MicrobiologyOpen

Microorganisms inhabiting geothermal springs and volcanic systems experience fluctuating temperatures that can periodically exceed their upper thermal limits, but the demographic consequences of such exposure remain poorly understood. Here, we investigated demographic responses of the thermophilic archaeon Sulfolobus acidocaldarius to an extreme temperature (94.1{degrees}C) under two regimes: sustained exposure varying in duration, and episodic exposure interspersed with recovery at a permissive temperature (75{degrees}C). Under sustained exposure, populations showed no detectable loss of viability after 15 min but declined thereafter, decreasing by approximately five orders of magnitude after 120 min. Under episodic exposure, populations remained viable across nine exposure-recovery cycles but declined in density with successive cycles. Similar responses were observed for three strains, including a DNA mismatch repair knockout ({Delta}nucS), indicating that mismatch repair deficiency did not affect viability or recovery. Together, these results demonstrate that S. acidocaldarius can withstand brief and repeated exposure to near-boiling temperatures, with mortality determined primarily by cumulative exposure duration rather than a fixed thermal threshold.

Cytolysin is a toxin-bacteriocin commonly encoded by Enterococcus faecalis that can target erythrocytes, neutrophils, and macrophages as well as other Gram-positive bacteria. Cytolysin can lyse human, horse, rabbit, and mouse erythrocytes, but not sheep erythrocytes (SEs). It is hypothesized that SEs are resistant to cytolysin because they lack phosphatidylcholine (PC) in their membranes, while susceptible erythrocytes have PC-rich membranes, suggesting PC as a cytolysin binding target. Here, we isolated a urinary E. faecalis strain C33 that can efficiently lyse SEs and found that this activity was conserved among several E. faecalis clinical isolates. We demonstrated that although C33 cytolysin was necessary for SE lysis, it was not sufficient, suggesting a secondary factor enhances cytolysin activity against SEs. Through liposome assays we found that while PC was required for the activity of DS16 cytolysin, PC was dispensable for C33 cytolysin activity. To assess the biological significance of C33 cytolysin in the bladder microenvironment, we assessed C33 cytolysin activity against bladder epithelial cells and the urinary microbiota. We found that C33 cytolysin was more cytotoxic to human bladder epithelial cells than the canonical DS16 cytolysin. We also found greater inhibition of co-isolated urinary microbiota species Streptococcus parasanguinis, Enterococcus raffinosus, Staphylococcus capitis, and Corynebacterium coyleae by C33 cytolysin. Interestingly, E. faecalis C33 also inhibited the protective urogenital taxa Lactobacillus crispatus and Bifidobacterium longum. Together, these results reveal a new PC-independent cytolysin activity among clinical E. faecalis isolates that may support survival and competition in the urinary microenvironment.

Prevotella melaninogenica is a Gram-negative bacterium that has been implicated in numerous disorders such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis, and despite its abundance in the human microbiome, there is still limited knowledge on the fundamental physiology describing Prevotella melaninogenica. To properly advance Prevotella biology, it is critical to report and document such characterizations of Prevotella; therefore, the purpose of this study is to examine and define the phenotypic and growth characteristics of P. melaninogenica ATCC 25845. In this study, we found that Prevotella melaninogenica ATCC 25845 is a Gram-negative bacterium that is predominantly short and medium rod-shaped, with occasional filamentous rods. SEM imaging revealed that this filamentation may serve as division centers, points from which multiple daughter cells form. We also describe the antibiotic profile, which showed susceptibility to four classes and resistance to six classes of antibiotics. Carbon source growth kinetic characterization revealed a preference for carbohydrate sources.

A subgroup of membrane-anchored bacterial Alkaline Phosphatase superfamily members transfers phospholipid headgroups from phospholipids to diverse acceptor molecules. As part of type II and hybrid type I/II cellulose biosynthesis gene clusters BcsG transfers phosphorylethanolamine pEtN from phospholipid to the emerging 1,4 {beta}-D-glucan chain synthesized by the BcsABC cellulose biosynthesis nanomachine. BcsG homologs throughout the phylogenetic tree possess a uniquely structured tripartite linker that connects the fifth transmembrane domain with the periplasmic catalytic domain. A N-terminal unstructured sequence of highly variable length and biased amino acid composition, the N-linker, is followed by an -helix and an unstructured sequence of high similarity and constant length leading into the first {beta}-strand of the catalytic domain. With BcsG from Salmonella typhimurium synthesizing pEtN cellulose as a model, linkers were shown to be promiscuous and linker length positively correlated with transfer efficiency of the pEtN headgroup and protein stability. As the donor substrate, the phosphatidylethanolamine content of the membranes affects pEtN transfer efficiency. With Alkaline Phosphatase superfamily members to be promiscuous enzymes the catalytic domain of OpgE transfers pEtN to osmoregulated periplasmic glucan. BcsG-OpgE hybrid proteins cause colony morphology alterations thus potentially transferring pEtN to the BcsA synthesized glucan chain. With BcsG with shortest linkers not associated with a cellulose biosynthesis gene cluster, multiple hypotheses can be proposed that select for different length of the N-linker in BcsG homologs located within a cellulose biosynthesis gene cluster in other Pseudomonadati.

Carbohydrate inducible promoters on expression plasmids are widely used for biotechnology and basic science research. However, using carbohydrates for inducers has drawbacks including influencing bacterial metabolism, the inducer being used as a carbon source by the bacteria, high background expression, and only a subset of cells experiencing promoter activation. Furthermore, some bacteria including the pathogen Acinetobacter baumannii currently lack good carbohydrate-inducible promoters. To address this issue, a tetracycline-inducible promoter, PtetB, isolated from A. baumannii was cloned and evaluated in a number of important bacteria: Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas fluorescens, and Serratia marcescens. A highly sensitive luxCDABE reporter was used to evaluate promoter activity. S. marcescens strains with elevated and reduced cyclic-AMP levels were used to evaluate the impact of cAMP on PtetB function. The PtetB promoter was induced over 800-fold in A. baumannii and 10-750-fold inducible in the other tested species. Background expression was lower from PtetB in the pseudomonads than previously described carbohydrate inducible promoters. Data indicated that the PtetB promoter system may be a useful tool for future basic science research with the tested bacteria.

Chlorella sorokiniana is an industrially important microalga, but its genetic manipulation remains limited by poor transformation efficiency. Although several barriers have been proposed, the role of intracellular nuclease activity has received little attention. Here, we compared the degradation of exogenous double-stranded DNA in whole-cell lysates from transformation-recalcitrant (Chlorella sorokiniana and Tetradesmus obliquus) and transformation-proficient (Phaeodactylum tricornutum, Microchloropsis gaditana, and Nannochloropsis oceanica) microalgae. C. sorokiniana rapidly degraded both linear and plasmid DNA, while T. obliquus exhibited intermediate activity. In contrast, no detectable degradation was observed in the transformation-proficient species. DNA degradation in C. sorokiniana occurred within minutes, and nuclease activity in both recalcitrant species was inhibited by EDTA and Zn2+, consistent with a metal ion-dependent endonuclease. These findings suggest that intracellular nuclease activity may represent an underexplored barrier to exogenous DNA stability during transformation and provide a potential target for improving genetic transformation of recalcitrant microalgae.

A significant fraction of archaeal genomes have their genomic content distributed across two or more DNA molecules, yet the characteristics and evolution of these different replicons remain poorly understood. Here, we investigated the genomic and functional organization of secondary replicons in Halobacteriota using 211 complete genomes, comprising 766 replicons, including 211 primary chromosomes (chr1), and 555 secondary replicons. Comparative analyses of genomic features, including replicon size, GC content, and codon usage, revealed a continuous distribution of secondary replicons from plasmid-like to chromosome-like characteristics. Larger secondary replicons showed progressively more chromosome-like genomic and functional properties, including greater protein-family overlap with chr1 and a higher proportion of conserved gene families. At the phylum level, functional profiles substantially overlapped among replicon types, providing further support for a continuum rather than discrete functional classes. Larger replicons also showed increased protein-family duplication, while pangenome analysis revealed a greater proportion of conserved gene families, with accessory families most abundant at intermediate sizes and rare and singleton families declining with increasing replicon size. Together, these results support a model in which secondary replicons in Halobacteriota span a genomic and functional continuum from plasmid-like to chromosome-like states , with increasing size associated with greater conservation, gene duplication and progressively more chromosome-like gene content and functional profile.

Stratified benthic deposits offer crucial modern analogs for Precambrian biogeochemical dynamics, yet whether the vertical organization of deep sedimentary columns reflects depositional history, present-day geochemical conditions, or both remains poorly understood, particularly under extreme oligotrophy. Here, we present a depth-resolved exploratory survey of a continuous 34-cm sediment core partitioned into 11 discrete horizons (L1 to L11) from Poza Roja 2, an alkaline, sub-hypersaline pond in the Cuatro Cienegas Basin, Mexico. Integrating physical stratigraphy, organic carbon and nutrient stoichiometry, and 16S rRNA gene metabarcoding, we evaluated down-core microbial community structure alongside acute phosphorus scarcity. Molar C:Po and N:Po ratios exceeded canonical Redfield proportions (106:16:1) in every layer, by up to 48.5-fold and 87.8-fold, respectively, both peaking in layer L4. Unconstrained ordination (PCoA) and hierarchical clustering grouped the layers into three compositional zones: upper (L2-L4), intermediate (L5-L6), and basal (L8-L11), with L1 and L7 occupying transitional positions. Archaea accounted for 31.0% of prokaryotic reads, ranging from 8.3% (L5) to 43.7% (L9), and were dominated by SM1-50 (Thermoplasmatota) and Halobacteriota. The phototrophic heliobacterium Heliorestis peaked in the intermediate zone (47.6% of bacterial reads in L6). Constrained Redundancy Analysis models lacked statistical significance (P >= 0.12), highlighting the exploratory nature of a single core (n = 11). This pilot characterization provides a baseline for understanding deep biogenic archives under severe nutrient limitation.

Jeongeupia sacculi (HS-3) is a novel {beta}-proteobacterium with a unique multicellular life cycle. The genetic tractability and mechanisms underlying differentiation are unknown. Here, we adapt a method of homologous recombination for genetic modification in HS-3 and generate a disruption mutant of rpoS, which encodes the alternative sigma factor{sigma} S commonly associated with general stress response or major behavioral switches. We then characterize phenotypes associated with rpoS disruption. Under low-nutrient conditions, rpoS disruption reduced Congo red binding, consistent with rpoS acting as a regulator of low-nutrient response by upregulating production of biofilm-associated materials. Surprisingly, rpoS disruption did not significantly alter survival in response to other stressors in general stress response assays tested. Our work establishes a method for genetic manipulation in HS-3 and enables future investigation into the life cycle of HS-3, including whether rpoS has a role in differentiation triggered by low-nutrient response.

Background: Cyanobacteria are the major phototrophs in many endolithic ecosystems, yet their genomic diversity and metabolic adaptations remain poorly characterized. Here, we investigated cyanobacterial diversity in endolithic dolomite rocks from the Piora Valley in the southern Swiss Alps using genome-resolved metagenomics. Results: We reconstructed 138 medium-quality metagenome-assembled genomes (MAGs) from four neighboring endolithic rock communities, including 15 cyanobacterial MAGs distributed across six order-level lineages. The cyanobacterial communities showed limited taxonomic overlap among rocks, and most recovered MAGs represented potentially novel species. Only one Gloeobacter lineage occurred in all sampled rocks, which we propose as Gloeobacter dolomitensis sp. nov., representing the first Gloeobacter genome recovered from an endolithic environment. Comparative genomic analyses identified a cynS gene encoding cyanase unique to G. dolomitensis among the Gloeobacter. Phylogenetic analyses support its horizontal acquisition from Pseudomonadota. Interestingly, the genome of G. dolomitensis lacks a canonical CynABD cyanate transporter and instead contains a distinct ATP-synthase binding cassette (ABC) transporter adjacent to cynS. Although the available genomic and structural evidence does not establish cyanate transport, its genomic association with cynS suggests a potentially non-canonical mechanism. We further show that horizontal gene transfer was unevenly distributed among cyanobacterial lineages, with Acaryochloridales acting predominantly as donors, Chroococcidiopsidales as recipients, and Gloeobacterales showing a comparatively high frequency of interphylum gene exchange. Conclusions: The Piora Valley dolomite environment represents an underexplored reservoir of cyanobacterial diversity, including basal and previously undescribed lineages. The discovery of G. dolomitensis and its unusual cyanate-associated genomic architecture expands our understanding of Gloeobacterales diversity and highlights the potential importance of horizontal gene acquisition and metabolic innovation in endolithic microbial communities.

The molecular epidemiology of Acinetobacter, which is almost exclusively culture-dependent, has centered mainly on clinical isolates of A. baumannii. Yet, this overlooks sources not amenable to microbial culture and many non-A. baumannii species. Here, by analysing metagenomic projects from public transport systems worldwide, we show that metagenome-assembled genomes (MAGs) are highly relevant for assessing the unrecognized diversity of Acinetobacter. We recovered 839 Acinetobacter MAGs, with 269 being high-quality MAGs. The high-quality MAGs were across more than 20 well-defined Acinetobacter species; however, five species (A. nosocomilalis, A. ursingii, A. radioresistens, A. schindleri, and A. variabilis) had the most MAGs. Pangenome and phylogenetic diversity analyses of 685 genomes plus the 269 high-quality MAGs from those 5 species showed that MAGs are an important source of both lineage and gene diversity. Also, FST analyses and genome phylogenies show that for some species there is no genetic differentiation between MAGs and clinical isolates, implying transmission between hospitals and public transport settings. In addition to finding the antibiotic resistance genes (ARGs) in MAGs, we implemented an ad hoc strategy (leveraging the use of genomes) to recover considerably more ARGs. PCoA and PERMANOVA analyses show that, whereas in A. nosocomialis and A. ursingii, the ARG profiles of MAGs and clinical genomes are different, in A. radioresistens and A. schindleri, the ARG profiles are not different. Finally, some clinically important ARGs, such as sul1 and sul2, aadA, and blaOXA23, were found in all or some of the 5 species. Our study shows how combining MAGs and regular genomes is a powerful strategy for better appraising the genomic diversity (including the resistome) of Acinetobacter. On a broader level, our study showcases the importance of MAGs for genomic surveillance of man-made environments with clear public health implications.

Metals are essential for all forms of life and are therefore the subject of tight competition at the host-pathogen interface. Manganese (Mn) is an important cofactor for enzymes involved in various processes in bacteria, including the response to oxidative stress, and the ability to scavenge Mn through the expression of high-affinity Mn transporters has been associated with virulence of many extracellular and vacuolar pathogens. While the cytosol has been established to be restrictive for growth of non-adapted pathogens, the extent to which metal limitation contributes to this is not well understood. Here, we investigate whether high-affinity Mn acquisition is important for intracellular adaptation and virulence of the cytosolic pathogen Listeria monocytogenes (Lmo). To do so, we generated a strain of Lmo in which both of its predicted high-affinity Mn transporters, MntH and MntABC, are disrupted. We found that this strain was severely defective in growth in vitro under Mn-limited conditions and had lower intracellular Mn content. Additionally, we observed Mn-dependent transcriptional regulation of the mntH and mntA genes. Together, these data strongly support that MntH and MntABC are high-affinity Mn transporters and are likely to be the only such dedicated Mn transporters expressed in Lmo. Using the {Delta}mntH {Delta}mntA strain, we provide evidence that Mn limitation sensitizes Lmo to oxidative stress in vitro, consistent with the known role of Mn in mitigating reactive oxygen species. Ultimately, we demonstrate that expression of high-affinity Mn transporters is essential for intracellular replication of Lmo in macrophages, plaquing on fibroblast monolayers, and for virulence in an animal model of systemic infection. Our work, therefore, suggests that Lmo is challenged by Mn limitation in the cytosol and requires Mn scavenging to adapt to its intracellular niche.

Methanogens, a diverse group of archaea that produce methane during energy metabolism, are prevalent in anoxic habitats where they are exposed to viral infections. The head-tailed virus {Psi}M2 is one of few virus isolates that infect a methanogen host and was described as a lytic virus that lyses its host, Methanothermobacter marburgensis. Here, we present evidence that {Psi}M2 does not exclusively follow the classic lytic lifecycle but can also enter an alternative, chronic infection-like state. Growth experiments showed that infected cultures in minimal medium, optimized for the methanogen host (MS medium) did not exhibit complete lysis but continuously released infective virions. In contrast, in a suboptimal complex medium (SAB medium), the {Psi}M2-infected culture released virions in the exponential growth phase and eventually lysed after 14 hours post infection. Although the genome of {Psi}M2 encodes a putative integrase, a prerequisite for lysogeny, whole genome sequencing of {Psi}M2-infected M. marburgensis cultures revealed that the viral DNA remained extrachromosomal rather than integrating into the host genome, likely due to a mutation in the integrase gene. These findings suggest that {Psi}M2 enters a non-lytic lifecycle alternative to lysogeny under suboptimal host growth conditions. Sequencing further identified a heterogeneous viral population including previously undescribed variants. A high density of non-synonymous mutations and frameshift-inducing insertions was observed within ORFs predicted to encode a capsid protein and a major spike protein, suggesting these viral variants to differ in host recognition and infectivity. Overall, our results show more complex viral behavior and greater genetic variability than previously described.

Plasmids are important agents of prokaryotic evolution and yet existing reference databases remain biased towards cultivable species, limiting our knowledge of their function and ecology in the environment. Here we present a major expansion of known plasmid diversity, through curation and annotation of 8,744 topologically complete plasmids from 9,972 short- and 154 long-read metagenomes as part of the Microflora Danica project. These were found to increase plasmid taxonomic unit diversity by 15% and 99% relative to all and environmental plasmids, respectively, from public sources. In particular, we identify a novel lineage of plasmids in the genus Methanosarcina, encoding genes associated with methanogenesis and cell surface properties, expanding our understanding of chromosome-plasmid complementarity and its potential contribution to host biology. Combining our plasmids with public sources, we curated a set of 17 replication-associated proteins that could be found in 82% of known plasmids and can serve as hallmarks for future detection of plasmids. We present this resource, and a tool for confirming topological completeness of plasmid assemblies, as a toolkit for future plasmid discovery.

Genome streamlining is expected to favor the loss of costly biosynthetic functions, raising the question of what benefit justifies the continued maintenance of complex pathways in small genomes. Here, we investigated this question in an abundant Nitrosopumilus population from the Baltic Sea that combined a streamlined genome (1.4 Mbp) with substantial biosynthetic capacity for vitamins B1, B7, and B12. We found three lines of evidence that support the biological significance of this retention. First, phylogenomic analysis of 1,077 archaeal species representatives showed that B-vitamin biosynthesis is broadly conserved across ammonia-oxidizing archaea (AOA), indicating maintenance over deep evolutionary timescales. Second, genes associated with B-vitamin biosynthesis tended to be under stronger purifying selection than the genomic background across Baltic Sea metagenomes, consistent with selection preserving these functions in current environmental populations. Third, measurements in the cultured relative Nitrosopumilus maritimus showed B-vitamins and related precursors in both intracellular and extracellular fractions, providing a potential route through which these retained functions could influence metabolite availability beyond the producing cell. Together, our findings suggest that retained biosynthetic pathways in highly streamlined microorganisms reveal functions that are beneficial for the success of a natural population, while also pointing to their potential importance for other community members coexisting within the same environment.

Pre-prolipoprotein phosphatidylglycerol diacylglyceryl transferase (Lgt) catalyzes the first step in the lipoprotein modification pathway and is essential for growth and viability of Helicobacter pylori. We explored the fate of H. pylori cells under Lgt depletion conditions to obtain insight into Lgt function on morphology and physiology. Downregulation of lgt expression led to growth arrest and a heterogenous population of small rod-shaped cells and small round cells with spiky protrusions, a loose outer membrane and disintegrated flagella. H. pylori overcomes a defect in lipoprotein modification through alteration of its gene expression profile resulting in so-called revertants. Outer membrane protein synthesis, carbon metabolism and nutrient uptake was reduced, and protein translation and flagellar biogenesis increased. Lgt specific regulation was observed in phospholipid biosynthesis resulting in an initial increase in cardiolipin followed by an increase in lysophospholipids at the time of adaptation. Suppressors of {Delta}lgt that were selected on solid media under restrictive conditions either lacked chromosomal mutations or had mutations in the cagUT-tetR regulon controlling the expression of lgt. A suppressor without mutations had an altered expression profile that led to downregulation of a DNA methyltransferase and upregulation of HP_0014, a motility associated glycosyltransferase family protein. Transmission electron microscopy and single cell video microscopy showed that a subpopulation of cells is motile in the revertant while the suppressor regained full motility with higher average speed compared to the wild type strain. Overall, our data show that lipoprotein modification by Lgt is important for membrane and flagellar integrity and that the heterogenous character of H. pylori cultures allows individual cells to overcome this cell envelope stress and survive. Our findings are important for antibacterial development strategies targeting essential integral membrane enzymes involved in lipoprotein modification and stimulate exploration of synergetic activity between known antibiotics and novel antibacterial agents targeting novel cellular pathways.

Bacteria utilize specialized protein secretion systems, particularly the type VI secretion system (T6SS), to dominate interbacterial competitions and kill the prey bacteria in a contact-dependent manner. Here, we report that disruption of the thioesterase gene encoded in the gramibactin (a bacterial siderophore) biosynthetic gene cluster activates the T6SS in the Burkholderia gladioli strain NGJ1, even in the absence of prey. The thioesterase mutant exhibits enhanced antibacterial activity, and ectopic expression of the gene complements the phenotype. Notably, thio mutant as well as thioesterase-complement strains show enhanced sensitivity to oxidative stress. The qRT-PCR analysis showed that the thio mutant had upregulated expression of RpoN1, a sigma factor, which positively regulates T6SS functions. We demonstrate that rpoN1 mutant is hypersensitive to oxidative stress and defective in T6SS activation, whereas overexpression of RpoN1 enhances oxidative stress tolerance in NGJ1 and activates T6SS. We propose that, during interbacterial conflicts encountered during plant colonization, NGJ1 faces an oxidative-stress-enriched environment that modulates thioesterase expression, induces RpoN1, and activates T6SS. Overall, our study exemplifies the broader physiological role of Thioesterase in maintaining intracellular ROS homeostasis in bacteria and stimulating antibacterial activity.

The opportunistic pathogen Pseudomonas aeruginosa can store carbon by polymerizing (R)-3-hydroxy fatty acids into polyhydroxyalkanoates (PHA) and forming large intracellular carbonosomes. These structures primarily function as stores of excess carbon that can be employed during nutrient imbalances, though they have been increasingly reported to have secondary functions in stress responses. A range of Proteobacteria can form carbonosomes, and they have been well characterised in strains used in bioplastic production and wastewater treatment. However, the occurrence, regulation and functions of carbonosomes in pathogenic bacteria remain poorly understood. Using flow cytometry and fluorescence microscopy following staining of carbonosomes with BODIPY 493/503, we characterised carbonosome abundance and dynamics in growth-arrested P. aeruginosa starved of nitrogen but provided with excess carbon. We found that carbonosomes substantially impact the energetic and biosynthetic output of growth-arrested P. aeruginosa, affecting protein biosynthesis, flagellar motility, and ATP/NADPH pools. The production and use of carbonosomes was found to confer a strong fitness advantage to growth-arrested P. aeruginosa during fluctuating nutrient conditions. Small carbonosomes were observed during the lag phase of growth in rich media and the ability to produce these structures was conserved across several different laboratory and clinical strains, although with different dynamics and maximal amounts. For example, the Liverpool epidemic strain LESB58 showed very little BODIPY staining while the closely related LESB65 strain showed high levels of staining. Crucially, we found that growth-arrested cells depolymerize carbonosomes upon tobramycin exposure and that carbonosome depolymerization significantly increases P. aeruginosa tobramycin sensitivity. Together, our observations suggest that carbonosomes significantly impact the physiology, metabolism, and antibiotic susceptibility of P. aeruginosa.

Climate change is threatening coral reef ecosystems around the world, causing significant declines in reef organisms, including sponges. The health of sponges is intimately linked to their symbiosis with microorganisms, yet little is known about their integrated response to ocean warming. Here, we present a comprehensive view of the effect of elevated sea surface temperatures (SST) on the model sponge Ianthella basta. I. basta individuals were exposed to temperatures up to 32 {degrees}C, 2-3 {degrees}C above the mean annual maxima. Host gene expression profiles showed an initial response to heat stress after 2 days, and a decreased expression of genes involved in maintaining tissue integrity, pumping capacity, vitamin transport, and stable symbioses after 4 to 8 days. Congruently, heat stressed samples started to show tissue regression and a reduced abundance of the dominant symbionts after 4 days. A significant loss of the symbionts occurred after 8 days, together with a reduction in microbially driven nitrogen cycling and amino-acid transport and an introduction of opportunistic microbes, which could lead to nutrient imbalance and further destabilisation of the holobiont. Together, this work provides important insights into the avenues through which future SST can impact host-microbe interactions in coral reef sponges.

Extremophilic fungi are a relatively unexplored source of stress-resilient microbes for sustainable agriculture. Here, we describe Talaromyces pseudofuniculosus FAR, isolated from the highly acidic site of Piccolo Inferno in southern Italy and identified by ITS and {beta}-tubulin (BenA) sequencing. Despite its origin from an extreme environment, T. pseudofuniculosus FRA remained fully functional under non-stress conditions and displayed tolerance to salinity and heavy metals while maintaining key plant growth-promoting traits, including phosphate solubilization, auxin-related compound production and hydrolytic activities. In soybean (Glycine max L.), the live fungus promoted plant growth under both non-stress conditions and agriculturally relevant salinity and copper stress, and successfully colonized the roots. A cell-free extracellular extract (exCFS) promoted early seedling development in vitro, but its effects were not sustained in pot experiments. GC-MS analysis provided a qualitative characterization of the extracellular metabolite profile and revealed condition-dependent differences between control, NaCl and CuSO4; growth conditions, indicating environment-dependent modulation of fungal metabolism. Overall, these findings highlight the physiological and metabolic plasticity of T. pseudofuniculosus FRA and support its potential as a fungal biostimulant for saline- and metal-impacted agricultural soils.