Frontiers in Microbiology

Andes orthohantavirus (ANDV), the primary etiological agent of hantavirus pulmonary syndrome (HPS) in South America, is uniquely capable of limited human-to-human transmission, posing a significant challenge for outbreak control. Recent events, including the 2018-2019 Epuyen outbreak and the 2026 MV Hondius incident, underscore the need for rapid, lineage-specific molecular diagnostics. In this study, we present an artificial intelligence (AI)-driven framework for the design of diagnostic primers targeting the S genomic segment of the Epuyen lineage. Using an evolutionary algorithm integrated with thermodynamic evaluation via Primer3Plus, candidate primers were optimized to maximize classification accuracy while satisfying stringent biochemical constraints. The resulting primer set enables amplification of lineage-specific regions suitable for molecular characterization and surveillance. In silico validation demonstrates that the proposed primers achieve perfect discrimination between 2026 outbreak sequences and other ANDV variants. Furthermore, in silico comparison with standard protocol-based primers reveals substantially reduced sensitivity and specificity in the latter, highlighting the limitations of static diagnostic designs when applied to evolving viral populations. Overall, this work demonstrates that AI-assisted primer design provides a robust and adaptable strategy to improve viral detection, enhance outbreak tracking, and support timely public health interventions. Integrating computational optimization into diagnostic development is essential for strengthening preparedness against emerging zoonotic threats.

Quorum sensing (QS) influences biofilm formation, persistence and stress adaptation in Salmonella enterica. Although Salmonella does not synthesise acyl-homoserine lactones (AHLs), it can detect exogenous AHLs through the LuxR homolog SdiA, allowing it to respond to interspecies signalling cues in polymicrobial environments. This study investigated whether external QS stimulation and quorum-modulatory compounds reshape biofilm-associated transcriptional programmes in S. enterica serovar Enteritidis (SE) and S. Typhimurium ST14028. Biofilm formation was assessed using the crystal violet assay, while expression of QS-, biofilm-, adhesion-, motility- and invasion-associated genes (sdiA, csgD, flgG, fimA, rck, invA, bapA and hilA) was quantified using multiplex RT-qPCR and analysed by the {Delta}{Delta}Ct method, with 16S rRNA used for normalisation. In parallel, molecular docking was used to explore the predicted interaction of C8-HSL, established quorum-quenching agents and selected phytochemicals with the Salmonella SdiA ligand-binding region. Exposure to exogenous C8-HSL increased biofilm biomass and induced coordinated upregulation of QS- and biofilm-associated genes in both serovars, supporting the role of external AHL sensing in Salmonella biofilm regulation. In contrast, farnesol and furanone produced broad transcriptional repression accompanied by reduced biofilm biomass. Selected natural products, including epigallocatechin gallate (EGCG), thymoquinone, garlic extract, turmeric extract and aloe-emodin, produced moderate antibiofilm effects and partial downregulation of QS-associated transcriptional responses, suggesting possible interference with biofilm-regulatory signalling pathways. Molecular docking further supported this interpretation by identifying potential interactions between selected quorum-modulatory compounds and the predicted SdiA binding region, providing a plausible mechanistic basis for their observed biological effects. Notably, responses differed between SE and ST14028, indicating strain-dependent sensitivity to QS stimulation and quorum-modulatory treatments. Collectively, these findings suggest that exogenous AHL sensing contributes to strain-dependent transcriptional reprogramming of Salmonella biofilm-associated genes and that selected phytochemicals may act as preliminary quorum-modulatory candidates. This study supports further investigation of SdiA-mediated signalling as an anti-virulence target for reducing Salmonella persistence in food-associated and clinical environments.

Salmonella spp. remains one of the leading foodborne pathogens worldwide, and the circulation of multidrug-resistant strains in the poultry industry poses a significant challenge. In this study, five isolates from poultry litter swabs (commercial broiler chickens) belonging to the Salmonella Heidelberg and Salmonella Minnesota serovars were characterized using an integrated approach involving phenotypic resistance profiling, whole-genome sequencing, structural prioritization of molecular targets, and in silico screening of ligands. All isolates exhibited multidrug resistance phenotypes and genetic repertoires consistent with resistance to {beta}-lactams, sulfonamides, and tetracyclines, as well as determinants linked to efflux systems, virulence, and persistence. Genomic analysis allowed for the prioritization of five proteins for structural investigation: CTX-M-2, CMY-2, Sul2, AcrB, and SpvC. Sequence-structure validation revealed high correspondence between the proteins of the isolates and the experimental structures selected for CMY-2, Sul2, AcrB, and SpvC, while CTX-M-2 was modeled with high structural confidence. Molecular docking analyses with GNINA revealed distinct behaviors among the targets. Sul2 showed biological relevance but a more conservative structural response, with no significant gain after analog generation. In contrast, AcrB stood out as the most promising target, with analogs generated by BRICS yielding better scores and, in some cases, coherent international networks identified by PLIP. The results demonstrate that the integration of phenotype, comparative genomics, and structural prioritization constitutes a rational strategy for selecting targets and molecular candidates in multidrug-resistant avian strains of S. Heidelberg and S. Minnesota.

A challenge in using plant biomass is its highly recalcitrant nature, which makes it economically infeasible to utilize. In natural environments, various microbes, including bacteria and fungi, are reported to decompose plant cell wall materials such as cellulose and hemicellulose, and there may be undescribed microbes that contribute to the degradation of plant biomass. We focused on isolating novel plant biomass-degrading bacteria and screened more than 100 isolates from the Tomakomai experimental forest in Hokkaido, Japan. Among them, one novel Bacillus species was chosen for whole-genome sequencing. Comparative genomics and a carbon source utilization assay indicated that the isolate belongs to a subspecies of Bacillus subtilis, which we named B. sp. TTS1. Glucose, cellobiose, xylose, xylan, mannose, or mannan was used as the sole carbon source in the minimum medium, and the growth of this bacterium was determined. Furthermore, a proteomic analysis of B. sp. TTS1 was performed using culture supernatants from various polysaccharide-containing media. In the present study, several key enzymes involved in plant biomass degradation were identified, namely {beta}-1,4-mannanase and xylanase, and they were highly enriched in all tested polysaccharides.

Pseudomonas aeruginosa is a clinically significant bacterial pathogen that poses a serious threat to aquaculture. However, there are limited information on Massilia isolates against pathogenic P. aeruginosa in aquaculture. In the present study, a facultative predator, M. varians isolate P2-4, was isolated from aquaculture sediment using Chinese mitten crab Eriocheir sinensis-pathogenic P. aeruginosa as the prey bacterium, and its genomic feature, bacteriolysis-related genes, safety, bacteriolytic spectrum, and in vitro and in vivo antibacterial effects against pathogenic P. aeruginosa in E. sinensis were further characterized. Isolate P2-4 consisted of one chromosome and one plasmid (with a total of 75 tRNAs, 7 5S rRNAs, 7 16S rRNAs, 7 23S rRNAs, 34 sRNAs, 5,238 coding genes, 20 genomic islands, 1 prophage, 23 insertion sequences, and 102 repeat sequences), and harbored 19 bacteriolysis-related genes (pilA, pilB, pilC, pilD, pilF, pilG, pilH, pilM, pilO, pilP, pilQ, pilS, pilR, pilT, mltA, mltB, mltC, mltD, and dacB) associated with cellular motility and cell wall lysis. In addition, the isolate carried no virulence genes, was unable to produce haemolysin, hydrogen sulfide, nitrite and ammonia, and avirulent in E. sinensis with a 7-day acute intraperitoneal LD50 value of above 5.0 x 108 CFU/mL. Furthermore, the isolate possessed a wide bacteriolytic spectrum against pathogenic Shewanella algae, Aeromonas caviae, A. hydrophila, and Photobacterium damselae besides P. aeruginosa, exhibited bacteriolysis rates of 99.35% to 99.99% towards the pathogenic P. aeruginosa at 1.0x103 to 1.0x10{square} CFU/mL, and displayed relative percentage survivals of 42.31% to 73.08% against P. aeruginosa infection in E. sinensis at doses of 6.0 x 103 to 6.0 x 105 CFU/g diet. To our knowledge, this study for the first time demonstrates a M. varians strain as a potential biocontrol agent against pathogenic P. aeruginosa in aquaculture.

Close to 50% of all bird species are reservoirs of potentially pathogenic fungi, including those listed as priority by the World Health Organization. In Malawi, data on diversity, pathogenic potential, and ecological avian sources of medically important yeast are scarce. A cross-sectional study using a descriptive approach was conducted in Blantyre, Southern Malawi, to characterise medically important yeasts recovered from environments contaminated with excreta/guano from synanthropic pigeons. A total of 20 samples were collected from 4 peri-urban areas, which yielded 71 yeast isolates. To assess the pathogenic potential of the environmental isolates, we compared their phenotypic virulence traits with those of 21 clinical yeast isolates collected from referral hospital laboratories. Pichia kudriavzevii (39%) and Candida orthopsilosis (30%) were the commonly isolated species in the pigeon-guano-contaminated environments. Candida parapsilosis sensu stricto (29%) and Candida albicans (24%) constituted most of the clinical yeast isolates. Half of the species isolated in the pigeon-guano-contaminated environments were also identified among the clinical isolates. A majority of the environmental isolates showed virulence traits similar to or stronger than clinical isolates. The findings underscore the critical need for integrated surveillance under the One Health framework, especially in bird-inhabited spaces close to human settlements.

Urinary tract infections (UTIs) are the most prevalent bacterial infections globally, and their management increasingly challenged by antimicrobial resistance (AMR). Probiotics offer a promising approach to mitigate AMR by competitively excluding uropathogens and enhancing host immunity by producing immune modulators. Despite being potential, key gaps persist between the discovery of uroprotective probiotic strains and optimization of formulations for urinary tract delivery. Here, we analyzed the urinary microbiome of UTI patients and healthy individuals to identify potential probiotic candidates for the prevention and management of UTIs. Publicly available 16S rRNA amplicon sequencing data of the urinary tract were processed using a standardized pipeline for sequence quality assessment, taxonomic assignment, and microbial function prediction. Comparative analysis showed a significant shift in microbial composition between UTI patients and healthy controls. The dominated phyla identified included Acidobacteriota, Actinobacteriota, Bacteroidota, Campylobacterota, Cyanobacteria, Firmicutes, Fusobacteriota, Patescibacteria, Proteobacteria, and Synergistota. Overall differential abundance analysis revealed Escherichia coli as the predominant UTI-associated species, while Lactobacillus crispatus was enriched in healthy samples. Additionally, predictive functional analysis indicated that metabolic pathways associated with beneficial microbes were enriched in the healthy group. Overall, the study highlights the association of distinct urinary microbiome signatures with infection status, which supports L. crispatus as the most promising probiotic for UTI prevention and control.

Per- and polyfluoroalkyl substances (PFAS) are new pollutants in the environment whose effects on bacterias physiology is not well understood. In this study, we show that exposure to PFAS causes membrane depolarization in Salmonella enterica serovar Typhi. This works as a metabolic uncoupler that breaks down proton motive force without immediately killing the cells. This disturbance results in a significant elevation of intracellular NADH and NAD levels while preserving redox equilibrium, signifying an augmented metabolic flux. At the same time, we see that {beta}-oxidation pathways are turned on, which suggests that the cells are shifting toward breaking down fats to make up for the lack of energy. Even though there are more reducing equivalents, ATP levels go down, which is what happens when respiration is uncoupled. This puts the cells in a state of "pseudo-starvation." This metabolic stress triggers the SpoT-dependent stringent response, leading to the accumulation of (p)ppGpp. Genetic analysis employing {Delta}relA and {Delta}relA{Delta}spoT mutants confirm that SpoT is necessary for this adaptive response. Functionally, PFAS-treated populations show an enhanced proportion of persister-like cells, which connects exposure to environmental pollutant in the environment to antibiotic tolerance. Our findings reveal a previously unidentified mechanism by which PFAS alters bacterial metabolism and stress responses, facilitating persistence through membrane depolarization, metabolic reconfiguration, and stringent response activation. This study underscores the potential influence of environmental pollutants on bacterial survival mechanisms and antibiotic resistance.

Ammonia-oxidizing archaea (AOA) are frequently found in oxygen-depleted marine environments with permanent or temporal presence of sulfide (HS-). However, it remains unexplored how sulfide affects the activity of ammonia-oxidizing archaea. We studied the capability of Nitrosopumilus maritimus SCM1 to oxidize ammonia when exposed to HS-. Ammonia oxidation remained active even after exposure to sulfidic spikes in the lower micromolar range, albeit at reduced rates compared to the absence of HS-. However, 90 {micro}M HS- completely inhibited ammonia oxidation. We found no evidence of NO-dismutation under oxygen depletion and presence of HS- (20 {micro}M): the formation of O2, N2O and N2 did not occur. All in all, we confirmed ammonia oxidation in N. maritimus SCM1 under oxic conditions after sulfide additions, but no evidence of NO-dismutation under sulfidic conditions. Our findings suggest that AOA can recover ammonia-oxidation activity after oxygen re-exposure in regions with periodic sulfide accumulation. However, in permanently sulfidic areas, ammonia oxidation recovery seems unlikely, as NO-dismutation does not appear to be a viable mechanism.

The slightly-alkaline (pH [~]8.5), boiling ([~]90{degrees}C) vent-water of a Trans-Himalayan geothermal spring, moderately-rich in dissolved solids ([~]1500 ppm), was explored six times over a year. 11 archaeal and 46 bacterial species were detected consistently, while nine bacteria occurred intermittently, in the vent-epicenter featuring a largely-stable physicochemical milieu. All 11 archaea were detected as metagenome-assembled genomes ascribable to Thermoproteota. Of the total 55 bacteria detected, 32 were retrieved as MAGs, 20 as isolates, and three in both forms. Four bacteria could not be classified below the domain-level; three and four belonged to hyperthermophilic (Aquificia) and thermophilic (Thermaceae and Thermoflexaceae) taxa respectively; 27 belonged to taxa having some moderately-thermophilic members; 17 belonged to mesophilic taxa. According to metagenomics, an Aquificia, followed by two Thermoprotei and one Thermoproteales, dominated the microbiome overwhelmingly. Metatranscriptomically, however, the Thermoproteales was most active. Metatranscriptomic signatures envisaged the in situ metabolic status of the 66 species discovered as follows. Among the 18 putative hyperthermophiles and thermophiles identified, 17 rendered wide-ranging activities including growth; one Thermoproteota species had considerable activities sans growth. One new-phylum-level bacterium rendered wide-ranging activities including growth, while three such entities had considerable/minimal activities sans growth. Among the 27 potential moderate-thermophiles discovered, two Armatimonadota and one Thermosynechococcus species rendered wide-ranging activities including growth, 20 had considerable/minimal activities sans growth, whereas four had zero activities. Among the 17 mesophiles identified, 16 rendered considerable/minimal activities sans growth, whereas one had zero activity. Molecular drivers were envisaged from the metatranscriptomic data to explain the trends of inequitable population ecology.

Bacillus thuringiensis INTA Mo4-4 was characterized phenotypically, genomically, and for insecticidal activity against Alphitobius diaperinus. Microscopy revealed rare flat rectangular parasporal crystals, and SDS-PAGE identified a ca. 67 kDa protein, similar to B. thuringiensis serovar morrisoni strain tenebrionis DSM-2803, which was proteolytically processed to a ca. 55 kDa fragment. Genomic analysis showed a 5.99 Mb genome with 99.43% completeness, clustering phylogenetically with B. cereus and B. thuringiensis. High genomic similarity was observed with B. thuringiensis svar. morrisoni BGSC 4AA1, confirmed by MLST analysis assigning it to ST-23. The genome encodes an interesting arsenal of pesticidal proteins showing significant similarity to Cry3Aa, Mpp23Aa, Xpp37Aa, Mpp5Ab, Vpb1Ad, Vpb1Ae, Vpa2Ab, Vpa2Ba, Vpa2Bb and Spp1Aa, with demonstrated toxicity against coleopteran pests. Biosynthetic gene clusters for toyoncin, fengycin, and bacillibactin were identified. Dose-response bioassays showed that INTA Mo4-4 was nearly four times more toxic to A. diaperinus larvae (LC50 136.9 {micro}g/ml) than DSM-2803 (LC50 540.5 {micro}g/ml), with the difference being statistically significant. No teratological effects were observed on Musca domestica. These findings suggest that INTA Mo4-4 is a promising candidate for the biological control of A. diaperinus.

Regulation of nitrogenase, which converts nitrogen gas (N2) into ammonium (NH4+), typically involves a conserved set of regulatory proteins across diverse N2-fixing (diazotrophic) bacteria. However, the interactions and relative influence of these regulators can vary between species. Thus, one cannot make assumptions about nitrogenase regulation when working with uncharacterized diazotrophs like Vibrio natriegens, a {gamma}-proteobacterium of growing interest for synthetic biology. Little is known about V. natriegens nitrogenase regulation, which could be used to exploit inexpensive N2 for various applications, including NH4+ production. Here, we characterized the roles of several annotated V. natriegens nitrogenase regulatory proteins in response to NH4+ versus N2. Using functional genomics, targeted mutations, and reporter assays, we identified a typical regulatory hierarchy where the two-component system NtrBC governs a nitrogen-scavenging regulon that includes NifA, the transcriptional activator of nitrogenase genes. Unlike other diazotrophic {gamma}-proteobacteria, NifA was sufficient to activate nitrogenase gene expression, as a mutant lacking NtrBC grew normally with N2 after a lag phase. Thus, NtrBC was dispensable, but still important for timely nitrogenase expression. Furthermore, NtrBC was negatively regulated by the nitrogen-responsive PII proteins GlnB and GlnK; disruption of both PII proteins led to NtrBC-dependent nitrogenase overactivity, marked by NH4+ excretion. The redundant repression of NtrBC by GlnB and GlnK more closely resembles that of non-diazotrophic E. coli than other diazotrophic {gamma}-proteobacteria. Together, our findings provide a framework for V. natriegens nitrogenase regulation that can be leveraged for applications like NH4+ production. HIGHLIGHTSO_LIA genetic examination of Vibrio natriegens nitrogenase regulation is performed C_LIO_LINtrBC is important for early nitrogenase gene expression but is not essential C_LIO_LINifA autoactivation is sufficient for nitrogenase expression C_LIO_LIPII proteins GlnB and GlnK are redundant negative regulators of nitrogenase C_LIO_LIGenetic targets are identified that result in excretion of NH4+ C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/728972v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@aac4adorg.highwire.dtl.DTLVardef@1565c0corg.highwire.dtl.DTLVardef@b545a6org.highwire.dtl.DTLVardef@efb052_HPS_FORMAT_FIGEXP M_FIG C_FIG

Anaerobic gut fungi (AGF) are key members of the herbivorous gut microbiome. While AGF communities have been well-studied in foregut and hindgut fermenters, they remain poorly characterized in pseudoruminants such as camels. Here, we present a comprehensive culture-independent diversity survey of 142 fecal samples from all three extant camel species (Camelus dromedarius, Camelus bactrianus, and Camelus ferus). The AGF community in Camelus was highly diverse, with representatives of 42 AGF genera identified. However, this diversity was unevenly distributed, with three genera (Neocallimastix, Caecomyces, and Orpinomyces) accounting for 70.7% of sequences encountered, and only 12 genera exceeding 1% relative abundance in the entire dataset. While several of the genera identified as major components of the AGF community in camels are highly ubiquitous in all herbivores, others, such as Oontomyces, Aestipascuomyces, Liebetanzomyces, and the yet uncultured genera NY09, NY03, and JV-2025d are extremely rare in ruminants and hindgut fermenters, hinting at their preference and potential co-evolution with the Camelidae. Ordination approaches identified host species and biogeography as key determinants driving AGF community structure differences between various camel species. Comparative community structure analysis between AGF community in camels versus reference foregut and hindgut fermenters identified the relative enrichment of the genera Oontomyces and Aestipascuomyces in pseudoruminants datasets. Our results demonstrate a distinct AGF community composition in Camelidae, elucidate factors impacting AGF diversity and community structure variations in Camelus, and identify key distinct taxa differentially enriched in psuedoruminants compared to ruminants and hindgut fermenters. The ecological and evolutionary drivers of such patterns are discussed.

Cyclic diguanosine monophosphate (c-di-GMP) is a ubiquitous bacterial second messenger that regulates diverse cellular processes, including colony morphology, motility, biofilm formation, and virulence. It is synthesized by diguanylate cyclases (DGCs) containing the GGDEF domain and degraded by phosphodiesterases (PDEs) containing the EAL domain. However, studies on the genetic and physiological characteristics of c-di-GMP metabolism in Pantoea ananatis are lacking. In this study, we identified 26 predicted c-di-GMP metabolism-related genes in the P. ananatis PA13 genome: 9 encode GGDEF-only domain proteins, 5 encode dual GGDEF/EAL domain proteins, and 12 encode EAL-only domain proteins. We constructed overexpression strains and mutants of 26 DGC- and PDE-encoding genes, and then assessed their Congo Red binding, mucoid and rugose phenotypes, pellicle formation, and swimming motility. We identified 14 of 26 DGC and PDE proteins that affect phenotype changes. Among the 26 DGC- and PDE-overexpressing strains, 13 exhibited the phenotypic changes described above, with some showing alterations in multiple phenotypes simultaneously. Notably, overexpression of dgcM induced changes across all phenotypes. Among the 26 DGC and PDE mutants, the pdeC mutant increased pellicle formation and Congo red binding, the pdeM mutant reduced the mucoid phenotype, and the pdeS mutant, which shows high similarity to ydiV, an anti-FlhD factor, increased swimming motility. Overexpression strains and mutants of 14 DGC and PDE proteins that exhibited phenotypic changes had higher intracellular c-di-GMP levels than the wild type. This study provides important insight into the role of the c-di-GMP network in the plant pathogen P. ananatis. IMPORTANCEPantoea ananatis is a versatile bacterium that causes significant diseases in various economically important plants. To survive and infect hosts, bacteria use a key signaling molecule called c-di-GMP to switch between swimming freely and forming protective communities known as biofilms. Despite its importance, the specific genes governing this signaling network in P. ananatis remained unknown. In this study, we systematically identified and characterized 26 genes responsible for regulating c-di-GMP levels in P. ananatis PA13. By analyzing mutants and overexpressing these genes, we pinpointed 14 critical factors that control essential behaviors such as motility, pellicle formation, and colony appearance. Notably, we discovered specific genes, such as dgcM and pdeS, that act as master regulators of these traits. This comprehensive functional map of the c-di-GMP network provides essential insights into how this pathogen adapts to its environment, offering potential targets to control plant infections.

Burkholderia cepacia (B. cepacia) is an opportunistic pathogen with versatile virulence mechanisms. The pathogenesis of B.cepacia in the immunocompetent host following intranasal exposure largely remains ambiguous. Male BALB/c mice were intranasally inoculated with B. cepacia strain 20209 (1x10{square} CFU) and evaluated on days 3, 7, 14, and 21 post-infection. Histopathology of lung, liver, spleen, and kidney tissues were performed using H&E and PAS staining. Plasma cytokines were quantified using commercial multiplex assays and ELISA. Matrix metalloproteinase-2 (MMP-2) activity was assessed via gelatin zymography and metabolomic profiling by high-resolution mass spectrometry (HRMS). Histopathological analysis revealed organ-specific pathological indices such as interstitial pneumonitis, bronchitis, leukocyte infiltration, hepatic inflammation, as well as splenic hyperplasia. Similarly, MMP-2 activity revealed time-dependent modulation, reflecting dynamic proteolytic responses. Plasma and tissue IL-18 and IL-1{beta} levels demonstrated a temporal regulation, with IL-18 peaking on day 7 post-infection, while IL-1{beta} showed a biphasic expression peaking on day 3 and 14. Untargeted metabolomics revealed differential expression of lipid metabolism, and energy pathways, with higher expression of phospholipids and sphingolipids. Together, our study portrayed a physiologically relevant intranasal BALB/c model that captures both localized and systemic inflammatory responses to B. cepacia. Our findings highlight organ-specific pathologic progression and sustained inflammation providing key insights into host-pathogen interactions.

Adeno-associated virus (AAV) vectors are widely used in gene therapy, whereas low manufacturing efficiency and a large proportion of empty capsids are major obstacles. This study focused on the Yin Yang 1 (YY1) binding motif (YY1-motif) and investigated the effect of its presence or insertion at upstream of the Replicase (Rep)/Capsid Cap) gene on AAV vector production. We found that the YY1-motif incidentally presented in a Rep/Cap plasmid was associated with high vector production. We then designed several modified Rep/Cap (RC2) constructs. The YY1-motif insertion at the upstream of Rep/Cap gene increased vector yield in a repeat-number-dependent manner, and similar effects were not observed with other promoters insertion. Furthermore, the insertion of the YY1-motif reduced the amount of Cap protein per the same amount of full particle in supernatants on multiple serotypes, indicating the improvement in the empty/full capsid ratio. The YY1-motif insertion did not affect the AAV vector infectivity. These results denote that the YY1-motif has a universal regulatory function that optimizes the Rep/Cap expression balance, and simultaneously improves the production efficiency and full particle formation of AAV vectors. This finding could contribute to the development of highly efficient and high-quality AAV manufacturing processes.

Ionizing radiation induces DNA damage and oxidative stress; however, the genes and molecular mechanisms involved in bacterial stress responses have not been sufficiently identified. In this study, we used Limnobacter thiooxidans strain CS-K2, which is the closest relative to the bacteria detected in torus room water at the Fukushima Daiichi Nuclear Power Plant according to 16S rRNA gene sequences, and evaluated its response to {gamma}-ray irradiation using integrated transcriptomic and proteomic analyses. We identified three previously uncharacterized genes (LT3105, LT3115, and LT3126) that were strongly induced at the mRNA and protein levels. These genes exhibited low basal expression but were markedly upregulated by {gamma}-ray irradiation. Notably, LT3126 encodes a protein containing VIT (vault protein inter--trypsin) and VWA (von Willebrand factor type A) domains and showed the strongest induction. Overexpression of LT3126 increased survival after 500 Gy irradiation by approximately 200-fold compared with the control bacteria, demonstrating a direct contribution to survival under high-dose stress. Comparative genomic analysis showed that these genes are not widely conserved across bacteria but are unevenly distributed among specific lineages. Taken together, this study identified a novel set of {gamma}-ray-responsive genes and demonstrated a functional role for LT3126 in radiation resistance, providing new insights into molecular adaptation in radiation-associated environments. IMPORTANCEWe identified a novel set of {gamma}-ray-responsive genes (LT3105, LT3115, and LT3126) in the non-model bacterium Limnobacter thiooxidans. These genes are located in relatively close genomic proximity and are coordinately induced upon irradiation, suggesting a shared functional role in stress response. Overexpression of LT3126 increased survival by approximately 200-fold after 500 Gy irradiation compared with the control bacteria, demonstrating a substantial contribution to survival under high-dose stress. These genes were also induced by heat shock and oxidative stress, indicating that their function extends beyond radiation-specific responses to broader environmental stress adaptation. Consistent with this, comparative genomic analysis showed that these genes are not widely conserved across bacteria but are unevenly distributed among specific lineages. Taken together, these findings highlight previously unrecognized molecular strategies that may support bacterial survival in radiation-associated environments.

Ethambutol (EMB) is a vital drug for treating Mycobacterium avium-intracellulare (MAI) infections; however, the genomic mutations underlying EMB resistance in MAI remain unclear. Herein, we evaluated eight sets of MAI clinical isolates, each containing at least two serial isolates collected from the same patient who received EMB in Japan. In four sets, the isolates independently increased EMB MIC by 4-fold, coinciding with mutations in the upstream region of embA or those corresponding to Mycobacterium tuberculosis (Mtb) embB Met306Val and Gln497Arg. Based on the increased EMB MIC values, we defined normal and elevated EMB MICs as [≤]8 {micro}g/mL and [≥]16 {micro}g/mL, respectively. In the other four sets, all of the isolates had elevated EMB MICs. In silico promoter prediction and expression analysis indicated that the upstream region of embA corresponds to the embA-embB promoter region, and mutations in this region increased the transcription of embA and embB, increasing EMB MICs. Furthermore, the analysis of 60 epidemiologically unrelated strains revealed that isolates with mutations in the embA-embB promoter and at embB codons 306/497 exhibited significantly higher EMB MICs compared with those without mutations. Publicly available genomic data demonstrate the worldwide occurrence of these mutations in clinical isolates. These results establish an association between elevated EMB MICs and mutations at embB codons 306/497 and the embA-embB promoter and are expected to predict EMB resistance.

Colibacillosis, caused by Avian Pathogenic Escherichia coli (APEC), result in substantial economic losses in global poultry production. The emergence of multidrug-resistant (MDR) APEC poses zoonotic risks through horizontal transfer of antimicrobial resistance (AMR) genes. Bacteriophage therapy emerges as a safe alternative to antibiotherapy; however, comprehensive characterization of phages targeting MDR-APEC from diverse geographical regions remains limited. We isolated five lytic bacteriophages from poultry fecal samples collected from five Indian states and characterized them through morphological analysis, physiological stability testing, whole-genome sequencing, and in vivo efficacy assessment. Host range was determined against APEC isolates, and therapeutic potential was validated in Galleria mellonella infection model. All five phages showed Myovirus-like morphology and stability across physiologically relevant temperatures (up to 55-70{degrees}C) and pH conditions (3-11). Their genome size ranges from 170 to 356 kb, belonging to three distinct genera; Dhakavirus, Gaprivervirus, and Asteriusvirus. Genomic analysis confirmed absence of antimicrobial resistance, virulence, toxin, or lysogeny genes. 51 APEC strains were isolated, of which 23 (45.1%) were MDR. Individual phages lysed 37-51% of tested APEC and 17-39% of MDR strains. Three Escherichia phages (fBSZT1, fUAMT1, fPKPT2) significantly improved larval survival to 60-80% at MOI 10 in G. mellonella infection models compared to untreated controls. This study establishes a well-characterized phage bank targeting MDR-APEC strains, providing foundation for developing phage-based interventions to reduce antibiotic dependency and mitigate AMR transmission risks under One Health framework.

Bacterial pathogens must adapt to dynamic host tissue environments to proliferate. Accordingly, elegant regulatory systems evolved to overcome challenges presented by the host and satisfy nutritional requirements. Sulfur is an essential macronutrient and Gram-positive bacteria such as Staphylococcus aureus balance this nutritional requirement by employing the transcriptional repressor, CymR. Previous investigations defined the S. aureus CymR regulon by comparing transcripts generated in a cymR mutant cultured in cystine replete, rich medium to wild type cells. This study defines the S. aureus CymR-dependent and -independent sulfur-starvation response in chemically defined growth conditions. Results demonstrate that the sulfur starvation and sulfur replete CymR regulons exhibit considerable overlap, including previously noted connections between iron acquisition, oxidative stress, and sulfur metabolism. The link between iron acquisition, oxidative stress, and sulfur metabolism is validated further by the finding that sulfur-containing glutathione (GSH) mitigates heme and peroxide toxicity. In addition to GSH, Cys and thiosulfate fulfill the S. aureus sulfur requirement. Transcriptional responses to organic (cysteine, cystine, reduced and oxidized GSH) or inorganic thiosulfate were quantified, revealing sulfur source-specific expression patterns. Thiosulfate induced the largest number of differentially expressed genes. Consequently, the thiosulfate transporter (SAUSA300_RS10985) has been confirmed as essential for S. aureus growth when thiosulfate is the sulfur source. Furthermore, we demonstrate that a hypothetical protein operonic with SAUSA300_RS10985, SAUSA300_RS10980, supports maximal growth on thiosulfate. Collectively, a resourceful transcriptomics framework is provided which underscores the dynamic nature of S. aureus sulfur metabolism.