Microbiological Research

Currently, the fungal class Archaeosporomycetes consists of one order, Archaeosporales with four families: Archaeosporaceae, Ambisporaceae, Geosiphonaceae, and Polonosporaceae. In the present study, the objective was to re-analyze the phylogeny and morphology of the Archaeosporomycetes from order to genus level. The different ecological strategies and, consequently, distinct evolutionary patterns of these taxa, as well as their morphological characters and other data updated here, suggest the need to divide Archaeosporales into four orders: (i) the type order Archaeosporales, (ii) Ambisporales ord. nov., both with four genera, (iii) Geosiphonales and (iv) Polonosporales ord. nov., both with single families and genera. Remarkably, the order Geosiphonales was described in the past, but was not considered in the Archaeosporomycetes until now. Phylogenetically, the four main clades (orders here proposed) of Archaeosporomycetes are well supported, with bootstrap values higher than 95% in all analyses, except Ambisporales/Ambisporaceae for RAxML-NG FBP analysis in the SSU tree (75%). Ecologically, this class includes three orders of arbuscular mycorrhizal fungi (AMF) forming symbiotic associations with plants, while Geosiphonales form an endocytobiosis with the cyanobacterium Nostoc. Morphologically, there are at least two AMF orders with spore bimorphism, which has not (yet) been described for Polonosporales. The only known species of Polonosporales, Polonospora polonica, forms spores directly on the neck of sporiferous saccules and the spores can morphologically be differentiated from all other taxa in Archaeosporomycetes by the formation of three permanent, rather thick spore walls, of which two form de novo during spore formation. The outer spore wall of Archaeosporales and Ambisporales are semi-permanent, evanescent or even short-lived, or show multiple fissures during aging, when it is more resistant. Ambisporales can easily be differentiated from Archaeosporales for instance by larger spores of the acaulosporoid morph and thicker spore walls. Our phylogenetic analyses suggested that Archaeosporales can be divided into two families: Antiquisporaceae that was described to form intraradical hyphae, vesicles and spores, staining darkly in Trypan blue, and Archaeosporaceae whose hyphae generally do not or only faintly stain in this reagent, and vesicles and intraradical spores have been rarely, if ever reported.

Histoplasma spp. is a dimorphic fungal primary pathogen that infects people worldwide and frequently affects immunosuppressed patients. Previous studies have identified the AMY1 gene product, the -amylase Amy1p, as essential for -glucan production and virulence in Histoplasma capsulatum. We identified two new genes (AMY2 and AMY3) in the Histoplasma genome that encode putative -amylases and made mutants using CRISPR/Cas9 technology, followed by evaluation of their role in -glucan biosynthesis and virulence. We also searched for AMY gene copies in 19 fungal genomes with the goals of identifying orthologs for AMY2 and AMY3, and establishing how many AMY copies existed across different fungi. We found that the number and type of -amylases vary depending on the fungal species; that all -amylases related to Histoplasma Amy1p belong to the GH13_5 subfamily, and all orthologs related to Histoplasmas Amy2p and Amy3p belong to the GH13_1 subfamily. We performed phylogenetic analyses of the three paralogs and revealed that the Histoplasma AMY duplications are ancient. We further established Amy2 is an ortholog of Aspergillus niger AgtA, and Aspergillus nidulans AmyD, and that it is partially involved in Histoplasma -glucan biosynthesis and virulence, while Amy3p is an ortholog of Aspergillus flavus Amy1, and it is dispensable for -glucan biosynthesis and virulence.

Rock-inhabiting fungi thrive in subaerial oligotrophic environments such as desert rocks, solar panels and marble monuments where organic carbon and nitrogen are scarce. We tested whether the rock-inhabiting fungus Knufia petricola showed a preference regarding nitrogen ([Formula] or [Formula]) and carbon (glucose or sucrose) sources and whether it was sensitive towards carbon and nitrogen limitation. As this fungus produces the carbon-rich, nitrogen-free 1,8-dihydroxynaphthalene (DHN) melanin, we tested whether a melanin-deficient mutant would be less sensitive to carbon limitation. The carbon and nitrogen concentrations were the primary predictors of growth, with a broad optimum partially explained by an optimal fungal C:N ratio. Limiting carbon or nitrogen supply decreased biomass formation, CO2 production and biofilm thickness but promoted substratum penetration through filamentous growth. The nitrogen content of the biomass was flexible within limits, increasing upon increasing nitrogen supply or decreasing carbon supply. The carbon use efficiency was fairly constant, whereas melanization correlated with a higher nitrogen content of the biomass despite melanin being nitrogen-free. In conclusion, in vitro, K. petricola switches to explorative growth under nutrient limitations, like fast-growing fungi, revealing universal fungal resource-acquisition patterns. Graphical abstract text and imageCarbon and nitrogen availability affect biofilm growth and morphology of the extremotolerant fungus Knufia petricola Abolfazl Dehkohneh, Julia Schumacher, Bastiaan J. R. Cockx, Karin Keil, Tessa Camenzind, Jan-Ulrich Kreft, Anna A. Gorbushina, Ruben Gerrits Growth of the rock-inhabiting fungus Knufia petricola was studied by varying carbon and nitrogen sources and concentrations. Overall, growth was best predicted by the carbon and nitrogen concentrations. Carbon and nitrogen limitation promoted substratum penetration through filamentous growth. O_FIG O_LINKSMALLFIG WIDTH=158 HEIGHT=200 SRC="FIGDIR/small/712823v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@6d98bdorg.highwire.dtl.DTLVardef@146aac5org.highwire.dtl.DTLVardef@757fa8org.highwire.dtl.DTLVardef@ff709_HPS_FORMAT_FIGEXP M_FIG C_FIG

Candida auris (Candidozyma auris) is an emerging multidrug-resistant fungal pathogen that poses a significant global health threat. However, the molecular mechanisms underlying its virulence remain incompletely understood. In this study, we performed in vivo transcriptome analysis using an immunosuppressed mouse gastrointestinal infection model to identify genes associated with host-adaptation and virulence during infection. By comparing fungal transcriptomes obtained from colonization and dissemination sites with those from in vitro cultures, we identified genes that were consistently upregulated during infection. Among these genes, the unfolded protein response regulator HAC1 was selected as a candidate virulence-associated gene for further analysis. RT-PCR and sequencing analyses revealed that HAC1 mRNA in C. auris undergoes an unconventional splicing event of 287 bp that is enhanced under ER stress conditions. The excised region spans the annotated open reading frame boundary, suggesting that the translated region of HAC1 may require re-evaluation. Notably, a proportion of HAC1 transcripts appeared to be spliced even under non-stress conditions, indicating a detectable basal level of UPR activation. Differences in splicing dynamics were also observed among clade strains. Functional analyses demonstrated that deletion of HAC1 increased sensitivity to ER stress and heat stress. The HAC1 deletion mutant also exhibited reduced virulence in both Galleria mellonella and immunosuppressed mouse infection models, as evidenced by delayed host mortality and decreased fungal burdens, respectively. These findings indicate that HAC1 contributes to ER stress adaptation, thermotolerance, and survival in the host environment, and identify HAC1 as a virulence-associated gene in C. auris.

Endophytic microbiomes of crop wild relatives (CWRs) adapted to extreme environments, such as halophytes, are promising sources of plant-beneficial bacteria and secondary metabolites for sustainable food production. Here, we analyzed 25 Bacilli isolates obtained from CWRs, halophytes, and other plant species in Crete, Greece. Using a hybrid Illumina-PacBio sequencing approach, we generated high-quality genomes and performed comparative genomics, phylogenetic, and pangenome analyses, complemented by in vitro assays. We identified 312 biosynthetic gene clusters (BGCs), nearly 60% of which showed no similarity to known clusters, revealing extensive unexplored biosynthetic potential. These unique BGCs may constitute an adaptive feature enabling endophytic Bacilli to colonize and interact with host plants. The isolates spanned diverse genera (Bacillus, Paenibacillus, Peribacillus, Neobacillus, Cytobacillus, Rossellomorea), including three novel species. Phenotypic assays of our isolates demonstrated high salinity tolerance (up to 17.5% w/v NaCl) and strong antagonism against major bacterial and fungal phytopathogens. Genome mining further revealed a broad array of putatively plant-beneficial traits related to growth promotion, stress adaptation, host interaction and inhibition of pathogens. Together, these findings show that Bacilli endophytes from wild and halophytic plants possess exceptional phylogenetic novelty, functional diversity, and biosynthetic capacity, providing new genomic and ecological insights into Bacilli associated with plants inhabiting extreme environments.

Aspergillus fumigatus is a world-wide saprophyte filamentous fungus which released conidia, its infectious morphotype, in the atmosphere. These conidia are inhaled daily by humans and can colonize the respiratory tract, where they may develop into hyphae, the invasive morphotype. We previously showed that bronchial epithelial cells (BECs) restrict A. fumigatus virulence by inhibiting conidial germination and filament formation through a process requiring PI3K signaling and the conidial fucose-specific lectin FleA. In the present study, we are looking to identify host factors and cellular partners involved in the BEC antifungal response and to define the molecular interactions underpinning FleA recognition. For this, we analyzed transcriptome of BECs infected with A. fumigatus in the presence or absence of the PI3K inhibitor LY294002. Functional involvement of candidate genes was assessed by siRNA knockdown and readouts of fungal filamentation (microscopic scoring and galactomannan release). FleA-interacting host proteins were identified by biotin-FleA affinity co-precipitation coupled to Tandem mass spectrometry, and validated by surface plasmon resonance and biolayer interferometry. The spatiotemporal dynamics of FleA and candidate partners were analyzed by confocal microscopy and proximity ligation assay We demonstrated that BEC antifungal activity involves at least two complementary pathways: a PI3K/laminin-332 axis promoting conidial adhesion, and a FleA-dependent pathway engaging ITGB1 and MRC2 consistent with lectin uptake and trafficking toward LAMP1-positive compartments. These findings nominate FleA-host receptor interactions as attractive targets for anti-adhesive strategies against A. fumigatus. Author summaryFungal pathogens are an increasing threat to public health, as they are becoming more common and harder to treat due to rising drug resistance. Among them, Aspergillus fumigatus has been classified as a critical pathogen by the World Health Organization (WHO). This filamentous fungus delivers spores in the air daily, which are constantly inhaled by humans. In people with weakened immunity, these spores can cause a range of lung diseases known as aspergillosis, with severity ranging from mild to life-threatening. Lung epithelial cells are the first cells of the respiratory tract to encounter inhaled spores. In a previous study, we showed that bronchial cells can prevent spore from developing into filaments, the invasive form of A. fumigatus that is responsible for tissue damage. This protective effect depends of on the recognition of a fungal protein called FleA. In the present study, we identified host cell proteins that bind to FleA and transport it into intracellular compartments. Our findings suggest that these proteins help bronchial epithelial cells to internalize fungal spores, thereby blocking their transformation into the invasive filamentous form.

The mitochondrion is a versatile organelle involved in diverse processes, such as cell death, metal homeostasis, plasma membrane and cell wall integrity, stress response, oxygen concentration, temperature, and metabolic adaptation, in addition to its role in generating energy. Consequently, mitochondrial fitness is essential for the pathogenicity of various organisms, including fungi. Cryptococcus neoformans is a fungal pathogen responsible for over 180,000 HIV-related deaths each year. In this study, we analyzed C. neoformans metabolic plasticity when grown with non-fermentable carbon sources and their impact on virulence and mitochondrial homeostasis. Growth on non-fermentable carbon sources increased thermotolerance, glucuronoxylomannan (GMX) content in the capsule, melanization rate, urease activity, biofilm formation, and virulence. Moreover, cells grown on non-fermentable carbon sources manifested increased mitochondrial number and activity. Conversely, mutants of the master regulator of mitochondrial biogenesis, the Hap complex, the catalytic subunit 1 of protein kinase A, or media supplementation with antioxidants, decreased the use of alternative carbon sources, capsule formation, melanin synthesis, urease activity, mitochondrial number, and resistance to both fluconazole and macrophage killing. Our results implicate mitochondrial homeostasis in virulence regulation via the PKA pathway, suggesting that targeting fungal mitochondrial homeostasis could be a therapeutic approach for cryptococcosis.

Despite the successful cultivation of many microbes from rich bacterial communities inhabiting alkaline soda lakes, members of the bacterial phylum Verrucomicrobiota have so far been detected only through metagenomics. Here, we used alginate as a selective substrate to enrich and isolate two strains of haloalkaliphilic Verrucomicrobiota. The isolates share identical 16S rRNA gene sequences representing a new genus lineage, and, together with other metagenome assembled genomes, a new family within Opitutales. Cells of strains AB-alg1T (from soda lakes) and AB-alg4 (from soda solonchak soils) are small and motile cocci forming submerged colonies in soft alginate agar. They are saccharolytic heterotrophs growing aerobically on polysaccharides (alginate, starch and inulin) and sugars (glucose, fructose, mannose, sucrose, melezitose, maltose and cellobiose). They also grow anaerobically by fermentation of alginate and D-mannose and by coupling incomplete denitrification to oxidation of alginate. Both isolates are obligately alkaliphilic and moderately salt-tolerant. The dominant membrane phospholipids include phosphatidylcholines and diphosphatidylglycerols (cardiolipins). The genome of AB-alg1T features polysaccharide lyases of the PL6, 7, 15, 17, 38, and 39 families for depolymerization of alginate. Based on distinct phenotype and phylogeny, we propose classification of strains AB-alg1T (JCM 35393T=UQM 41574T) and AB-alg4 as Verruconatronum alginivorum gen. nov., sp. nov. within a new family Verruconatronumaceae. ImportanceThe presented isolates are the first isolated representatives of an environmental family of Opitutales, part of the core microbiome of alkaline soda lakes. These bacteria feed on polysaccharides. We present the key enzymatic machinery for the polysaccharide breakdown. These enzymes are high-pH tolerant and have potential for industry applications, for example in washing powders and biomass waste recycling.

During host-pathogen interactions, fungal pathogens secrete effector proteins into host cells to manipulate the host immune system and facilitate infection. Although many effector genes are highly expressed during infection stages, there is limited information on the mechanisms regulating their in planta expression. Here, we characterize the promoter of MoHTR1, a nuclear effector gene of the rice blast fungal pathogen, to elucidate its in planta-specific expression. Using promoter deletion and mutation analyses, we identified a core cis-element (TATTTCGT) within the MoHTR1 promoter, designated the in planta active (IPA) element, which is crucial for in planta-specific expression. The IPA element is responsible for the expression of not only MoHTR1, but also other effector genes including a known effector Slp1. Furthermore, the IPA element enables the in planta expression of MobZIP14, a gene specifically expressed during vegetative growth. The IPA element plays a critical role in fungal virulence by enabling MoHTR1 expression and regulating host immune responses. Bioinformatic and DNA-protein interaction analyses revealed that RGS1, a transcription factor containing a winged-helix binding domain, acts as a transcriptional regulator of MoHTR1 by directly binding to the IPA element. Our findings provide new insights into the regulatory mechanisms driving the in planta-specific expression of fungal effector genes.

Nowadays, finding new sustainable ways to combat plastic pollution is a pressing challenge. Here, we provide a comprehensive genome mining analysis of 284 publicly available Stutzerimonas genomes for potential PET-active enzymes (PETases). While Stutzerimonas is a relatively newly established genus, it emerges as an interesting candidate in the search for novel biocatalysts. Hence, the first pangenome assessment of this genus based on its high-quality publicly available genomes was performed. An increasingly open pangenome was revealed, suggesting the versatility and adaptability of these strains to a variety of ecological niches. Moreover, functional characterisation of a new isolate, Stutzerimonas frequens VG-9, was carried out, confirming that enzymes found via in silico analyses may indeed display activity towards different polyesters. In summary, this study provides insights into the diversity of PETase homologues within still underexplored bacterial hosts, offering new perspectives for enzyme discovery in the Pseudomonadaceae family. Impact StatementMicrobial enzymes known as PETases have emerged as promising candidates for the biological degradation of PET. This study investigated the potential of underexplored bacterial genera by genome mining of PETase homologues. Our findings provide new insights into the distribution of PETase-like enzymes in the Pseudomonadaceae family, offering a more comprehensive view of their plastic degradation capacity. These results hold practical implications for the development of optimized enzyme discovery strategies, while also highlighting the vast genetic plasticity of Pseudomonadaceae. We also provided the first report on the Stutzerimonas pangenome and insights into the enzymatic activity towards polyesters of a newly isolated strain. Hence, the role of this genus as a highly adaptable and versatile entity was reinforced, further disclosing it as a potential source of novel biocatalysts. Data SummaryThe genome of S. frequens VG9 has been deposited in Genbank under the accession number SAMN49487720. The accession numbers of all analyzed genomes are listed in Tables S2 and S3 (available in the online Supplementary Material).

The environmental bacterium Pseudomonas protegens PBL3 has antagonistic activity against the bacterial pathogen Burkholderia glumae, the causal agent of the rice disease Bacterial Panicle Blight (BPB). The antimicrobial activity of the P. protegens PBL3 was found in the bacteria-free secreted fraction (secretome), but the specific molecules, as well as the genetic basis conferring activity, have not been identified. To elucidate the genes and biosynthetic pathways governing the antimicrobial activity of the P. protegens PBL3 secretome, we used comparative genomics, by leveraging six Pseudomonas spp. strains, with available genomic sequences and exhibiting contrasting antimicrobial activities against B. glumae. We hypothesized that Pseudomonas spp. strains with antimicrobial activity against B. glumae have conserved genes with P. protegens PBL3 and those genes are absent in the strains lacking activity. To test this hypothesis, we performed comparative genomics analysis across the six strains using two complementary approaches, anvio and progressiveMauve, and using P. protegens PBL3 as the reference genome. This analysis revealed 188 genes uniquely present in antimicrobial-producing strains. Seven of those genes were associated with biosynthetic gene clusters predicted to encode secondary metabolites; additional genes were grouped into twenty-five contiguous clusters with functions associated with secretion, signal transduction, regulation, transport/efflux, carbohydrate metabolism and one with additional uncharacterized function. Altogether, this study provided a complex and multi-functional network of candidate genes in antimicrobial-producing strains, suggesting that P. protegens PBL3 employs not only classical biosynthetic pathways but also integrated regulatory, metabolic, and export modules to synthesize and deploy antimicrobials. ImportanceBacterial plant diseases can be controlled through biological control, a strategy wherein beneficial microorganisms known as biological control agents (BCAs) interfere with the biological activities of pathogens through several mechanisms. One of these mechanisms is antibiosis, by which antimicrobial molecules produced by the BCA prevent pathogen multiplication or actively kill the pathogen. While this mechanism of antibiosis has been widely recognized, the specific molecules associated with the antimicrobial activity are not always identified, given their diverse and complex chemical structures as well as the unique and intricate biosynthetic pathways. This study unraveled different pathways underlying the antimicrobial activity in the environmental bacterium Pseudomonas protegens PBL3 to advance the discovery of effective antimicrobials to control bacterial plant diseases.

The role of microbial strains in regulating natural stresses and their impact on plant health is well-established. However, the role of microbial tolerance mechanisms in plant response to unnatural or anthropogenic stresses is less understood. Examination of these interactions impact our deeper understanding of plant-microbe interactions and our ability to enhance beneficial functions. In this study we use the model plant Brachypodium distachyon and its prominent root colonizer Paraburkholderia graminis OAS925 to investigate mechanisms of tolerance to alcohol and salt stress. We examined the ability of OAS925 to reduce root growth inhibition during exposure to short chain alcohols and salt. We also examined the tolerance mechanism for OAS925 towards these stresses using RB-TnSeq fitness assays. The most prominent tolerance systems in OAS925 are genes specifically involved in membrane transport (such as the Mla operon), efflux systems (e.g., RND efflux systems), signaling and regulation (PrtR/PrtI, NtrY/NtrX, and EnvZ/OmpR), and oxidative stress response (GshB). Our findings provide a model where bacterial membrane integrity, active solvent efflux, and stress signaling are crucial not only for bacterial survival but also for maintaining the root colonization and biofilm formation that confer protection to the host plant.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense race TR4 (Foc), is one of the most destructive diseases threatening global banana production, particularly the Cavendish cultivar. Conventional control strategies, including chemical treatments and quarantine, remain largely ineffective and unsustainable, underscoring the urgent need for alternative approaches. Biological control using rhizosphere-associated microorganisms offers a promising and environmentally friendly strategy. In this study, we isolated 436 bacterial strains from the rhizosphere of healthy banana plants and screened them for antifungal activity against Foc. Out of the screened isolates, 93 exhibited significant in-vitro inhibitions, and 64 of these were subsequently evaluated in greenhouse assays. We found that 22 strains reduced Fusarium wilt severity by 45-85% compared to untreated controls. Among them, two isolates, DDC20 and DDC_NEW2, consistently demonstrated strong biocontrol activity. In addition, cell-free culture media (CFCM) and crude extracts inhibited spore germination in fluorescence-based assays, indicating the involvement of secreted antifungal metabolites. Microscopy and confocal observations of GFP-tagged Foc revealed hyphal abnormalities in the presence of bacterial treatments, including swelling, irregular branching, and distortion, accompanied by excessive sporulation characterized by abundant microconidia, macroconidia, and chlamydospores. Whole-genome sequencing and comparative analyses placed both isolates within the genus Bacillus. Genome mining using antiSMASH identified multiple biosynthetic gene clusters encoding known antifungal compounds such as surfactin, fengycin, bacillibactin, and difficidin, as well as putative novel clusters. LC-MS confirmed the presence of surfactin and fengycin in bacterial extracts, supporting the genomic predictions. Collectively, these findings highlight the potential of DDC20 and DDC_NEW2 (related to Bacillus spp.) from the banana rhizosphere as effective biocontrol agents against Foc TR4. This integrated approach, combining phenotypic assays, microscopy, and genome mining, provides a strong foundation for the development of sustainable strategies to manage Fusarium wilt in banana cultivation.

Non-pathogenic Xanthomonas (NPX) from a diverse plant hosts are being reported on an increasing basis. There are also reports of multiple species forming communities on a single host plant, such as rice, and, given their role as core endophytes in protecting plants from pathogens, it is essential to isolate and characterization of more NPX species from diverse host plants. Using phylogenomic analysis of publicly available Xanthomonas genome sequences, we identified a novel clade comprising NPX strains from diverse hosts. One of the strains previously reported from our lab is from healthy rice seeds and was reported to be non-pathogenic, with bio-protection function against the bacterial leaf blight pathogen. Genomic investigation confirmed the lack of type III secretion system and its effectors, consistent with their non-pathogenic nature. These strains also harbour core and unique biosynthetic loci identified in other non-pathogenic Xanthomonas (NPX) strains. Further investigation using multiple genomic-based taxonomic indices indicates that these strains represent a potential new species. Hence, we propose Xanthomonas imtechensis sp. nov. as a new species of the genus Xanthomonas, with the type strain being PPL568 = MTCC 13186 = CFBP 9040 = ICMP 24395.

The genus Exiguobacterium comprises Gram-positive, non-spore-forming, facultative anaerobic bacteria known for their remarkable adaptability to extreme environments, including soils, hot springs, glaciers, and the gastrointestinal tracts of certain organisms. Despite their unique adaptations for surviving in extreme environments, their pathogenicity is well documented. Here, we analyzed the phenotypical traits of two Mexican strains of Exiguobacterium--JVH47, isolated from contaminated urban sediments in Mexico City, and P4526, from the less human-impacted Cuatro Cienegas Basin. Furthermore, strains were related via comparative genomics using publicly available genomes. Phenotypic characterization demonstrated that both strains thrive across a wide range of temperatures (20-50 {degrees}C), pH (7-11), and salinity (up to 7% NaCl). Although sensitive to erythromycin, the JVH47 strain exhibited higher erythromycin resistance and harbored antibiotic resistance genes. This study underscores the ecological versatility of Exiguobacterium and its potential role as a reservoir for antibiotic resistance genes. While rarely associated with human infections, its ability to survive in extreme conditions and form biofilms raises concerns for immunocompromised individuals. These findings highlight the need for careful consideration of Exiguobacterium in biotechnological applications and its implications under the One Health framework.

Chronic wounds are difficult to treat because underlying medical conditions can impair the mechanical and physiological first-line innate immune defenses, leading to persistent microbial infections. We report here the isolation, molecular and phenotypic characterization of seven E. coli strains that were isolated concomitantly with Enterococcus faecalis after an open foot fracture caused by the 2004 tsunami resulting in a 10-year chronic bone and joint infection. Initially present antimicrobial resistant E. coli ST405 and ST940 isolates were followed by host adapted isolates of ubiquitous ST131 clone presumably acquired from the environment already upon initial foot fracture. The E. coli ST131 clade C1 strains showed genomic alterations associated with virulence and persistence including large chromosomal inversions and, subsequently, a large deletion to cause small colony variants and higher susceptibility to formaldehyde and other stress provoking In this context deletion of hemB catalyzing an early step in the pathway for heme biosynthesis was the major, but presumably not the only cause of small colony variant emergence. Surprisingly, ST131 isolates did not display pronounced biofilm formation in conventional biofilm assays suggesting unconventional modes of persistence. In summary, the genomes of ST131 clone members are highly plastic which enables their persistence in novel ecological niches. In individuals with underlying metabolic diseases such as diabetes wound infection can prepare for colonization with ST131 E. coli isolates. FundingThis work was partially funded by ALF, the Petrus and Augusta Hedlunds Foundation and the Karolinska Institutet.

Filamentous fungi, such as Aspergillus species, use microtubule transport to move early endosomes. Other cargos, such as peroxisomes and mRNAs, "hitchhike" on early endosomes to move throughout the long hyphae of these organisms. In Aspergillus nidulans, peroxisomes hitchhike on early endosomes using the endosomal protein PxdA and the peroxisomal protein AcbdA. The HookA adaptor protein links endosomes to microtubule motors. Here, we set out to explore the physiological functions of peroxisome hitchhiking and endosome motility. A. nidulans has a complex life cycle that includes asexual and sexual reproduction. A. nidulans and other fungi within the Pezizomycotina subphylum are also notable for the vast number of secondary metabolites they produce. Light and other environmental conditions influence developmental decisions and secondary metabolite production. Here, we found that sexual reproduction is favored in the absence of endosome motility, even in the light, which normally promotes asexual reproduction. RNA sequencing of strains lacking endosome motility showed altered expression of genes involved in development. Unexpectedly, we observed altered expression of genes involved in secondary metabolism in strains lacking endosome motility and peroxisome hitchhiking. Using mass spectrometry, we found that the loss of endosome motility affected the biosynthesis of secondary metabolites, including sterigmatocystin, a carcinogenic mycotoxin that is a food contaminant. Finally, in a pathogenic species, Aspergillus fumigatus, we found that deletion of its PxdA homolog also significantly altered secondary metabolite production. Our work uncovers an unexpected link between organelle motility, developmental decisions in response to light, and secondary metabolite production in filamentous fungi.

To shed light into the biology of bacteria belonging to the genus Xylophilus, including the grapevine pathogen Xylophilus ampelinus, we scrutinized all available genomes of the genus for the presence of type III secretion and flagellar systems. We found three different flagellar systems in the genus, one of which was present in all twelve strains with good-quality genome sequences were available. The other two flagellar systems were only detected in one or two strains. We also identified two types of type III secretion systems, likely under control of the AraC-type transcriptional activator HrpX. One system with resemblance to systems from plant-pathogenic bacteria was only found in the grapevine pathogen. The other system was found in three strains of Xylophilus, all isolated from plant material. We predicted genes that are co-regulated with the type III secretion systems, as supported by the presence of strongly conserved HrpX-binding promoter elements. We identified about 40 type III effectors in the grapevine pathogen with homologs in plant pathogenic bacteria. In contrast, a rhododendron flower isolate had only two type III effector gene candidates with conserved HrpX-binding promoter elements but many genes without homologs beyond the species. Finally, we predicted and confirmed three novel effector candidates from X. ampelinus to contain a functional type III secretion signal using an AvrBs1 reporter approach. The presence of type III effectors suggests that effector-triggered immunity may exist in grapevine or non-host plants and that strategies targeting type III effectors for resistance engineering may contribute to suitable control measures.

N-glycosylation is an essential post-translational modification required for proper protein folding, stability, trafficking, and secretion in eukaryotes. In such organisms, an efficient endoplasmic reticulum (ER) quality control, such as the ER-associated degradation (ERAD) pathway, is critical for maintaining cellular homeostasis. During ERAD, terminally misfolded glycoproteins undergo N-deglycosylation prior to proteasomal degradation, a process typically mediated by peptide N-glycanase (PNGase). However, in the filamentous fungi, the PNGase seems to be catalytically inactive, indicating evolutionary divergence from the canonical PNGase pathway. Filamentous fungi also encode endo-{beta}-N-acetylglucosaminidases (ENGases), particularly members of glycoside hydrolase family 18 (GH18), which may compensate for the loss of canonical PNGase activity. Here, we investigated the roles of the cytosolic GH18 ENGase and a putative acidic PNGase in N. crassa using transcriptomic and functional approaches. Our results demonstrate that the cytosolic GH18 ENGase is an active deglycosylating enzyme likely associated with the ERAD pathway, whereas no deglycosylation activity was detected for the acidic PNGase. Deletion of the ENGase severely compromises tolerance to diverse stress conditions and induces substantial transcriptomic reprogramming, including upregulation of a GH20 exo-{beta}-N-acetylhexosaminidase under ER stress. These findings identify cytosolic ENGase as a key component of fungal proteostasis and suggest that N. crassa activates alternative compensatory mechanisms to maintain protein quality control when canonical deglycosylation pathways are impaired.

The Bacillus genus comprises physiologically versatile, endospore-forming bacteria widely distributed in natural environments. In this study, we report the isolation and genomic characterization of strain Bva_UNVM-123, recovered from agricultural soil in Pergamino, Argentina. Whole-genome sequencing using Illumina technology yielded a 5.1 Mbp draft genome assembled in 67 contigs with a GC content of 36%. Comparative genomic analyses using the TYGS server and digital DNADNA hybridization (dDDH) values supported its classification as a potentially novel species within the Bacillus sensu lato (s.l.) group. Genome annotation revealed 4,866 protein-coding genes, including multiple determinants conferring resistance to antibiotics (e.g., fosfomycin, tetracycline, beta-lactams) and toxic heavy metals (e.g., arsenic, cadmium, mercury), supporting its potential application in bioremediation. Additionally, PathogenFinder predicted a low probability of human pathogenicity (0.207), reinforcing its safety for environmental use. Functional classification based on Swiss-Prot further supported a metabolically versatile profile and revealed the presence of resistance-related categories associated with environmental adaptation. This study adds to the growing knowledge of environmental Bacillus species and their biotechnological potential