During wood degradation, fungi have to deal with toxic and stressful compounds called wood extractives. The identification of the various detoxification strategies developed by fungi, and the molecular targets of these compounds is limited in Basidiomycetes because of the lack of genetic tools. To circumvent this problem, we have developed a direct genetic strategy in the white-rot fungus Phanerochaete chrysosporium. A library of P. chrysosporium UV mutants was generated and screened to isolate mutants resistant to Bagassa guianensis wood extractives (BWE). Wood extractives contribute to B. guianensis wood durability, and resistance to those extractives confers to the mutants a better ability to mineralise wood sawdust. This resistance phenotype is due to causal mutation(s) in the gene coding for an ortholog of the human DENND6, a protein involved in endocytic recycling. Using in silico and invivo assays, we identified that moracin N, found in BWE, has antifungal activity likely by binding onto the wild type PcDENND6 protein but not onto the mutated variant.

Sea ice is a crucial, yet declining, habitat in high latitude ecosystems. Here we present a high-temporal resolution amplicon sequence data set collected during the spring ice-algal bloom near Utqiaġvik, Alaska in 2021 to study sea-ice microbial dynamics. The ice-algal bloom peaked on May 8th, reaching 46.6 mg chlorophyll a m-2 and thereafter became limited by nitrate availability. A massive bloom of the oil-degrading bacterium, Oleispira (> 80% relative abundance), coincided with the algal bloom raising questions about hydrocarbon exposure. The sea-ice algal bloom was dominated by diatoms, particularly, Nitzschia spp. and transitioned into a flagellate-dominated postbloom community which aligned with melt-associated changes to the physicochemical environment. We explored the relationship between putative parasitoids, Chytridiomycetes, Thecofilosea (Cercozoa), Oomycetes, Syndiniales (Dinoflagellata) and Labyrinthulomycetes (Bigyra) and potential microalgal hosts. Chytrids peaked periodically suggesting synchronised infections and Cryothecomonas (Thecofilosea) was observed parasitizing Nitzschia spp. for the first time in Arctic sea ice. Co-occurrence analysis suggested that diatoms, especially Nitzschia, were the primary hosts of Pacific-Arctic parasitoids and that top-down parasitoid control may dramatically alter community composition over short timescales, such as days. These results provide important insights into the drivers of spring bloom timing and maintenance of microalgal diversity in sea ice.

ABSTRACTGetting insights into the quantitative and qualitative contribution of different DNA states, i.e., extracellular (exDNA) and intracellular DNA (iDNA), to the total environmental DNA (eDNA) pool requires reliable methods for their separation. Even though a multitude of respective extraction protocols has been published, their validation is often missing. Here, we selected four protocols for the state‐specific extraction of eDNA and traced the separation of exDNA and iDNA within natural environments using previously designed spike‐and‐recovery controls. Besides accounting for the different eDNA states, the spike‐ins also distinguished different bacterial origins (gram‐positive, gram‐negative). Following their quantification by digital PCR, the recovery of exDNA and iDNA spike‐ins in both the target as well as nontarget eDNA states differed among the selected extraction protocols and environmental matrices, albeit the effect of the former was far more decisive. While the recovery of exDNA spike‐ins was mainly affected by the chemical composition of the washing buffer and the duration of each washing step, the lysis method determined the recovery of spiked iDNA. These aspects were further combined within an optimised protocol, providing a valuable step towards a more concise understanding of factors governing the state‐specific extraction of eDNA and hence their relevance in molecular microbial ecology.

ABSTRACTIn cold seeps, anaerobic methanotrophic archaea (ANME) and sulphate‐reducing bacteria (SRB) oxidise methane to inorganic carbon (IC) coupled to sulphate reduction. While catabolic pathways are well resolved, carbon flow into biomass as well as the functional roles of lipid biomarkers remain unclear. We conducted lipid stable isotope probing (lipid‐SIP) experiments with Astoria Canyon sediments dominated by ANME‐2/SRB consortia and incubated samples with either 13C‐labelled methane (13CH4) or dissolved IC (DI13C). Lipid‐specific δ13C analysis showed higher 13C incorporation from DI13C than from 13CH4. After 30 days, δ13C values were up to +417‰ in SRB‐specific fatty acids (e.g., C16:1ω5c, cyC17:0ω5,6) and +126‰ in ANME‐2‐specific isoprenoid lipids (e.g., archaeol, crocetane). Based on these values, we calculated carbon assimilation rates and found that both partners primarily assimilate IC. Remarkably, IC assimilation in SRB lipids was eight times higher than in ANME lipids, suggesting that ANME may use additional yet‐to‐be‐identified carbon sources, potentially produced by their partner SRB. By examining the stepwise 13C‐enrichment of ANME‐ and SRB‐derived lipids, we further delineate biosynthetic pathways for archaeal and bacterial diether lipid formation and highlight crocetane as a bilayer‐modulating isoprenoid hydrocarbon potentially affecting membrane fluidity and proton permeability.

ABSTRACTArctic warming is leading to permafrost thawing which modifies, in cascade, hydrosystems at diverse levels. This study aimed to compare prokaryotic community structure and methane (CH4) dynamics across 16 sub‐Arctic waterbodies, and to assess how these features are shaped by permafrost thaw. The sampled waterbodies, located in an ice‐rich discontinuous permafrost region (Southwestern Yukon, Canada) differed in size, depth, stratification and degree of thaw influence. Prokaryotic communities were characterised through 16S rRNA gene sequencing and qPCR targeting mcrA (methanogenesis) and pmoA (methanotrophy) genes. Community structures differed significantly between shallow stratified, deep stratified and non‐stratified waterbodies. Methanogens, predominantly represented by the Methanobacterium genus, were mostly detected in shallow non‐stratified waterbodies. Methanotrophs, primarily represented by the Methylacidiphilaceae family, were more abundant in oxic layers whereas bacteria of Crenothrix and Methylobacter genera dominated in anoxic conditions. Our results showed that non‐stratified waterbodies directly affected by permafrost thaw harboured distinct prokaryotic communities, including specific methanogens and methanotrophs. The two sites with the highest CH4 emissions were affected by permafrost thaw, with fluxes reaching up to 1.7 × 10−1 mg m−2 s−1. Future investigations should address gaps in CH4‐related processes in thaw‐affected systems, as they are hotspots for methane emissions and harbour different microbial communities.

ABSTRACTThe dairy starter Lactococcus lactis shifts its metabolism from mixed‐acid fermentation to homolactic fermentation under anaerobic conditions as growth rates increase. Although its metabolism at acidic and neutral pH values is well‐researched, knowledge about lactococcal physiology under alkaline conditions remains limited. Here, we investigated how L. lactis subsp. lactis biovar diacetylactis FM03 adapts its metabolism and morphology at alkaline pH using lactose‐limited chemostat cultures at pH 6, 7 and 8. At alkaline pH, L. lactis FM03 shifted from energetically more favourable mixed‐acid fermentation towards homolactic fermentation at lower growth rates compared to pH 6, resulting in a 20% lower biomass yield despite an unchanged maintenance coefficient and maximum biomass yield per ATP. Proteome analysis revealed a 1.5 to 13.5‐fold downregulation of enzymes in the mixed‐acid fermentation pathway at alkaline pH, thereby reducing its metabolic capacity. Morphologically, L. lactis became more spherical at alkaline pH, reducing the surface‐to‐volume ratio and did not enlarge upon higher dilution rates. This morphological shift potentially limits substrate uptake, contributing to the lower maximum growth rate at pH 8. Our findings reveal new insights into pH‐driven metabolic plasticity and resource allocation in L. lactis and highlight opportunities for optimising fermentation processes under varying pH conditions.

ABSTRACTHypersaline microbial mats at Guerrero Negro harbor a stratified, highly diverse community with diel metabolic changes. While oxygenic photosynthesis and sulfate reduction are the dominant bacterial metabolic processes, methylotrophic methanogenesis is the main archaeal pathway. Although these metabolic processes have been biochemically characterized, the identity and encoded metabolism of the microorganisms have been inferred only from gene‐marker data. Here, a genome‐resolved approach in both environmental, as well as experimental dark condition samples (control, H2/CO2, TMA, and H2/CO2‐TMA) was used to stimulate less‐known anaerobic strategies, determine the metabolic potential of the main microbial players, and analyze the community. Representative metagenome‐assembled genomes (170 MAGs) were obtained, encompassing 25 bacterial and 4 archaeal phyla. The metabolic analyses of three basic elements (carbon, sulfur, nitrogen) encoded in the MAGs suggested that in environmental samples, phototrophic taxa were the main source of the organic matter that fueled most of the community. Different sulfur species acting as electron acceptors led to the metabolism of partially degraded organic matter in the lower layers of the mat. These results link and clarify the biochemical processes and microbial players, adding a novel genomic component for the ecological understanding of the microbial mats of Guerrero Negro.

Heterotrophic nanoflagellates (HNF) are a key component of the microbial food webs, playing an essential role in nutrient recycling and energy transfer in aquatic ecosystems. They have been typically considered to be bacterivores, but they can also be omnivorous (feeding on prokaryotes and other eukaryotes) and predatory grazers (feeding on other eukaryotes). Here, we combine CARD-FISH with both short and long-amplicon sequencing to resolve the dynamics of key HNF groups during two high-frequency sampling campaigns in spring (March-May) and autumn (September-November) phytoplankton blooms in the coastal waters of the Baltic Sea. This approach allowed us to resolve the microbial food web dynamics within HNF communities at the phylotype level at time scales relevant to HNF duplication times. Omnivorous katablepharids and predatory MAST-2 dominated the HNF community, especially in spring. Bacterivorous groups (e.g., MAST-1, CRY1) were less abundant. Long-read sequencing revealed distinct seasonal shifts in dominant phylotypes, with Katablepharis sp. and MAST-2D peaking in spring, while other lineages became more prominent in summer and autumn. The high abundance of omnivorous HNF, compared to bacterivores, highlights their key role both as grazers of bacteria and flagellates, and as a food source for predatory and omnivorous ciliates.

Viruses play essential roles in shaping the structure and function of microbial communities, including those inhabiting extreme environments. The Qaidam Basin is a unique Mars-analog desert characterised by hyperaridity, oligotrophy and high soil salinity. We hypothesise that viruses contribute to microbial adaptability and biogeochemical cycling through virus-host interactions in desert ecosystems. Here, we investigated viral diversity, biogeography, life strategies and interactions with soil microbiomes across the Qaidam Basin. Soil properties, such as water content, pH and mineral assemblages, significantly influenced the distribution patterns of both viral and prokaryotic communities. Broad host range may confer fitness advantages for viruses in deserts where host biomass is limited. Most viruses were characterised as lytic and infected dominant microbial phyla, supporting the "Kill-The-Winner" model, which suggests viral regulation in microbial community diversity and stability. We identified over 32,000 potential viral auxiliary metabolic genes (AMGs), including key genes involved in carbohydrate metabolism, carbon fixation, photosynthesis and phosphorus cycling. Moreover, AMGs related to the biosynthesis of antibiotics, pigments and alkaloids may enhance the adaptability of hosts under extreme conditions. This study unveils the enigmatic virosphere of the Mars-analog Qaidam Basin and underscores the roles of viruses in promoting microbial adaptability and driving biogeochemical cycling.

Endosymbionts play pivotal roles in driving ecological and evolutionary diversification of many insects, yet the morphogenesis and evolutionary origin of their specialised symbiotic organs (e.g., bacteriomes) remain poorly understood. Here we investigated the bacteriome morphogenesis in Cicadidae using microscopy-based methods. We revealed that bacteriomes originate either from both the original bacteriocytes that emerged after anatrepsis and the novel bacteriocytes that appeared during katatrepsis, or solely from the latter. Bacteriomes expand via "budding" proliferation to increase the bacteriome unit number, and bacteriome developmental patterns closely correlate with the presence/absence of the yeast-like fungal symbionts (YLS) and their colonisation dynamics. The obligate endosymbiont Karelsulcia and YLS, coexisting in bacteriomes during early stages of host ontogeny, may compete for ecological niches, potentially resulting in translocation of YLS into fat bodies. This indicates that bacteriomes may have initially functioned as immune organs like fat bodies, but evolved specifically for accommodating bacterial endosymbionts. The translocation of YLS from bacteriomes to fat bodies during the later development of host cicadas indicates that immune-mediated regulation occurs in such symbiotic organs as host insects mature. This study sheds light on how symbiont-host interactions shape the symbiotic organogenesis, which provides insights into adaptive evolution of specialised symbiotic organs in plant sap-feeding insects.