Applied Microbiology and Biotechnology

In many tropical countries, fermentation of palm sap into palm wine is an important fermented beverage contributing to local economies, tradition, and culture. Traditionally made in villages and families, palm sap is not inoculated with starter cultures and fermentation commences spontaneously. It is therefore possible that fermentation is influenced by multiple ecological factors, which affect microbial dynamics and thus flavour outcomes. Here, we studied microbial communities during fermentation of palm sap from three different palm tree species (palmyra, coconut, and sugar palm) on the island of Bali, Indonesia in both the wet and dry seasons. Our results suggest that season of collection has a strong influence on microbial dynamics and succession, and these changes positively correlate to metabolite concentration. The change of the season from the dry to wet season led to the loss of microbial diversity with lower richness in the dry season. The dominance of Saccharomyces cerevisiae was not affected by season and fermentation time and was dominant in all samples. Potential spoilage species, such as Candida tropicalis were negatively correlated to ester production and more abundant in the dry season. As microbial species varied in incidence and thus biochemical activity, the chemical groups of esters from their metabolism related to the change of season and fermentation time, while volatile compounds and small molecules were highly discriminated by season in the resultant wines. Ethyl octanoate was consistently different across all variables through comparison by three-way ANOVA and is proposed as a biomarker of seasonal variation in palm sap fermentation. These findings improve our understanding of microbial dynamics in palm sap fermentation, revealing flavour differentiation within season and suggests that strategies for microbial management, product development and quality assurance will elevate this traditional product into the future.

The purification of industrial enzymes typically relies on costly, multi-step chromatographic protocols. To address this, we developed a novel platform termed Coated Bacterial Enzymes (CBEs), which enables one-step purification and immobilization of recombinant proteins fused to the SlpA cell wall binding domain. As a proof of concept, we used a {beta}-galactosidase from Bifidobacterium bifidum of dairy relevance. The chimeric enzyme BbgII-SlpA was expressed in Escherichia coli and captured from crude lysate onto glutaraldehyde-inactivated Bacillus subtilis cells via SlpA domain. Binding was characterized by a dissociation constant (Kd) of 16.2 {micro}M and maximum binding capacity (Bmax) of 144 {micro}mol/g. The resulting CBE biocatalyst exhibited optimal activity at pH 6.0 for ONPG and lactose, with a broader pH profile than the free enzyme. Optimal temperatures were 60 {degrees}C for ONPG and 50 {degrees}C for lactose, and CBE retained >80% activity after 390 min at 45 {degrees}C, compared to 20% for the free enzyme. Catalytic efficiencies (kcat/Km) were 2.62 x106 M-1{middle dot}s-1 for ONPG and 4.40 x102 M-1{middle dot}s-1 for lactose. Moreover, CBE showed improved tolerance to cations such as Ca2+ and Fe2+. These results suggest that the CBE platform offers a cost-effective alternative for producing high-purity, immobilized enzymes for diverse industrial bioprocesses.

Nutrient-rich food-processing wastewaters represent valuable yet under-utilised side streams for sustainable protein production in the form of microbial biomass. Here we present an integrated dual-loop bioprocess that converts soybean-processing wastewater into microbial single-cell protein (SCP) while achieving substantial nutrient removal and product refinement. In the first loop, previously enriched microbial consortia were inoculated and cultivated in four parallel sequencing batch reactors (SBRs) for 44days at a hydraulic retention time (HRT) of 3days. This bioprocess configuration demonstrated features that support future scale-up while maintaining process stability, achieving a protein content of 33.3{+/-}3.2%, doubling the protein yield (15.32{+/-}3.49g dry weight per g soluble TKN) and quadrupling the production rate (0.29{+/-}0.06g dry weight L-1 d-1) compared to operating reactors without inoculation (HRT: 7.2days). Effluent treatment was stable, with 84% carbon and 78% nitrogen removal efficiencies, demonstrating efficient nutrient recovery. The SCP biomass was enriched in functional taxa, including Acidipropionibacterium, Lactococcus, Megasphaera, and Azospirillum, suggesting that reactor conditions and inoculum selection promoted a stable, protein-productive microbial community with potential probiotic benefits. In the second loop, bioreactor effluent was reused as aqueous matrix for heat treatment (60{degrees}C) of the SCP biomass, reducing the RNA content from 8.6% to 2.6%, with a 39% biomass loss accompanied by a 30% increase in total amino acid concentration. Hence, our valorisation approach integrates microbial biomass production, effluent reuse, and product refinement within a circular framework. The system provides a resource-efficient pathway for converting food-sector side streams into high-quality microbial community-based SCP, highlighting its potential scalability for sustainable nutrient and water management.

Municipal wastewater constitutes a major reservoir of unutilized reactive nitrogen, representing a significant opportunity for biological valorization. The biopolymer cyanophycin is promising as a means of nitrogen capture and recovery, but current production strategies are not optimized for the physicochemical constraints of municipal wastewater systems. Here, we engineered the naturally competent soil bacterium Acinetobacter baylyi ADP1 ISx to synthesize cyanophycin from carbon and nitrogen sources prevalent in municipal wastewater and over a range of wastewater-relevant temperatures. To overcome the recurring problem of arginine availability limiting cyanophycin synthesis, we engineered an arginine-producing strain (AP1) which accumulated cyanophycin when grown on acetate and ammonium (19% CDW), nitrate (9% CDW), or urea (29% CDW) and without arginine supplementation. During this work, we observed that conditions associated with reduced cell fitness correlated with increased intracellular cyanophycin content. As temperature strongly influences cell growth but cannot be realistically modulated in wastewater contexts, we investigated the potential of induced fructose-auxotrophy to modulate cell growth independently from temperature. This intervention, accomplished with a single knockout (gap), expanded the effective range of cyanophycin accumulation from 12 C up to 30 C. Collectively, these results establish the relevance of arginine-producing strains for cyanophycin biosynthesis and position A. baylyi as a promising chassis for continued development under real-world wastewater conditions.

Caldimonas thermodepolymerans is a thermophilic polyhydroxyalkanoate (PHA)-producing bacterium with strong potential for sustainable bioplastic production. Besides serving as intracellular carbon and energy storage compounds, PHAs are increasingly associated with bacterial stress resistance and cellular robustness. This study aimed to investigate the physiological and transcriptomic response of C. thermodepolymerans to osmotic stress induced by elevated NaCl concentrations. Growth analysis demonstrated tolerance up to a supplementation of 2% NaCl, while moderate salt concentrations enhanced PHA accumulation, reaching 65% cell dry weight at 1.5% NaCl supplementation. To better understand the bacterial response to osmotic stress, RNA sequencing was performed under sublethal salt stress conditions. Differential expression analysis revealed major changes in genes related to osmoprotection, trehalose metabolism and type VI secretion systems, whereas motility and chemotaxis genes were strongly repressed. Phenotypic assays confirmed increased biofilm formation and reduced swarming motility under salt-induced osmotic stress. Although canonical PHA biosynthesis genes were not significantly differentially expressed, increased polymer accumulation suggests other underlying mechanisms linked to osmoadaptation. Together, these findings demonstrate that osmotic stress induces metabolic, physiological and regulatory responses in C. thermodepolymerans, highlighting the importance of PHA in stress adaptation besides its industrial applicability.

Rare earth elements (REEs) are critical components of green technologies, but current mining and purification methods remain environmentally unsustainable due to their high energy consumption and intensive chemical requirements. Bio-hydrometallurgical processes have the potential to concentrate and recover REEs at a circumneutral pH. Work presented here uses bacteria at neutral pH to concentrate REEs from solution and subsequently recover those REEs using sodium citrate. Shewanella oneidensis MR-1 was incubated anaerobically in a culture media solution spiked with 14 REEs and yttrium for one to six days. REE concentrations remaining in solution were then compared to REE concentrations on cell pellets. For these same timepoints, the loosely bound extracellular polymeric substance (LB-EPS) was removed from cells prior to quantifying REEs on pellets to narrow down the location of REE binding. Moreover, cell pellets collected after 5 days in REE spiked solution were subjected to a time series desorption assay using sodium citrate. Shewanella oneidensis at a starting OD600 of 0.6 adsorbed 1.18mg/g of REE after 3 days. 80% of these REEs were located in the LB-EPS. In 10 minutes, 0.5 M sodium citrate desorbed about 75% of REEs from cells and over 95% after 24 hours. This method was also applied to Alaskan coal and showed that 68-86% of REEs were desorbed form S. oneidensis. This study elucidates the REE binding location and capacity of S. oneidensi, REE removal efficiency of sodium citrate overtime, and the application of this sustainable biotechnology for REE recovery at a circumneutral pH from Alaskan coal.

Simultaneous removal of organic waste and industrial gypsum was assessed in continuous flow-through bioreactors that treat sulfate-rich sewage sludge. Metabolic fluxes, the composition of microbial communities, and profiles of organic matter in the presence of different organic loads were tracked over [~]190 days. The addition of a pre-enriched microbial community enhanced the rates of sulfate reduction during the establishment of the sludge blanket, but microbial diversity in established reactors depended primarily on organic loading. Organic removal rates were comparable to those in standard anaerobic digesters, but methane production accounted for [~]1% of electron flow compared to >70% in traditional systems. Stoichiometric analyses revealed that molar COD: sulfate ratios below [~]1 favored complete oxidation of acetate by sulfate-reducing bacteria (SRB) and those above [~]2.1 permitted either complete or incomplete oxidation, allowing sulfate reduction and methanogenesis to co-occur. Sequencing of the 16S rRNA confirmed these trends by revealing that the faster-growing SRB that do not oxidize acetate were more abundant at higher organic loads and during the establishment of the sludge blanket, whereas complete oxidizers became more abundant when the molar COD: sulfate ratio was [≤]3.2. In reactors that had been seeded with the pre-enriched communities, acetate-oxidizing SRB became prevalent over the incomplete oxidizers 25-50 days earlier. These results enable targeted design and control of microbial processes and bioreactors that remove waste organics and gypsum while producing less methane due to the competition for acetate between methanogenic archaea and SRB that oxidize acetate.

Culture conditions were optimized for the production of melanin nanoparticle by the bacterial strain Pseudomonas stutzeri BTCZ 305. Response surface methodology was employed for determining the most significant fermentation conditions using variables including, pH, temperature and L-tyrosine concentration identified through one-factor-at-a time approach. Box-behnken design consisting of 17 different combinations of all these factors were performed. Using this methodology, a quadratic regression model was built and the optimal combinations of media constituents for maximum melanin production 1192.27 microg/mL were determined as temperature (32.5 degreeC), pH (8.5) and L-tyrosine concentration (7 g/L). Melanin production was obtained experimentally coincident with the predicted value and the model was proven to be adequate. The nanostructural distribution, its stability in colloidal suspension and particle size were also characterized with the help of TEM, particle size analysis and Zeta potential. The potent applicability of this molecule in anti-inflammation and wound healing was also elucidated.

Past few decades witnessed a boom in pharmaceutical and bioproduct industry with the help of bioprocess technology. Industrially important bioproducts can be produced in large scale for commercialization with the help of fermenters. Here in, pharmaceutically valuable bioproduct melanin, synthesized from Pseudomonas stutzeri strain BTC109 by using two different sized bioreactors. Under controlled conditions the bacteria were allowed to synthesis melanin nanoparticles. The important parameters to be monitored here are pH, dissolved oxygen, agitation, aeration, melanin production and cell biomass concentration. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=163 SRC="FIGDIR/small/737634v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@d92fe5org.highwire.dtl.DTLVardef@d76c07org.highwire.dtl.DTLVardef@f5516forg.highwire.dtl.DTLVardef@1b5864b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIPharmaceutical and bioproduct development industries witnessed a shoot up due to bioprocess technology. C_LIO_LIIndustrially important bioproduct like melanin can be produced in large scale with the help of industrial fermentation technology. C_LIO_LIThe product thus obtained was found to be nano sized and it can be commercialized. C_LI

Background: Biochemical, biophysical and structural characterisation of isozymes from the ubiquitous family of iron- or manganese-dependent superoxide dismutases (SodFMs) requires the purification of high-quality preparations of recombinant enzymes. Determination of their key biochemical parameter, their catalytic metal-preference, requires the comparison of the catalytic turnover of samples loaded exclusively with iron versus samples loaded exclusively with manganese. Both of these aims are inhibited by the potential contamination of recombinant preparations of SodFMs, prepared by heterologous overexpression inside Escherichia coli cells, by even low levels of endogenous SodFMs from the host, both of which show very high turnover with either manganese (E. coli MnSOD) or iron (FeSOD). To overcome this problem, we created a strain of E. coli lacking the endogenous SodFMs. Here, we characterised this E. coli BL21 (DE3) {Delta}sodA{Delta}sodB strain, determining the physiological effects of SodFM deletion and demonstrating its utility for producing recombinant SodFMs for in vitro characterisation and use. Results: Genomic analysis verified the targeted gene deletions, without off-target effects. Growth, expression, elemental analysis, and proteomic data confirmed a lack of physiological defects of the strain except for a known inability to grow on glucose, which is overcome by heterologous SodFM expression. We demonstrate the utility of the strain for the efficient production of diverse recombinant SodFMs, including highly divergent, understudied isozymes, including the ability to precisely control the metal-loading of the heterologously expressed protein. Conclusions: The E. coli strain described herein is a useful microbial cell factory for production of recombinant SodFMs, which should find widespread utility as expression host of choice, enabling more efficient production of protein for studies of the biochemical, biophysical and structural properties of this remarkable family of metalloenzymes.

Melanins are biological macromolecule with immense functionality synthesised by a wide spectrum of living organism. It is mainly synthesised by the oxidative polymerization of indolic and phenolic compounds through several enzymatic process. It has wide spread application in agriculture, cosmetic and therapeutic industry due to its various properties including antioxidation ability, UV protection efficiency and anticancer activity. Because of this wide range of application in different sectors, large scale production and commercialization attains enormous consideration. The present study deals with the effect of 12 different process parameters on melanin production viz., production media, incubation time, inoculum concentration, pH, temperature, agitation, carbon source, phosphate and magnesium source, CuSO4.5H2O, sodium chloride and L-tyrosine on melanin production by Pseudomonas stutzeri strain BTCZ 109 obtained from Arabian sea sediments was evaluated. After optimizing the important process parameters, the bacteria showed about ~4.65 fold increase in melanin production compared to unoptimized cultural conditions. The melanin optimized through this method was found to be nano sized. The Nano sized DOPA melanin in treating Skin cancer cell line SK ML28 which showed a dose-dependent activity with an IC50 value of 164 g/mL. All these results highlight the therapeutic efficiency of DOPA melanin Nano particle as promising bioactive molecule.

BackgroundThe corn smut fungus Ustilago maydis is an important microbial model organism representing a genetically amenable and readily cultivable basidiomycete. Research in this fungus addresses a broad range of fundamental questions and its biotechnological exploitation is on the rise. Although genetic engineering in principle is well established, efficient methodology for synthetic biology approaches such as metabolic engineering or pathway transplantation has remained limited. ResultsHere, we present a comprehensive toolbox for U. maydis based on modular cloning and the characterization of more than 20 promoters. Careful comparative evaluation of insertion loci and terminator as well as reporter effects was conducted and a novel color-based strategy for straightforward genome integration was implemented. Moreover, the cloning and subsequent one-step integration of four transcriptional units into U. maydis was demonstrated by creating a "rainbow" strain producing four fluorescent proteins. ConclusionOverall, this next generation toolkit strongly advances genetic engineering and systems biology approaches in U. maydis, fostering its development into a valuable and competitive fungal chassis and prime model, particularly in applied research.

Background: Human activities such as mining generally lead to increased heavy metal concentrations in the environment. While traditional remediation techniques are often costly, the use of fungi as bioremediators, known as mycoremediation, is increasingly gaining attention as a sustainable approach for removal of heavy metals. Here, we evaluated heavy metal levels inside the Kiirunavaara iron ore mine in Northern Sweden and analysed fungal responses to various metal concentrations by comparing growth and metal uptake in mine-derived isolates and closely related control isolates. Results: Sediments inside the mine were enriched in heavy metals compared to those from the outlet of the mine to natural lakes. Six Fusarium isolates were recovered from contaminated mining environments: five isolates from inside the mine were identified as Fusarium oxysporum, and one isolate from the outlet was identified as Fusarium tricinctum. Isolates from the mine and outlet showed overall higher survival and biomass production in presence of copper, iron, and zinc across a range of concentrations (up to 1000 mg/L) compared to control isolates. At the same time, these isolates often exhibited reduced relative metal uptake. As a result, mycoremediation potential, assessed as total uptake in the grown mycelium, was isolate-dependent. Conclusions: Based on these results, we conclude that Fusarium isolates from the Kiirunavaara mine show increased growth in media enriched with heavy metals compared to closely related control isolates. We additionally show that mycoremediation potential is not necessarily associated with environmental origin. Instead, mycoremediation potential should be evaluated on a case-by-case basis for each isolate and based on specific needs for mycoremediation.

Gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae, poses a growing global public health threat due to the rapid emergence of multidrug-resistant strains and the limited availability of effective treatments. Since there are no known lytic gonophages, we explored prophages present in the genome of N. gonorrhoeae FA1090, with a particular focus on prophage-encoded endolysins. In this study, we evaluate antigonococcal properties of prophage-encoded endopeptidases with the NlpC/P60 enzymatic domain. Recombinant endolysin Phi1gp518 exhibits intrinsic bactericidal activity against non-permeabilized N. gonorrhoeae FA1090 cells. Furthermore, it shows an expanded host range against clinical gonococcal isolates. The gonolysin remains stable across all human body temperatures, a pH range of 5-10, and shows no cytotoxic effects toward human cervical epithelial cells, supporting its potential safety for therapeutic applications. Additionally, Phi1gp518 impairs the formation of gonococcal microcolonies and prevents proper biofilm establishment. The antigonococcal properties of Phi1gp518 endopeptidase make it a good candidate for further protein engineering and development as an alternative treatment strategy for drug-resistant N. gonorrhoeae infections.

Recent advances in the discovery, characterisation, and engineering of urethanases provide new opportunities for the sustainable biocatalytic degradation of polyurethane waste. A mechanistic understanding of enzyme-plastic interactions is essential for structure-based engineering to enhance urethanase activity. However, the extremely complex and hydrophobic nature of polyurethane makes it challenging to elucidate the structural basis of enzyme-plastic interactions. Here, we used a fragment-based approach to characterise the active sites of two novel urethanases with different catalytic scaffolds, employing both a crystallographic fragment-screening (FASE) campaign and soluble fragments of plastic-like analogues that mimic the substrate, transition state, or product. FASE identified new substrate-binding subpockets while interactions of plastic mimetics in the active site provided a mechanistic understanding of the recognition and binding of polyurethane fragments by these subpockets. These results highlight a diversity of binding modes among urethanases toward different polyurethane fragments.

Compact poultry raising has turned poultry litter into an environmental problem, as it may all be packed with heavy metals and drug-resistant germs. Of all the metals, chromium contamination not only disturbs the general environment but is also a source of concern for public health. Poultry litters were taken from 14 farms in different places, and the bacteria characters from different places were tested for their capacity to tolerate Cr(VI). A total of 31 bacterial isolates were initially screened, and three of them (AH-2, AZ-1, and AMF-3) appeared to be very resistant to chromium. The isolates were able to survive at the highest concentration, 800 mg/L of the Cr(VI); however, AH-2 was the most resistant one (MIC: 900 mg/L; MBC: 1000 mg/L). Chromium reduction tests showed that AMF-3 at high concentration showed the maximum chromium reduction, while AH-2 achieved higher chromium reduction at medium concentration. Phenotypic and biochemical analysis showed that the isolates were Staphylococcus spp., which was confirmed by 16S rRNA gene sequencing as S. cohnii, S. saprophyticus, and S. gallinarum. Moreover, chromium was detected at higher levels in poultry litter compared to the feed, with the highest accumulation in AZ farm litter (4464.0 {micro}g/kg). The highlighting feature of our article is the presence of chromium-tolerant and reducing bacteria in poultry environments. Besides that, the level of chromium in poultry litter is really high, and it points to the need for better waste management.

Enzymatic depolymerization of polyethylene terephthalate (PET) has advanced rapidly, alongside a growing volume of publicly available metagenomic data from microbial communities under sustained selective pressure from plastic exposure. Reasoning that such environments may harbor underexplored polyester-active enzymes, we developed a targeted mining workflow that screens exclusively plastic-associated datasets through multi-step bioinformatic filtering--integrating catalytic-motif screening, disulfide-topology validation, structural-similarity scoring, and phylogenetic profiling--to recover high-confidence PETase candidates. Applied to 271 plastic-associated metagenomes, the pipeline yielded 21 non-redundant candidates, several of which combine the Type I catalytic motif (GHSMGGGG) with Type II-like extended loops and secondary disulfide bonds. Two candidates were experimentally confirmed as PET hydrolases; the more active, PET-KR1, is a thermostable enzyme (Tm = 66.5 {degrees}C) that depolymerizes PET across a broad temperature range, with markedly higher productivity on powdered than on film substrate. PET-KR1 achieved optimal depolymerization at 50 {degrees}C, yet at 60-65 {degrees}C, where total yields declined, the product pool was more strongly enriched in the terminal monomer TPA, suggesting that thermostability and substrate accessibility are the primary targets for further engineering. Molecular dynamics simulations revealed a conserved hydrophobic binding network around the catalytic serine, consistent with established PETase substrate-recognition modes, and rational disulfide engineering raised the melting temperature by 3.5 {degrees}C, confirming amenability to further optimization. Overall, PET-KR1 expands the scaffold space available for PETase engineering, while the discovery workflow, built entirely on publicly available tools and open-access data, provides a reproducible strategy for metagenomic mining of novel PET-degrading enzymes toward biocatalytic PET recycling.

Endosymbiosis is widespread throughout the tree of life. Understanding how the transition of a bacterial endosymbiont from facultative to host-dependent obligate life occurs is an important question for defining the origin of endosymbiosis. A novel gram-positive bacillus, Brevibacillus sp. TJ4 was isolated from the nitrogen-fixing yeast Rhodotorula mucilaginosa JGTA-S1, which houses several endobacteria within its cells. TJ4 can survive independently of yeast but exhibits genomic and metabolic features characteristic of an evolving endosymbiont, slowly assuming a host-dependent, obligate lifestyle. The TJ4 genome contains several incomplete pathways for carbohydrate, amino acid, vitamin, and cofactor metabolism, which is reflected in its increased reliance on host-derived nutrients and auxotrophy compared with that of other Brevibacillus spp. Comparative genomics revealed widespread genome rearrangements, loss of synteny, and multiple cross-genus and inter-kingdom horizontal gene transfer (HGT) events in TJ4 compared to other Brevibacillus spp. These HGTs include the acquisition of genes from bacteriophages and co-resident endobacteria of JGTA-S1. One such horizontally acquired gene, Type II 3-dehydroquinate dehydratase (AroQ), appears to have originated from the Rhodotorula host itself. This acquisition functionally restores the shikimate pathway in strain TJ4, as evidenced by the phylogenetic placement of AroQ from TJ4 within the clade of fungal AroQ homologs. Potential exploitation of the host JGTA-S1 appears to be a probable mode of endosymbiosis of TJ4, an evolving endosymbiont that we named Brevibacillus rhodotorulae sp. nov.

Carbapenem-resistant Acinetobacter (CRA) has been associated with increased morbidity and mortality in clinical settings. In this study, we explored the transfer potential of a mobilizable plasmid-harboring blaOXA-72 gene between Acinetobacter species originating from patient, municipal wastewater, and pig farm wastewater. PCR-based evidence suggested putative transfer of blaOXA-72 from Acinetobacter pittii to Acinetobacter baumannii. In this pair, the apparent frequency of PCR-marker-positive putative transconjugants varied depending on temperature and meropenem supplementation, with higher number observed at 27{degrees}C compared to 17{degrees}C and 37{degrees}C. Likewise, the presence of antibiotic pressure yields to higher apparent conjugation frequency, however this observation was limited to a singled donor-recipient pair. Further, we revealed a phenotypic conversion in terms of meropenem susceptibility and a fitness cost in the putative transconjugants. While whole genome sequencing did not conclusively verify the presence of blaOXA-72 or fully resolved plasmid configuration, Oxford Nanopore read mapping consistently detected the chromosomal strA gene in all isolates. In contrast, only a limited number of reads aligned with blaOXA-72 gene, traC, or the complete plasmid sequences. Comparative analyses further revealed variations in the surface-associated factors and defense systems composition of the recipient strains, which could be considered as barriers in conjugation. Lastly, the persistence of PCR-detectable marker genes in putative transconjugants was variable and generally unstable over a 30-day period. Overall, these findings provide preliminary insights into the factors that may influence horizontal gene transfer and short-term maintenance of blaOXA-72.

Filamentous phages infecting Gram-positive bacteria remain largely unexplored. Notably, only two filamentous phages, B5 and Philemon infecting Propionibacterium freudenreichii, have been described to date in the phage-rich dairy ecosystem. Although both were genomically characterized, only B5 was confirmed to be an infective filamentous single-stranded DNA phage. The aim of this study was to isolate and characterize new filamentous phages from Swiss-type cheese to investigate their diversity, structural features, host specificity, and potential adaptation to the dairy environment. Thirty raw and pasteurized milk cheeses from France were screened for phages infecting P. freudenreichii strains. Eleven phages were isolated, nine of which displayed a filamentous morphology. Named MINOG1 to MINOG9, these filamentous phages exhibited genomic features typical of this morphotype, including small single-stranded DNA genomes with collinear genes organized into functional modules. Comparison with B5 and Philemon revealed sequence divergence ranging from 0.1% to 7%. These phages also exhibited a diverse host range. To further explore phage-P. freudenreichii interactions, we screened the genomes of the strains used in this study, as well as additional genomes retrieved from the NCBI database, for CRISPR spacers predicted to target these filamentous phages. Numerous strains contained CRISPR spacers showing 79 to 100% identity to genomic regions of these phages. Two P. freudenreichii strains displayed markedly different phage resistance levels despite exact spacer-protospacer matches with phages B5, MINOG1, MINOG2, and MINOG8. Conversely, several strains were resistant to nearly all tested phages despite lacking CRISPR spacers targeting them suggesting the presence of additional defense systems in P. freudenreichii. IMPORTANCEFilamentous phages can play important roles in bacterial ecology by modulating host physiology, population dynamics, and bacterial adaptation to specific environments. However, filamentous phages infecting Gram-positive bacteria remain among the least explored bacterial viruses, and their diversity, ecology, and interactions with their hosts are still poorly understood. This knowledge gap is particularly relevant in dairy ecosystems, where phages are abundant and can influence microbial communities and fermentation processes. In characterizing nine new filamentous phages infecting Propionibacterium freudenreichii from Swiss-type cheeses, this study expands the known diversity of filamentous phages associated with Gram-positive bacteria and provides new insights into phage-host interactions and bacterial defense strategies in dairy-associated bacteria.