest relative abundance in door seal samples. However, only a few statistically significant differences were found in overall community composition (alpha- and beta-diversity) between odorous and non-odorous machines. To investigate whether odorous machines might contain a higher microbial load, bacterial DNA was quantified by qPCR. Odorous machines showed higher median bacterial DNA concentrations across compartments, but odour category had no significant effect on bacterial abundance in a mixed-effects analysis. In summary, odorous and non-odorous machines did not differ greatly in bacterial community composition. Differences in bacterial activity or subjective odour perception may be responsible for perceived odour. Hence, future studies should include metatranscriptomic analyses and larger sample sizes.