Environmental Microbiology Reports

In this study we used high throughput DNA sequencing and ICP-MS to compare the microbiome of the common earthball fungus, Scleroderma citrinum (Pers.) to that of its sister taxon in the Sclerodermataceae, Pisolithus arhizus (Scop.). ICP-MS analysis demonstrates that S. citrinum is enriched in silica, sulfur and zinc relative to P. arhizus, while P. arhizus is enriched in arsenic, calcium, cadmium, cobalt, copper, lithium, magnesium, molybdenum, nickel, potassium and vanadium. Statistical analysis of molecular data indicates that the microbiome of P. arhizus is both richer and more diverse than that of S. citrinum, and that the microbiomes are significantly different with that of S. citrinum being enriched in Cyanobacteria represented by the chloroplast of a photosynthetic, cryptoendolithic red alga, Saccharibacteria (TM-7), and Planctomycetes, while that of P. arhizus is enriched in Gemmatimonadetes, Latescibacteria, Elusomicrobia, and Tectomicrobia. Further, the P. arhizus microbiome is enriched in anaerobes relatives to that of S. citrinum, probably reflecting anaerobic zones previously measured in P. arhizus. Together, the data indicate diverse microbiomes comprised of aromatic hydrocarbon-degrading, metal- and radiotolerant bacteria, indicating that these fungi may provide a rich source of novel microbes suitable for bioremediation strategies.

Associations between hosts and their microbial symbionts are considered mutualistic when both partners benefit. While the advantages received by eukaryotic hosts from association with bacterial symbionts are frequently examined, benefits to the bacteria are rarely experimentally tested. Here, we consider whether the bug-Caballeronia symbiosis is truly mutualistic by measuring the effect of a leaffooted bug (Leptoglossus phyllopus) on the abundance of its horizontally acquired symbiont, Caballeronia grimmiae. We predicted that the free-living Caballeronia population would increase over time in the presence of its insect partner. We quantified Caballeronia titer in soil microcosms (i) in the presence and absence of L. phyllopus over time, and (ii) at different bug densities. Insect presence resulted in higher soil Caballeronia titer over time. As bug density increased, the soil Caballeronia population also increased. Additionally, soil moisture affected Caballeronia abundance, with moister soil supporting a larger population. These results demonstrate that the relationship between Caballeronia and L. phyllopus is truly mutualistic and add to a small but growing body of literature that has quantified the effects of eukaryotic hosts on their bacterial partners.

The Candidate Phyla Radiation (CPR) is a very large group of bacteria with no pure culture representatives, first discovered by metagenomic analyses. Within the CPR, candidate phylum Parcubacteria (previously referred to as OD1) within the candidate superphylum Patescibacteria is prevalent in anoxic sediments and groundwater. Previously, we had identified a specific member of the Parcubacteria (referred to as DGGOD1a) as an important member of a methanogenic benzene-degrading consortium. Phylogenetic analyses herein place DGGOD1a within the Candidate clade Nealsonbacteria. Because of its persistence over many years, we hypothesized that Ca. Nealsonbacteria DGGOD1a must serve an important role in sustaining anaerobic benzene metabolism in the consortium. To try to identify its growth substrate, we amended the culture with a variety of defined compounds (pyruvate, acetate, hydrogen, DNA, phospholipid), as well as crude culture lysate and three subfractions thereof. We observed the greatest (10 fold) increase in the absolute abundance of Ca. Nealsonbacteria DGGOD1a only when the consortium was amended with crude cell lysate. These results implicate Ca. Nealsonbacteria in biomass recycling. Fluorescent in situ hybridization and cryogenic transmission electron microscope images revealed that Ca. Nealsonbacteria DGGOD1a cells were attached to larger archaeal Methanothrix cells. This apparent epibiont lifestyle was supported by metabolic predictions from a manually curated complete genome. This is one of the first examples of bacterial-archaeal episymbiosis and may be a feature of other Ca. Nealsonbacteria found in anoxic environments.

Petroleum hydrocarbon (PHC) contamination poses widespread environmental risks in Canada, where it is known to contribute to 60% of existing contaminated sites. It requires effective remediation strategies in the boreal ecozone due to the cold climate resulting in slow site weathering processes and soil nutrient cycling. This study utilized PHC-contaminated field soil (25,700 mg/kg total petroleum hydrocarbons - TPH) and background soil (<120 mg/kg TPH) collected from a Canadian boreal site where PHCs had weathered in place for 25+ years. Additionally, a 1:1 diluted soil was prepared (12,600 mg/kg TPH), and with the field contaminated soil, used to investigate the effects on rhizobacterial community composition. In two-year greenhouse experiments (2023 - 2025), the soils were planted with native or na turalized plant species and inoculated with i) arbuscular mycorrhizal fungi (AMF), ii) the biosurfactant-producing and plant-growth-promoting rhizobacterium (PGPR) - Bacillus subtilis ATCC 21332, or iii) both AMF and B. subtilis 21332. PHC contamination was determined to exert a dominant influence on rhizobacterial community composition. Alpha- and beta-diversity analyses revealed that neither plant species nor microbial inoculants significantly altered bacterial diversity or community structure beyond the overriding effect of PHC concentration. Proteobacteria, Actinobacteriota, Acidobacteriota, and Chloroflexi dominated across all soils, with hydrocarbon-degrading genera such as KCM-B 112 and Sphingomonas significantly enriched in the 25,700 mg/kg TPH soil. Functional gene profiling identified widespread aerobic (e.g., alkB, CYP153, assA, nahAc, pheA, xylM, xylE, todC1, bphA1, and pahE) hydrocarbon-degradation pathways across phyla, suggesting extensive horizontal gene transfer and functional redundancy across treatments and plant species. Exogenously introduced B. subtilis ATCC 21132 established only in the background soil, suggesting competitive dominance of indigenous PHC-degraders. These findings underscore the magnitude of PHC concentration as the primary driver of rhizobacterial dynamics and indicate that augmenting native microbial capacity through soil carbon enhancement, rather than bioaugmentation, may significantly impact soil bacterial community composition during rhizodegradation of boreal soils.

Frankia cluster-2 are diazotrophs that engage in root nodule symbiosis with host plants of the Cucurbitales and the Rosales. They are rarely found in the soil in the absence of their hosts. Previous studies have shown that an assimilated nitrogen source, presumable arginine, is exported to the host in nodules of Datisca glomerata (Cucurbitales), but not in the nodules of Ceanothus thyrsiflorus (Rosales). To investigate if an assimilated nitrogen form is commonly exported by cluster-2 strains, and which nitrogen source would then be exported to C. thyrsiflorus, gene expression levels, metabolite profiles and enzyme activities were analysed. We found that the export of assimilated nitrogen in symbiosis is a common feature for Frankia cluster-2 strains, but which source is host-plant dependent. We also identified several gene losses. The ammonium assimilation via the GS/GOGAT cycle for export to the host, entails a high demand of 2-oxoglutarate from the TCA cycle. This specialised metabolism seems to have led to genome reduction: we show that Frankia cluster-2 strains have lost the glyoxylate shunt and succinate semialdehyde dehydrogenase, leading to a linearization of the TCA cycle. This could explain the low saprotrophic potential of Frankia cluster-2.

JF2 08-2F Crusty is a novel melanized polyextremotolerant fungus isolated from a biological soil crust, which we believe harbors Methylobacterium spp. endosymbionts, called Pinky. Crusty is capable of utilizing many sources of carbon and nitrogen and is resistant to multiple metals and UV-C due to its melanized cell wall. We were unable to recover a Pinky-free culture of Crusty via usage of antibiotics. However, when exposed to antibiotics that kill or stop the growth of the Pinky, growth of Crusty is significantly stunted, implying that actively growing Pinky symbionts are needed for Crustys optimal growth. The Crusty-Pinky symbiosis also seems to be able to perform active metabolism in carbonless and nitrogenless medium, which we believe is due to Pinkys ability to perform aerobic anoxygenic photosynthesis. Finally, Pinky was identified as being capable of growth stimulation of the algae Chlorella sorokiniana, indicating that Pinky likely produces cytokinins or auxins which Methylobacterium are known for. Features of this symbiosis provide us insight into the ecological roles of these microbes within the biological soil crust.

Purple nonsulfur bacteria (PNSB) use light for energy and organic substrates for carbon and electrons when growing photoheterotrophically. This lifestyle generates more reduced electron carriers than are required for biosynthesis, even during consumption of some of the most oxidized organic substrates like malate and fumarate. Excess reduced electron carriers must be oxidized for photoheterotrophic growth to occur. Diverse PNSB commonly rely on the CO2-fixing Calvin cycle to oxidize excess reduced electron carriers. Some PNSB also produce H2 or reduce terminal electron acceptors as alternatives to the Calvin cycle. Rhodospirillum rubrum Calvin cycle mutants defy this trend by growing phototrophically on malate or fumarate without H2 production or access to terminal electron acceptors. We used 13C-tracer experiments to examine how a Rs. rubrum Calvin cycle mutant maintains electron balance under such conditions. We detected the reversal of some TCA cycle enzymes, which carried reductive flux from malate or fumarate to -ketoglutarate. This pathway and the reductive synthesis of amino acids derived from -ketoglutarate are likely important for electron balance, as supplementing the growth medium with -ketoglutarate-derived amino acids prevented Rs. rubrum Calvin cycle mutant growth unless a terminal electron acceptor was provided. Flux estimates also suggested that the Calvin cycle mutant preferentially synthesized isoleucine using the reductive threonine-dependent pathway instead of the less-reductive citramalate-dependent pathway. Collectively, our results suggest that alternative biosynthetic pathways can contribute to electron balance within the constraints of a relatively constant biomass composition.

The competence of bacteria to colonize different environmental niches is often determined by their ability to form biofilms. This depends on both cellular and extracellular factors, such as individual characteristics of a strain, type of colonized surface (abiotic or biotic) or availability and source of nutrients. Pseudomonas donghuensis P482 efficiently colonizes rhizosphere of various plant hosts, but a connection between plant tissue colonization and biofilm formation has not been verified for P482 up to date. Here we demonstrate that the ability of P482 to form biofilm on abiotic surfaces and the structural characteristics of the biofilm are connected to the type of carbon source available to the bacteria, with glycerol promoting formation of developed biofilm at early stages. Also, the type of substratum, polystyrene or glass, significantly influences the ability of P482 to attach to the surface, possibly due to hydrophobic effects. Moreover, mutants in genes associated with motility or chemotaxis, synthesis of polysaccharides, and encoding proteases or regulatory factors, affected in biofilm formation on glass were fully capable of colonizing root tissue of both tomato and maize hosts. This indicates that the ability to form biofilm on distinct abiotic surfaces does not simply correlate with the efficient colonization of rhizosphere and formation of biofilm on plant tissue by P482.

Root exudates are composed of primary and secondary metabolites known to modulate the rhizosphere microbiota. Glucosinolates are defense compounds present in the Brassicaceae family capable of deterring pathogens, herbivores and biotic stressors in the phyllosphere. In addition, traces of glucosinolates and their hydrolyzed byproducts have been found in the soil, suggesting that these secondary metabolites could play a role in the modulation and establishment of the rhizosphere microbial community associated with this family. We used Arabidopsis thaliana mutant lines with disruptions in the indole glucosinolate pathway, liquid chromatography-tandem mass spectrometry (LC-MS/MS) and 16S rRNA amplicon sequencing to evaluate how disrupting this pathway affects the root exudate profile of Arabidopsis thaliana, and in turn, impacts the rhizosphere microbial community. Chemical analysis of the root exudates from the wild-type Columbia (Col-0), a mutant plant line overexpressing the MYB transcription factor ATR1 (atr1D) which increases glucosinolate production, and the loss-of-function cyp79B2cyp79B3 double mutant line with low levels of glucosinolates confirmed that alterations to the indole glucosinolate biosynthetic pathway shifts the root exudate profile of the plant. We observed changes in the relative abundance of exuded metabolites. Moreover, 16S rRNA amplicon sequencing results provided evidence that the rhizobacterial communities associated with the plant lines used were directly impacted in diversity and community composition. This work provides further information on the involvement of secondary metabolites and their role in modulating the rhizobacterial community. Root metabolites dictate the presence of different bacterial species, including plant growth-promoting rhizobacteria (PGPR). Our results suggest that genetic alterations in the indole glucosinolate pathway cause disruptions beyond the endogenous levels of the plant, significantly changing the abundance and presence of different metabolites in the root exudates of the plants as well as the microbial rhizosphere community.

Endomyxans are a poorly sampled and incompletely resolved aggregate of Rhizarian lineages that fall outside Filosa and Retaria. Among them, "Novel Clade 12" (NC12; Bass et al. 2009) is an environmental clade comprised primarily of sequences derived from anoxic sediments, hitherto lacking a morphologically-characterised representative. We have cultivated a marine anaerobic eukaryotroph, SSF, that we identify as the first representative of NC12. SSF is a teardrop-shaped cell with two unequal flagella emerging a third of the way down the cell behind a distinctive row of refractile globules. The posterior end of the cell is filled with food vacuoles. There is a surface thickening discernible in light microscopy. We also describe another distinct anaerobe eukaryotrophic lineage, also cultivated from marine sediment: PG. It consists of large pyriform cells with a substantial trailing "tail" and two unequal flagella, the posterior exceptionally long. In small subunit ribosomal RNA gene phylogenies, it falls outside the characterised clades and forms a distinct novel rhizarian lineage in its own right. Together, SSF and PG represent two additional independent adaptations to anoxic conditions within Rhizaria.

Symbiosis with bacteria is widespread among eukaryotes, including fungi. Bacteria that live within fungal mycelia (endohyphal bacteria) occur in many plant-associated fungi, including diverse Mucoromycota and Dikarya. Pestalotiopsis sp. 9143 is a filamentous ascomycete isolated originally as a foliar endophyte of Platycladus orientalis (Cupressaceae). It is infected naturally with the endohyphal bacterium Luteibacter sp. 9143, which influences auxin and enzyme production by its fungal host. Previous studies have used transcriptomics to examine similar symbioses between endohyphal bacteria and root-associated fungi such as arbuscular mycorrhizal fungi and plant pathogens. However, currently there are no gene expression studies of endohyphal bacteria of Ascomycota, the most species-rich fungal phylum. We developed methods for assessing gene expression by Pestalotiopsis sp. and Luteibacter sp. when grown in co-culture and when each was grown axenically. Our assays showed that the density of Luteibacter sp. in co-culture was greater than in axenic culture, but the opposite was true for the Pestalotiopsis sp. Dual RNA-seq data demonstrate that growing in co-culture modulates developmental and metabolic processes in both the fungus and bacterium, potentially through changes in the balance of organic sulfur via methionine acquisition. Our analyses also suggest an unexpected, potential role of the bacterial type VI secretion system in symbiosis establishment, expanding current understanding of the scope and dynamics of fungal-bacterial symbioses. TWEETWhen in co-culture, Luteibacter downregulates motility and upregulates a T6SS. Gene expression changes in its host, Pestalotiopsis, suggest the bacterium impacts fungal cell structure and methionine availability. IMPORTANCEInteractions between microbes and their hosts have important outcomes for host- and environmental health. Foliar fungal endophytes that infect healthy plants can harbor facultative endosymbionts called endohyphal bacteria, which can influence the outcome of plant-fungus interactions. These bacterial-fungal interactions can be influential but are poorly understood, particularly from a transcriptome perspective. Here, we report on a comparative, dual RNA-seq study examining the gene expression patterns of a foliar fungal endophyte and a facultative endohyphal bacterium when cultured together vs. separately. Our findings support a role for the fungus in providing organic sulfur to the bacterium, potentially through methionine acquisition, and potential involvement of a bacterial type VI secretion system in symbiosis establishment. This work adds to the growing body of literature characterizing endohyphal bacterial-fungal interactions, with a focus on a model facultative bacterial-fungal symbiosis in two species-rich lineages, the Ascomycota and Proteobacteria.

Copiotrophic Bacillus and related taxa grow rapidly and are commonly isolated from soil. Despite their growth rate, Bacillus sensu lato (BSL) constitute less than one percent of soil bacterial communities, and the nutrient-enriched rhizosphere contains even fewer. Amendment of bulk soil with synthetic root exudate did not lead to increase in Bacillus culturable counts. We hypothesized that BSL populations in soil enriched with growth-supporting carbon are suppressed by various soil microbes. A screen using B. pseudomycoides as tester strain yielded 124 growth inhibiting isolates, aligning by 16S rRNA genes to 3 Alphaproteobacteria, 6 Betaproteobacteria, 5 Gammaproteobacteria, 3 Streptomyces, and 19 Bacillaceae. Most antagonists also suppressed four other BSL, and over 70% of the BSL isolates suppressed each other. The 11 sequenced BSL genomes encoded between 2 and 10 antibiotic biosynthetic gene clusters. Incubation of multiple isolates in artificial soil microcosms resulted in population growth restraint through a high percentage of endospores formed. This indicated that growth suppression by antagonists was due primarily to induction of sporulation. These results support our hypothesis that Bacillus populations in soil enriched with growth-supporting carbon are suppressed by various soil microbes.

Bacteria of the genus Achromatium are known for their large cell sizes and intracellular calcium carbonate deposits. Achromatium inhabit freshwater, brackish, and marine sediments where they accumulate to high abundances at the oxic-anoxic interface. These bacteria alter their vertical position in the sediment along with daily fluctuations in oxygen concentrations. Yet, the mechanism behind their migration in the sediment remains unknown. In this study, we used chemotaxis assays and time-lapse microphotography to analyze the motility and chemotactic behavior of Achromatium oxaliferum. Microscopic observations revealed that rolling and gliding were the main forms of locomotion exhibited by Achromatium. In absence of any stimulant, the movement appeared to be mostly random and changes in direction frequently occurred. Chemotaxis assays showed a negative chemotaxis of Achromatium to oxygen, sulfide, and nitrate, as evidenced by the change from undirected to directed locomotion against the respective chemical gradient. For periods of more than 1 hour, Achromatium cells moved continuously towards regions of low concentration. We further investigated whether the genetic repertoire of Achromatium corresponds to our observations. Based on lab experiments and bioinformatic analyses we conclude that Achomatium motility is propelled by type IV pili guided by a plethora of chemo- and photoreceptors. We conclude that Achromatium uses negative chemo- and phototaxis to confine their distribution in aquatic sediments between opposing oxygen and sulfide gradients. This allows Achromatium to dynamically adjust its position in redox gradients, and thus is likely to have a major contribution to its success in the global colonization of diverse aquatic sediments.

Microorganisms are important catalysts for the oxidation of reduced inorganic sulfur compounds. One environmentally important source of reduced sulfur is metal sulfide minerals that occur in economic mineral deposits and mine waste. Previous research found that Sulfuriferula spp. were abundant and active in long-term weathering experiments with simulated waste rock and tailings from the Duluth Complex, Northern Minnesota. We therefore isolated several strains of Sulfuriferula spp. from these long-term experiments and characterized their metabolic and genomic properties to provide insight into microbe-mineral interactions and the microbial biogeochemistry in these and other moderately acidic to circumneutral environments. The Sulfuriferula strains are all obligate chemolithoautotrophs capable of oxidizing inorganic sulfur compounds and ferrous iron. The strains grew over different pH ranges, but all grew between pH 4.5-7, matching the weathering conditions of the Duluth Complex rocks. All strains grew on the iron-sulfide mineral pyrrhotite (Fe1-xS, 0 < x < 0.125) as the sole energy source, as well as hydrogen sulfide and thiosulfate, which are products of sulfide mineral breakdown. Despite their metabolic similarities, each strain encodes a distinct pathway for the oxidation of reduced inorganic sulfur compounds as well as differences in nitrogen metabolism that reveal diverse genomic capabilities among the group. Our results show that Sulfuriferula spp. are primary producers that likely play a role in sulfide mineral breakdown in moderately acidic to circumneutral mine waste, and the metabolic diversity within the genus likely explains their success in sulfide mineral-rich and other sulfidic environments. ImportanceMetal sulfide minerals such as pyrite and pyrrhotite are one of the main sources of reduced sulfur in the global sulfur cycle. The chemolithotrophic microorganisms that break down these minerals in natural and engineered settings are catalysts for biogeochemical sulfur cycling and have important applications in biotechnological processes such as biomining or bioremediation. Sulfuriferula is a recently described genus of sulfur oxidizing bacteria that are abundant primary producers in diverse terrestrial environments, including waste rock and tailings from metal mining operations. In this study, we explored the genomic and metabolic properties of new isolates from this genus, and the implications for their ecophysiology and biotechnological potential in ore and waste from economic mineral deposits.

O_LIEctomycorrhizal (EcM) fungi play key roles in ecosystem functioning, in particular temperate ones. Recent findings suggest that they can endophytically colonize the roots of non-EcM plants. Here we aim at (i) providing new evidence of colonization of non-EcM hosts by EcM fungi, (ii) exploring factors driving such colonization (plant identity, site, root filter), and (iii) providing direct microscopical evidence for endophytism. C_LIO_LIUsing amplicon sequencing (ITS2), we described the root fungal communities of 42 plant species collected at nine locations in France. In two of those sites, we also compared rhizosphere and root fungal communities to identify a potential root filter. Finally, we investigated endophytism in Russula spp. at two Russula-rich sites using fluorescence in situ hybridization (FISH) paired with confocal microscopy. C_LIO_LIWe find a large but variable share of EcM sequences in roots of non-EcM plant species, in particular nearby EcM hosts, suggesting that endophytism is a secondary ecological niche. Though EcM fungi were more abundant in the rhizosphere compared to roots, their composition was similar to that of roots, suggesting a poor root filter. We observed metabolically active hyphae of Russula spp. endophytically colonizing the apoplast of two non-EcM plant species. C_LIO_LIAs shown for other EcM fungi (e.g., Tuber spp., Ascomycota) we demonstrate the dual EcM/endophyte niche for Russula (Basidiomycota). The ecological consequences of this duality still need to be addressed. The ability to colonize two ecological niches may be a trait kept by EcM fungi which evolved from endophytic fungi, as stipulated by the "waiting room hypothesis". C_LI

The genus Denitromonas is currently a non-validated taxon that has been identified in several recent publications as members of microbial communities arising from marine environments. Very little is known about the biology of Denitromonas spp., and no pure cultures are presently found in any culture collections. The current epitaph of Denitromonas was given to the organism under the assumption that all members of this genus are denitrifying bacteria. This study performs phenotypic and genomic analyses on three new Denitromonas spp. isolated from tidal mudflats in the San Francisco Bay. We demonstrate that Denitromonas spp. are indeed all facultative denitrifying bacteria that utilize a variety of carbon sources such as acetate, lactate, and succinate. In addition, individual strains also use the esoteric electron acceptors perchlorate, chlorate, and iodate. Both 16S and Rps/Rpl phylogenetic analyses place Denitromonas spp. as a deep branching clade in the family Zoogloeaceae, separate from either Thauera spp., Azoarcus spp., or Aromatoleum spp. Genome sequencing reveals a G+C content ranging from 63.72% to 66.54%, and genome sizes range between 4.39-5.18 Mb. Genes for salt tolerance and denitrification are distinguishing features that separate Denitromonas spp. from the closely related Azoarcus and Aromatoleum genera.

Studying the minor part of the uncultivated microbial majority ("rare biosphere") is difficult even with modern culture-independent techniques. The enormity of microbial diversity creates particular challenges for investigating low-abundance microbial populations in soils. Strategies for selective sample enrichment to reduce community complexity can aid in studying the rare biosphere. Magnetotactic bacteria, apart from being a minor part of the microbial community, are also found in poorly studied bacterial phyla and certainly belong to a rare biosphere. The presence of intracellular magnetic crystals within magnetotactic bacteria allows for their significant enrichment using magnetic separation techniques for studies using a metagenomic approach. This work investigated the microbial diversity of a black bog soil and its magnetically enriched fraction. The poorly studied phylum representatives in the magnetic fraction were enriched compared to the original soil community. Two new magnetotactic species, Candidatus Liberimonas magnetica DUR002 and Candidatus Obscuribacterium magneticum DUR003, belonging to different classes of the relatively little-studied phylum Elusimicrobiota, were proposed. Their genomes contain clusters of magnetosome genes that differ from the previously described ones by the absence of genes encoding magnetochrome-containing proteins and the presence of unique Elusimicrobiota-specific genes, termed mae. The predicted obligately fermentative metabolism in DUR002 and lack of flagellar motility in the magnetotactic Elusimicrobiota broadens our understanding of the lifestyles of magnetotactic bacteria and raises new questions about the evolutionary advantages of magnetotaxis. The findings presented here increase our understanding of magnetotactic bacteria, soil microbial communities, and the rare biosphere.

Bacterial interactions-whether positive or negative - are crucial for the functioning of microbial communities. Though bacterial interactions are mainly expected to be negative, the sign and strength of interactions are predicted to be context dependent, with interactions typically being more positive in more stressful and nutrient-poor conditions. However, systematic studies investigating how the environment affects interactions between multiple taxa are lacking. Here, we determine if interactions between a panel of natural soil isolates change in response to the environment in which they are grown, with two different artificial media used (one simple and one complex) and a more ecologically relevant soil wash. To maximise natural variation in interactions, we collected multiple isolates from multiple sites: co-occurring (sympatric) isolates were predicted to show more negative interactions than allopatric isolates because of greater overlap in resource use. Pairwise interactions were in general negative, but more negative when grown in a complex lab-derived medium (Tryptic Soy Broth). Mutually beneficial interactions were most common in a simple resource medium (M9 minimal media) and exploitative interactions were most frequent in a soil broth. These patterns were independent of whether species originated from the same or a different site. The study supports the prediction that nutrient rich environments promote more negative interactions, and that measuring interactions of soil isolates in standard lab media is likely to misrepresent interactions occurring in natural environments.

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

The study of microbial volatile organic compounds (mVOCs) is a growing area of research, with applications ranging from agriculture to human health. The majority of the mVOC data are from in vitro liquid cultures, while few analyses of bacterial and fungal volatilomes on solid media cultures exist. Studies comparing liquid versus solid cultures of bacteria and fungi show significant changes to the soluble metabolites that are produced, suggesting that large differences would be observed for mVOCs based on the culture conditions. To test this idea, we characterized the volatilomes of Chromobacterium violaceum (strain ATCC(R) 12472) and C. vaccinii (strain MWU328), and those of their isogenic cviR- quorum sensing mutants cultured on solid versus liquid Kings Medium B media. VOCs were sampled using thin-film solid-phase microextraction (TF-SPME) and analyzed by two-dimensional gas chromatography-time-of-flight mass spectrometry (GCxGC-TOFMS). Of the three variables examined - Chromobacterium species, media type, and quorum sensing ability - growth on liquid versus solid media caused the most significant differences in the volatilomes. Bacterial species and quorum sensing ability were also influential, but to a lesser degree. Our findings indicate the importance of growth conditions in microbial volatilomics, and therefore, more consideration should be given to how microorganisms are cultured for volatilome analyses. ImportanceThe purpose of this work is to elucidate the differences in the volatile metabolic profiles of Chromobacterium spp. by exploring them through the lens of three variables: growth conditions, species, and the ability to quorum sense. Work on organismal metabolic differences stemming from factors such as liquid versus solid media types remains broadly overlooked. Understanding these effects will allow future researchers to design more robust experiments that better translate to native microbial ecosystems such as rhizosphere and phyllosphere, where volatile compounds may influence plant-pathogen or plant-saprobe interactions.