Environmental Microbiology Reports

Organosulfur compounds are the predominant sulfur source in terrestrial environments, requiring bacteria to use enzymes for their assimilation. Most described organosulfur-assimilating enzymes require oxygen, and enzymes that function under anoxic conditions remain poorly understood. Recently, methylthio-alkane reductase (Mar), a nitrogenase-like enzyme that reduces the volatile organic sulfur compounds (VOSCs) methylthio ethanol (MT-EtOH), dimethyl sulfide (DMS), and ethyl methyl sulfide (EMS) under anoxic conditions, was identified in the purple nonsulfur bacterium Rhodospirillum rubrum. However, another purple nonsulfur bacterium, Rhodopseudomonas palustris, has three loci of nitrogen fixation-like (NFL) genes with high sequence similarity to Mar, suggesting additional Mar-like enzymes with distinct roles. Here, we tested whether these NFL genes are required for VOSC assimilation in R. palustris. RNA-seq analysis revealed that all three NFL loci are upregulated under sulfur limitation, supporting a role in sulfur assimilation. Only disruption of the NFL genes encoded by RPA2634-37, renamed marBHDK1, caused fitness defects with EMS, DMS, and dimethylsulfoniopropionate (DMSP) as sulfur sources, indicating a functional Mar enzyme. The NFL genes RPA2347-48 and RPA2353-54, renamed marKD2 and marHB2, were required for activity with MT-EtOH or ethanethiol but not DMS, EMS, or DMSP. No activity was observed for the third locus, RPA2363-64, renamed nflDK. Overall, two Mar homologs in R. palustris are capable of VOSC reduction, one specialized for simple VOSCs and the other preferring a substrate with an additional functional group.

Myxococcus xanthus bacteria form aggregates when starved on solid surfaces and some cells differentiate into spores. Studies of mutants in monoculture have advanced knowledge of this multi-cellular developmental process, but our understanding of the genetic determinants is incomplete. To assess gene function genomewide, we generated a pool of barcoded transposon insertion mutants, subjected it to starvation, and separated developmental samples into non-aggregated cells, aggregated cells, and spores. We also subjected our pool to chemically-induced unicellular sporulation. Evaluation of changes in the abundance of mutants in samples allowed identification of 200 genes in which insertions reproducibly caused distinct patterns of depletion and/or accumulation over time. Many of these genes have well-established roles in development, validating our approach, while many others have not previously been associated with development. Genes involved in type IV pili (T4P)-dependent motility were more important than gliding motility genes for aggregation and sporulation in the mixed population. Although exopolysaccharide (EPS) synthesis genes are required for aggregation in monoculture, most were dispensable for aggregation in our pool, consistent with EPS sharing between cells, yet these genes were required cell-autonomously for efficient sporulation. Genes for positive regulators of EPS synthesis were important for aggregation as well as sporulation, suggesting functions beyond EPS production. Insertions in several novel genes impaired both starvation- and chemically-induced sporulation. Many genes increased the efficiency of starvation-induced sporulation. Some of these mutants, which we call "developmental winners", are novel cheaters. Our results demonstrate the power of using the newly-created mutant library to elucidate M. xanthus biology.

Intertidal mudflats are low hydrodynamic energy environments hosting microphytobenthic communities that experience strong spatiotemporal variability in light regimes, including changes in spectral quality and light intensity that can lead to cellular photooxidative stress. To cope with these fluctuations, autotrophs exhibit diverse and highly plastic adaptations that are often species-dependent and shaped by their ecological niches. This study investigates photophysiological responses and metabolic remodeling in a diatom assemblage originating from a natural winter microphytobenthic biofilm under contrasting red and blue light intensities. To this end, photosynthetic parameters were monitored alongside changes in lipophilic metabolites, including untargeted lipids and lipophilic pigments. While few metabolites showed temporal remodeling, rapid and contrasting changes were observed within 30 minutes in response to both spectral quality and light intensity. Red light treatments induced broader remodeling of lipophilic metabolites than blue light, whereas blue light appeared to have a greater impact on photosynthetic parameters. Moreover, red light induced xanthophyll-cycle responses comparable to those observed under blue light at equivalent incident intensity. We discuss these metabolic responses in relation to diatom photoadaptive strategies, placing these findings within the intertidal environmental framework. This work further underlines the importance of understanding rapid metabolic plasticity in coping with light fluctuations, providing new insights into the photoregulatory strategies of natural microphytobenthic communities.

Winogradsky columns are a classic model ecosystem for studying microbial biogeochemistry across steep gradients of oxygen and sulfide. They also remain widely used in microbiology education, introducing generations of students to microbial diversity. Yet, the genomic potential of their microbial communities remains uncharacterized. Here, we applied shotgun metagenomic sequencing to a Winogradsky column community at multiple depths, yielding 20 metagenome-assembled genomes (MAGs) representing diverse, largely uncultivated taxa. Genome-resolved analyses revealed metabolically diverse oxygenic and anoxygenic phototrophs that could potentially contribute to carbon and nitrogen fixation across all layers of the column. Most of these phototrophs also encoded one or more pathways for sulfur oxidation, which we speculated may support both energy conservation and/or sulfide detoxification by these populations. Complex carbon degradation capacity was also widespread across the MAGs, suggestive of the potential for the transformation of the column's amended organic matter (shredded coffee filters) into smaller depolymerization products and, through fermentation, organic acids. Together, these findings reveal how distinct microbial guilds might partition interconnected carbon, sulfur, and nitrogen transformations within redox-stratified systems.

Enterococcus faecalis is an opportunistic pathogen and facultative anaerobe that primarily relies on fermentative metabolism to colonize a wide range of aerobic and anaerobic environments. In the presence of exogenous heme, E. faecalis can assemble a minimal electron transport chain consisting of membrane-associated primary dehydrogenases, demethylmenaquinone, and the terminal cytochrome bd oxidase (CydAB). This respiratory chain is thought to generate a proton motive force to drive ATP synthesis via the F-type ATP synthase, thereby improving energy conservation under aerobic conditions. However, a cytosolic NADH oxidase (Nox) also consumes NADH and oxygen, potentially competing with the electron transport chain for reducing equivalents and terminal electron acceptors; but the relative physiological contributions of these two oxygen-reducing pathways remain poorly understood. To define the roles of CydAB and Nox under normoxic and hypoxic conditions, we constructed {Delta}cydAB and {Delta}nox mutants. Real-time, in situ measurements revealed {Delta}cydAB had no significant effect on oxygen utilization while in the {Delta}nox it was significantly reduced; revealing Nox as the major consumer of oxygen. Semi-untargeted metabolomic analysis further revealed oxidase-specific alterations in central metabolism with the {Delta}nox causing pronounced shifts in the ATP and NADH ratios; highlighting Nox as a key determinant of intracellular redox and energy homeostasis. Finally, single-cell fluorescence microscopy showed that membrane potential, a component of proton motive force, was substantially diminished only in the absence of both CydAB and Nox, or the F-type ATP synthase. These findings indicate that the F-type ATP synthase is a major generator of proton motive force, even upon aerobic growth, and demonstrate a complementary role for the electron transport chain and Nox in the bioenergetics of E. faecalis.

Diatoms are major contributors to marine primary production, yet current approaches for monitoring their abundance and function rely on coarse satellite chlorophyll estimates or sparse cell count and carbon fixation measurements. Molecular markers are a promising approach for high-resolution measurement of both abundance and metabolic activity through analysis of environmental DNA (eDNA) and RNA (eRNA). We present an isothermal quantitative recombinase polymerase amplification (qRPA) assay targeting rbcL gene copies and transcripts of marine diatoms, operating at low temperature and producing results in less than 15 min. We demonstrate specificity and calibration across diverse diatom taxa, then apply the assay to eDNA and eRNA samples from the Mare Chiara Long-Term Ecological Research site in the Bay of Naples, Italy, alongside microscopy, chlorophyll, physicochemical, and carbon-fixation data. Diatom rbcL DNA tracked abundance across five orders of magnitude despite seasonal shifts in community composition. Combining molecular and optical data revealed increased cellular rbcL copies and chlorophyll in low-light winter populations, suggesting enhanced photosynthetic capacity despite lower abundance. Furthermore, rbcL RNA reflected total carbon fixation rates and identified populations with differing carbon fixation activity. These results support rapid, RPA-based rbcL quantification as a robust approach for biomolecular ocean observing.

Candida auris (currently Candidozyma auris) is an emerging fungal pathogen responsible for dramatic global increase in invasive candidiasis with high mortality. Most concerning, C. auris has a high propensity to colonize patients and persist and develop multidrug resistance to main classes of antifungals. In this study, we investigated the genetic and phenotypic diversity and resistance mechanisms of C. auris clinical isolates recovered from hospitalized infected patients. A total of 53 isolates from 38 unique patients were recovered from various clinical sources and evaluated for susceptibility to routine antifungal drugs. Whole genome sequencing (WGS) and single nucleotide polymorphism (SNP) analysis were performed to generate a phylogenetic network to infer population structure and identify mutations associated with drug resistance development. Isolates were also phenotypically evaluated for ability to form biofilms and aggregate, and cell wall adhesins gene expression studies were performed to provide mechanistic insights into C. auris phenotypic plasticity. Except for one clade III isolate, all isolates belonged to clade I and all were resistant to fluconazole with incidence of resistance to amphotericin B, echinocandins or both. Non-synonymous SNPs were found in genes associated with antifungal resistance including ERG11, TAC1B, CDR1 and FKS1. Phenotypically, isolates varied in their ability to form biofilm and aggregate which correlated with expression of the Scf1 and Als4112 cell wall adhesins genes highlighting C. auris phenotypic plasticity in circulating clinical strains. These findings underscore the growing clinical threat posed by C. auris and reinforce the need for optimized surveillance and treatment strategies for controlling its spread.

The Bacillus cereus group comprises bacteria of biotechnological interest, but also raises health concerns. Some bacteria in this group are opportunistic human pathogens, mainly causing foodborne gastrointestinal infections. As of today, the presence, sequence variability, or expression of genes encoding toxins or other virulence factors are insufficient to predict the potential of a given isolate to cause the diarrheal form of the disease. To address this limitation, we developed a sandwich ELISA to quantify the NheA and Sphingomyelinase (SMase) proteins in culture supernatants to test them as markers of pathogenic potential. Application of the assay to a collection of B. cereus group isolates revealed that strains associated with food poisoning outbreaks produce significantly more NheA and SMase than those isolated from the environment or from commercial products. Statistical analyses show that the combined quantification of NheA and SMase provides robust discrimination between pathogenic and non-pathogenic (environmental and commercial) profiles. These results demonstrate that the quantitative assessment of both NheA and SMase production can serve as a reliable biomarker for distinguishing diarrheic food poisoning isolates from harmless strains.

Protists play important roles in food chains and symbioses in soil and aquatic environments, displaying an enormous morphological and functional diversity. While most commonly found protist species are well known to science, our global-scale environmental DNA survey across soil, water, and sediments reveals dozens of novel, phylum-level phylogenetic lineages that remain to be characterized for basic morphology and function. A vast majority of these undescribed taxa occur in marine water and sediments, but some are common in soil. Most of these novel taxa have distinct substrate and habitat preferences and biogeographic patterns. To accord these lineages scientific agency and enable unambiguous scientific communication, we propose formal names for 150 species to phylum-level taxa from 25 deep lineages based on eDNA and rRNA gene long-read sequence information.

Cutaneotrichosporon dermatis (formerly Trichosporon dermatis) is a basidiomycetous yeast-like fungus known to cause summer-type hypersensitivity pneumonitis, although its virulence in humans remains poorly understood. We performed morphological and molecular identification of an isolate from the sputum and blood cultures of an immunocompromised patient, together with pathogenicity assessment using a Galleria mellonella model, biofilm formation/eradication assays, antifungal susceptibility testing, drug combination effects, and the post-antifungal effect (PAFE), compared with Trichosporon asahii. The isolate was identified as C. dermatis by ITS/IGS1 sequencing, supported by phylogenetic analysis. Growth of C. dermatis increased more at 37 than at 25. In the Galleria mellonella assay, C. dermatis, T. asahii, and Candida albicans each showed dose-dependent pathogenicity at sufficiently high inocula, although Rhizopus oryzae was the most potent pathogen on a per-CFU basis. C. dermatis formed biofilms that were more completely inhibited by terbinafine (TRB) and amphotericin B (AmB) than azole agents, which showed only partial inhibitory activity even at high concentrations. Susceptibility testing showed relatively strong susceptibility to AmB and azole agents. In the TRB and azole combination assay, the fractional inhibitory concentration index (FICI) was below 0.5, indicating synergy. Isavuconazole (ISC) showed a markedly stronger PAFE than the other azole agents tested. These findings indicate that although azoles show only partial efficacy against its biofilm, C. dermatis can still cause invasive infection, and that azole monotherapy or TRB and azole combination therapy, aided by the potent PAFE of ISC, may represent effective treatment options.

Marine diatoms contribute to about 20% of global primary production. We present the first basin-scale, multiyear assessment of diatom communities in the North Pacific, combining taxonomically high-resolution RuBisCO large subunit gene (rbcL) metabarcoding with concurrent environmental measurements. Using a nine-year time series of daily samples resolved at the species level via ~500 bp rbcL fragments, we performed multivariate analyses across biogeographic provinces, identifying significant correlations between community structure and environmental drivers such as temperature and macronutrient availability. We report the prevalence of a previously overlooked centric diatom species in the North Pacific, Eunotogramma lunatum, which appears to be near-dominant even in subarctic high-nitrate, low-chlorophyll waters where pennate diatoms are typically favored. These results demonstrate the power of rbcL for large-scale ocean monitoring and provide a critical baseline for future studies of diatom population dynamics, climate change impacts, and ecosystem resilience in a key marine region.

Rinsing-dependent dental hygiene presents a significant public health challenge in water-scarce environments. This study investigated combinations of xylitol (Xyl), chitosan (Chi), glycyrrhizin (Gly), epigallocatechin gallate (EGCG), dicalcium phosphate (DCP), and nano-hydroxyapatite (nHA) on the primary bacteria behind dental caries, S. mutans. These combinations were assessed for markers of dental caries by biofilm reduction, bacterial killing, and acid buffering against S. mutans when applied to an in vitro simulated enamel model using glass bead surfaces for biofilm formation, and gene expression was subsequently examined via RT-qPCR. Separately, mineral retention was also quantified. The EGCG-DCP-Xyl film demonstrated the highest overall efficacy, achieving a significant reduction in biofilm concentration compared to the untreated control and performing similarly in magnitude to the positive toothpaste control. Dead fluorescence staining confirmed that the EGCG-DCP-Xyl film induced the highest rate of non-viable cells, followed by the Chi-Gly film and the Gly-Xyl film. During 10-day pH cycling, the EGCG-DCP-Xyl and DCP-Xyl formulations buffered pH the most, consistently maintaining mean pH levels safely above the demineralization threshold of pH 5.5. The EGCG-DCP-Xyl also optimized mineral stability with the highest retained calcium concentration, significantly outperforming the Chi-Xyl film. At the transcript level, the EGCG-DCP-Xyl film induced substantial downregulation of key virulence genes, yielding decreases in expression for glucosyltransferase B (gtfB), associated with biofilm synthesis, collagen-binding protein (cnm), associated with tissue invasion, and lactate dehydrogenase (ldh), associated with lactic acid production, compared to the untreated control, with effects comparable in magnitude to the positive toothpaste control. This research suggests that targeting bacterial pathways and mineral loss through a portable film may have potential for preventing dental caries, especially in environments where water is limited. However, additional studies are necessary to evaluate real-world effectiveness.

Microbial and biochemical properties of soil respond quickly with management practices, than chemical and physical properties. Moreover, impact of conservation agriculture (CA) on soil microbial properties is limited to microbial enumeration, but its effect on soil enzyme and microbial activity is little documented. To address these problems soil enzyme activities [dehydrogenase (DHA), {beta}-glucosidase (BGA), acid phosphatase (AcP) and alkaline phosphatase (AlP) and fluoresceine diacetate (FDA)], microbial activites ((Nitrogen fixation (NFBAct), Phosphate solubilization (PSBAct) & Cellulolytic activities (CDBAct)), microbial biomass ((Soil microbial biomass carbon (SMBC) & soil microbial biomass nitrogen (SMBN)) and available nutrient were studied to evaluate biological soil health in alluvial soil of lower Indo-Gangetic plain (IGP) under CA. Field experiment was conducted in split plot design (SPD), under 3 cropping systems (RMCp: rice-maize-cowpea; RWGg: rice-wheat- green gram; RCfBr; rice-cauliflower- bororice/summer rice). Tillage operations (CT: conventional; MT: minimum and ZT: zero tillage) was main plot and residue application as sub plot treatments [(R0 (no residue), R50 (50% residue) and R100 (100% residue)], treatments were replicated thrice. Biological soil health index (BSHI) indicated that among different degree of CA, ZT (0.464) and (MT=0.441) and R100 (0.464) treatment showed better response. Among different cropping system RMCp (0.359) & RWGg (0.343) outperformed RCfBr (0.609) cropping system with respect to (wrt) microbial and biochemical properties of the soil. Results indicated that for restoring microbial and biochemical properties of soil CA can be used as sustainable practice to restore agro-ecosystem. Keywords: Conservation agriculture, Cropping systems, Soil enzyme, Soil microbial properties, Residue application, Tillage operations.

Brettanomyces bruxellensis is a yeast species associated with diverse fermentation environments and characterized by extensive genetic diversity, including diploid, autotriploid, and allotriploid lineages resulting from independent hybridization events. These lineages are associated with distinct ecological niches and provide a framework for studying metabolic trait evolution in complex genomes. Nitrate assimilation is a relatively uncommon trait among yeasts and has been reported in B. bruxellensis, but its distribution and evolutionary history within the species remain poorly understood. Here, we combined phenotypic characterization of 151 strains with genomic analyses of 946 whole-genome sequences to investigate nitrate assimilation. Growth assays revealed that nitrate assimilation is widespread but unevenly distributed across genetic lineages, with some populations largely retaining the trait whereas others have frequently lost it. Genomic analyses identified extensive variation affecting the nitrate assimilation gene cluster composed of YNR1, YNI1, and YNT1. Nitrate assimilation was strongly associated with both gene copy number and predicted gene functionality, with nitrate-assimilating strains generally carrying more functional copies of the cluster. Leveraging the complex genomic architecture of the species, we independently analyzed primary and acquired genomes in allotriploid lineages and uncovered contrasting evolutionary trajectories following hybridization. While nitrate assimilation genes were generally maintained in primary genomes, acquired genomes showed a higher prevalence of gene loss and predicted loss-of-function variants, revealing asymmetric dynamics between subgenomes. Altogether, our results suggest that nitrate assimilation represents an ancestral trait that has been differentially maintained across B. bruxellensis lineages through a combination of copy number variation, gene degeneration, and genome-specific evolutionary dynamics. These findings provide new insights into how genome architecture and polyploid evolution shape the maintenance and loss of metabolic traits in an industrially relevant yeast species.

Heritable bacterial symbionts are pervasive in terrestrial arthropods, often imposing reproductive manipulations to promote their own spread within host populations. Co-infections are common, potentially allowing symbiont co-infectors to hitchhike through a host population. However, adverse thermal conditions can disrupt these communities, particularly when co-infectors vary in their thermal sensitivity. We used a multi-generation experiment to test whether warm (29 {degrees}C) conditions disrupted spread of heritable symbionts through uninfected populations of the spider, Mermessus fradeorum. We tested two common infection combinations: a single infection with a cytoplasmic incompatibility (CI) inducing Rickettsiella or a feminizing co-infection that included a feminizing Wolbachia, the same Rickettsiella, and up to three apparent hitchhikers (two additional Wolbachia strains and Tisiphia). We initiated replicate populations with 1/3 of one infection type and 2/3 uninfected spiders, evaluating population infection rate over 5 spider generations under different temperature regimes. Under cool (21{degrees}C) conditions, Wolbachia feminization drove co-infection to 88% and Rickettsiella CI drove single infection to 83% of host populations. Vertical transmission for all symbionts was high (97-99%) and hitchhiking symbionts also spread effectively. Under warm conditions, feminization and CI efficacy were reduced, and symbionts suffered variably reduced vertical transmission. Warm conditions ultimately destroyed the co-infecting symbiont consortium and impeded symbiont spread. On its own, though, Rickettsiella was still able to increase, despite reduced strength of CI. We hypothesize that contrasting tensions between feminizing spread of the symbiont consortium versus environmentally driven loss of function and transmission may explain observed patterns of mixed infections in field populations of this spider.

Antibiotic-induced disruption of plant-associated microbiomes has the potential to alter host health beyond the directly exposed generation, yet whether the effects of dysbiosis are transmitted through the seed microbiome remains unknown. Here, we investigated the intergenerational impacts of streptomycin-induced dysbiosis in tomato (Solanum lycopersicum), demonstrated that seed microbiome transfer (SMT) restores progeny microbiome function and disease resistance, and characterized the underlying physiological and genetic mechanisms. Parental streptomycin exposure altered the composition of progeny rhizosphere bacterial communities, reduced expression of defense-associated genes, and increased susceptibility to Xanthomonas perforans. Suppression of immune gene expression was strongly associated with increased disease severity, indicating that parental dysbiosis impaired progeny plants ability to mount effective immune responses. Transfer of the seed microbiome from healthy plant donors partially restored rhizosphere community composition, reduced disease severity and recovered defense gene expression of three genes. Together, our findings demonstrated that antibiotic exposure microbiome disturbance generates intergenerational legacy effects that influence plant immunity and disease susceptibility and seed microbiome transfer can counteract this dysbiosis across generations.

The continued emergence of Neisseria gonorrhoeae (Ng) isolates resistant to front-line antibiotics has focused efforts on understanding how alternative therapies, such as the expanded use of gentamicin (Gen), might counteract this global public health problem. Focusing on Gen as a viable alternative antibiotic for the treatment of gonorrheal infections, we previously used RNA-seq to determine if sub-lethal levels of Gen might impact gonococci on a transcriptional level and showed that expression of the putative HicA-HicB toxin-antitoxin (TA) system was increased in response to sub-lethal Gen. Importantly, loss of this TA system resulted in reduction of Ng biofilm formation in a strain specific manner. Focusing on this strain specificity, we found that the CpxR/CpxA two-component system (TCS) influences expression of the hicAB operon independently of HicB autoregulation. We now report that CpxR selectively binds to the hicAB operon to enhance expression of hicAB but does not interfere with binding of HicB to the promoter region. Furthermore, we show that single base pair differences in the intergenic region between hicA and hicB impact regulation by CpxR. Hence, the regulation of the HicAB TA in gonococcal strains is a highly coordinated response that can involve autoregulation by HicB and the CpxRA TCS. We propose that this dual regulatory scheme maximizes the ability of Ng to respond to Gen and hostile environmental conditions.

Edible mushrooms have been reported to have antimicrobial properties and other health benefits. This study aims to test the antimicrobial activities of several edible mushrooms from markets and test if co-culturing them with bacteria could induce stronger anti-bacterial properties. Commercial mushrooms, Hericium erinaceus (lions mane), Pleurotus ostreatus (oyster mushroom), Lentinula edodes (Shiitake) and Agaricus bisporus (button mushroom), were grown from strictly controlled/sterile substrates. Ethanol and water extracts from the mushrooms were prepared and tested against the bacteria Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis, and the fungus Candida albicans for antimicrobial activities. Shiitake water extract (SWE) showed strong antibacterial effects against all tested bacterial species, inhibitory effects on their biofilms, and antifungal activity. The antimicrobials in SWE seem to damage the cell wall and cell membrane of the bacteria, prefer weak acidic conditions, and are heat labile. Some antimicrobials are likely proteins and polysaccharides. In contrast, 3 other mushrooms displayed only weak antimicrobial effects. The fast-growing lions mane and oyster mushroom were co-cultured with different bacteria. The co-cultivation promoted the fruiting body development of lions mane. Co-culturing with S. aureus increased the anti-bacterial effects of lions mane against S. aureus, E. coli and particularly B. subtilis. Co-culturing the oyster mushroom with bacteria, especially B. subtilis and P. aeruginosa, boosted the mushroom growth. All tested bacteria, especially S. aureus, increased oyster mushroom anti-bacterial effect against E. coli and B. subtilis. The findings indicate that mushroom-bacteria co-culturing could have benefits both agriculturally and medicinally.

Water availability is critical for plants and their microbial communities, including pathogens. The plant pathogen Phytophthora cinnamomi persists in soils with fluctuating moisture, yet cellular responses to water limitation remain poorly understood in Phytophthora and oomycetes more broadly. Although we recently characterized the proteomic response of P. cinnamomi to NaCl-induced osmotic and ionic stress, its response to PEG-mediated water limitation remains poorly understood, leaving a critical gap in our understanding of drought-relevant stress adaptation. Here, we quantified mycelial growth and profiled time-resolved proteome dynamics of P. cinnamomi during polyethylene glycol (PEG-3350)-treatment, simulating moderate water limiting conditions. Treatment with 5% PEG-3350 enhanced radial mycelial growth relative to controls, with no early growth inhibition observed. Label-free proteomics identified 1,097 protein groups, with 880 proteins shared between conditions and an asymmetric abundance profile dominated by decreasing protein abundance over time. Only a small subset of proteins increased, mainly enzymes involved in redox buffering (e.g., thioredoxin and glutaredoxin-like proteins) and mitochondrial/metabolic regulation (e.g., alternative oxidase) and mitochondrial/metabolic regulation. Hierarchical clustering revealed a potential three-phase temporal program: early translational and regulatory remodeling (1-6 HPT), sustained metabolic adjustment (6-12 HPT), and delayed engagement of redox and proteostasis functions (12-24 HPT). Network analysis demonstrated that redox-associated function was integrated throughout this adaptation, with individual clusters further specialized by cofactor preference (NADP- versus NAD-dependent enzymes) and distinct metabolic roles (malate dehydrogenase, CoA-ligase activity). This coordinated, multi-phase reorganization sustained mycelial growth despite moderate osmotic stress, indicating that P. cinnamomi employs active proteomic adaptation rather than passive stress tolerance. These findings reveal the cellular mechanisms underlying drought persistence in this invasive pathogen and suggest molecular targets for disease management under water-limited conditions.

Sicyoidochytrium minutum DNA virus (SmDNAV) was isolated several years ago from a protist host of family Thraustochytriaceae of the class Labyrinthulomycetes. This virus shared little similarity to other viruses in gene content and protein sequences, albeit seemingly belonging to the phylum Nucleocytoviricota. By extensive searches in genomic and metagenomic sequence databases, we identified numerous long contigs related to the SmDNAV genome and analyzed proteins shared by these putative viruses. Phylogenetic analyses place these viruses within the class Megaviricetes, outside of all established orders, and as a sister group to the clade combining families Mamonoviridae and Manesviridae. Homologs of SmDNAV proteins were found in association (either integrated or co-sequenced) with other Labyrinthulomycetes and Rhodophyta protists from diverse marine and freshwater environments. Consequently, we propose SmDNAV as the prototype member of a new order, provisionally named Ariadnavirales, within class Megaviricetes, phylum Nucleocytoviricota. Members of Ariadnavirales have lost most of the genes encoding components of the replication and transcription systems that are otherwise conserved in nucleocytoviricots, suggestive of transition to genome replication and expression dependent on the host nucleus.