Microscopic Raman study of fungal pigment using the genetically amenable rock inhabitant Knufia petricola as a model organism.

Fungal pigments such as melanin and carotenoids are distinctive markers of animal and plant pathogenic fungi as well as their environmental relatives. These complex pigments play important roles in pathogenicity and stress tolerance while also being useful as biomarkers. Accordingly, it is important to be able to identify in situ the pigments in black fungi, a group of clinical and environmental importance. In this study, wild-type and genetically modified strains of Knufia petricola A95 and wild fungal cells attached to ancient rock were investigated for their spectroscopic and microscopic Raman features and morphological appearance. Knockout mutants of melanin synthesis genes pks1 (polyketide synthase), sdh1 (scytalone dehydratase), and both pks1 and the carotenoid synthesis gene phd1 (phytoene desaturase) were studied We applied two different Raman microscopes using two lasers, with 633 nm and 488 nm wavelengths. We analyzed and compared Raman spectra between the measured reference substances and the mutant and wild-type strains. In the wild strain WT:A95, the peaks close to melanin peals were found at 1353 cm-1 and 1611 cm-1. There are no characteristic melanin peaks at 1580-1600 cm-1 and around 1350 cm-1 at the spectrum of the Δpks1/Δphd1 mutant and the Δsdh1 mutant. The Δpks1 mutant spectrum has the peaks at the beta-carotene v2 C-C in-plane stretch at 1155 cm-1 and v3 C-CH3 deformation at 1005 cm-1. The peaks of carotenoids and melanin were found in all mutants and the wild strain, except the Δpks1/Δphd1 mutant. Raman spectra allow for discrimination between the various pigments. Hence, interactions between natural fungal melanin, as well as other protective pigments, and complex environmental matrices can be characterized on a range of spatial and temporal scales.

Genetic Engineering of the Rock Inhabitant Knufia petricola Provides Insight Into the Biology of Extremotolerant Black Fungi.

Black microcolonial fungi (Ascomycetes from Arthonio-, Dothideo-, and Eurotiomycetes) are stress-tolerant and persistent dwellers of natural and anthropogenic extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures and accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) melanin in the multilayered cell walls. To understand how black fungi live, survive, colonize mineral substrates, and interact with phototrophs genetic methods are needed to test these functions and interactions. We chose the rock inhabitant Knufia petricola of the Chaetothyriales as a model for developing methods for genetic manipulation. Here, we report on the expansion of the genetic toolkit by more efficient multiplex CRISPR/Cas9 using a plasmid-based system for expression of Cas9 and multiple sgRNAs and the implementation of the three resistance selection markers genR (geneticin/nptII), baR (glufosinate/bar), and suR (chlorimuron ethyl/sur). The targeted integration of expression constructs by replacement of essential genes for pigment synthesis allows for an additional color screening of the transformants. The black-pink screening due to the elimination of pks1 (melanin) was applied for promoter studies using GFP fluorescence as reporter. The black-white screening due to the concurrent elimination of pks1 and phs1 (carotenoids) allows to identify transformants that contain the two expression constructs for co-localization or bimolecular fluorescence complementation (BiFC) studies. The co-localization and interaction of the two K. petricola White Collar orthologs were demonstrated. Two intergenic regions (igr1, igr2) were identified in which expression constructs can be inserted without causing obvious phenotypes. Plasmids of the pNXR-XXX series and new compatible entry plasmids were used for fast and easy generation of expression constructs and are suitable for a broad implementation in other fungi. This variety of genetic tools is opening a completely new perspective for mechanistic and very detailed study of expression, functioning and regulation of the genes/proteins encoded by the genomes of black fungi.

Searching for biological feedstock material: 3D printing of wood particles from house borer and drywood termite frass.

Frass (fine powdery refuse or fragile perforated wood produced by the activity of boring insects) of larvae of the European house borer (EHB) and of drywood termites was tested as a natural and novel feedstock for 3D-printing of wood-based materials. Small particles produced by the drywood termite Incisitermes marginipennis and the EHB Hylotrupes bajulus during feeding in construction timber, were used. Frass is a powdery material of particularly consistent quality that is essentially biologically processed wood mixed with debris of wood and faeces. The filigree-like particles flow easily permitting the build-up of wood-based structures in a layer wise fashion using the Binder Jetting printing process. The quality of powders produced by different insect species was compared along with the processing steps and properties of the printed parts. Drywood termite frass with a Hausner Ratio HR = 1.1 with ρBulk = 0.67 g/cm3 and ρTap = 0.74 g/cm3 was perfectly suited to deposition of uniformly packed layers in 3D printing. We suggest that a variety of naturally available feedstocks could be used in environmentally responsible approaches to scientific material sciences/additive manufacturing.

An advanced genetic toolkit for exploring the biology of the rock-inhabiting black fungus Knufia petricola.

Microcolonial black fungi are a group of ascomycetes that exhibit high stress tolerance, yeast-like growth and constitutive melanin formation. They dominate a range of hostile natural and man-made environments, from desert rocks and salterns to dishwashers, roofs and solar panels. Due to their slow growth and a lack of genetic tools, the underlying mechanisms of black fungi's phenotypic traits have remained largely unexplored. We chose to address this gap by genetically engineering the rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales), a species that exhibits all characteristics of black fungi. A cell biological approach was taken by generating K. petricola strains expressing green or red fluorescent protein variants. By applying: (1) traditional gene replacement; (2) gene editing and replacement via plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9, and (3) silencing by RNA interference (RNAi), we constructed mutants in the pathways leading to melanin, carotenoids, uracil and adenine. Stable single and double mutants were generated with homologous recombination (HR) rates up to 100%. Efficient, partially cloning-free strategies to mutate multiple genes with or without resistance cassettes were developed. This state-of-the-art genetic toolkit, together with the annotated genome sequence of strain A95, firmly established K. petricola as a model for exploring microcolonial black fungi.

Shed Light in the DaRk LineagES of the Fungal Tree of Life-STRES.

The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the "Shed light in The daRk lineagES of the fungal tree of life" (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments.

Light sensing in plant- and rock-associated black fungi.

Fungi that share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. But to cope with sunlight-associated stresses [e.g. high temperatures, UV radiation with associated DNA damage, accumulation of reactive oxygen species (ROS), desiccation and osmotic stresses] it is important for fungi to accurately sense and respond to changes in light. To test the hypothesis that light is an environmental cue that Ascomycota use to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships, the photoreceptor (PR) distribution in species from different ecological niches was analysed. The genomes of black [dihydroxynaphthalene (DHN) melanin-containing] fungi from phyllosphere and exposed solid surfaces contain multiple photoreceptors (PRs). The plant pathogen Botrytis cinerea (Leotiomycetes) has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts. Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess equal numbers of PRs along with the same set of protective pigments. This similarity between black fungi from plant and rock surfaces suggests that photoperception and -regulation are important for fungi that receive nutrients through cooperation with phototrophs. Genetic tools for manipulating K. petricola exist and will be used to test this idea.

Magnesium Stable Isotope Fractionation on a Cellular Level Explored by Cyanobacteria and Black Fungi with Implications for Higher Plants.

In a controlled growth experiment we found that the cyanobacterium Nostoc punctiforme has a bulk cell 26Mg/24Mg ratio (expressed as δ26Mg) that is -0.27‰ lower than the growth solution at a pH of ca. 5.9. This contrasts with a recently published δ26Mg value that was 0.65‰ higher than growth solution for the black fungus Knufia petricola at similar laboratory conditions, interpreted to reflect loss of 24Mg during cell growth. By a mass balance model constrained by δ26Mg in chlorophyll extract we inferred the δ26 Mg value of the main Mg compartments in a cyanobacteria cell: free cytosolic Mg (-2.64‰), chlorophyll (1.85‰), and the nonchlorophyll-bonded Mg compartments like ATP and ribosomes (-0.64‰). The lower δ26Mg found in Nostoc punctiforme would thus result from the absence of significant Mg efflux during cell growth in combination with either (a) discrimination against 26Mg during uptake by desolvation of Mg or transport across protein channels or (b) discrimination against 24Mg in the membrane transporter during efflux. The model predicts the preferential incorporation of 26Mg in cells and plant organs low in Mg and the absence of isotope fractionation in those high in Mg, corroborated by a compilation of Mg isotope ratios from fungi, bacteria, and higher plants.

Roof-Inhabiting Cousins of Rock-Inhabiting Fungi: Novel Melanized Microcolonial Fungal Species from Photocatalytically Reactive Subaerial Surfaces.

Subaerial biofilms (SAB) are an important factor in weathering, biofouling, and biodeterioration of bare rocks, building materials, and solar panel surfaces. The realm of SAB is continually widened by modern materials, and the settlers on these exposed solid surfaces always include melanized, stress-tolerant microcolonial ascomycetes. After their first discovery on desert rock surfaces, these melanized chaetothyrialean and dothidealean ascomycetes have been found on Mediterranean monuments after biocidal treatments, Antarctic rocks and solar panels. New man-made modifications of surfaces (e.g., treatment with biocides or photocatalytically active layers) accommodate the exceptional stress-tolerance of microcolonial fungi and thus further select for this well-protected ecological group. Melanized fungal strains were isolated from a microbial community that developed on highly photocatalytic roof tiles after a long-term environmental exposure in a maritime-influenced region in northwestern Germany. Four of the isolated strains are described here as a novel species, Constantinomyces oldenburgensis, based on multilocus ITS, LSU, RPB2 gene phylogeny. Their closest relative is a still-unnamed rock-inhabiting strain TRN431, here described as C. patonensis. Both species cluster in Capnodiales, among typical melanized microcolonial rock fungi from different stress habitats, including Antarctica. These novel strains flourish in hostile conditions of highly oxidizing material surfaces, and shall be used in reference procedures in material testing.

Corrosive extracellular polysaccharides of the rock-inhabiting model fungus Knufia petricola.

Melanised cell walls and extracellular polymeric matrices protect rock-inhabiting microcolonial fungi from hostile environmental conditions. How extracellular polymeric substances (EPS) perform this protective role was investigated by following development of the model microcolonial black fungus Knufia petricola A95 grown as a sub-aerial biofilm. Extracellular substances were extracted with NaOH/formaldehyde and the structures of two excreted polymers studied by methylation as well as NMR analyses. The main polysaccharide (~ 80%) was pullulan, also known as α-1,4-; α-1,6-glucan, with different degrees of polymerisation. Αlpha-(1,4)-linked-Glcp and α-(1,6)-linked-Glcp were present in the molar ratios of 2:1. A branched galactofuromannan with an α-(1,2)-linked Manp main chain and a ß-(1,6)-linked Galf side chain formed a minor fraction (~ 20%). To further understand the roles of EPS in the weathering of minerals and rocks, viscosity along with corrosive properties were studied using atomic force microscopy (AFM). The kinetic viscosity of extracellular K. petricola A95 polysaccharides (≈ 0.97 × 10-6 m2 s-1) ranged from the equivalent of 2% (w/v) to 5% glycerine, and could thus profoundly affect diffusion-dominated processes. The corrosive nature of rock-inhabiting fungal EPS was also demonstrated by its effects on the aluminium coating of the AFM cantilever and the silicon layer below.

Mg Isotope Fractionation during Uptake by a Rock-Inhabiting, Model Microcolonial Fungus Knufia petricola at Acidic and Neutral pH.

The model rock-inhabiting microcolonial fungus Knufia petricola fractionates stable Mg isotopes in a time- and pH-dependent manner. During growth, the increase of 26Mg/24Mg in the fungal cells relative to the growth media amounted to 0.65 ± 0.14‰ at pH 6 and 1.11 ± 0.35‰ at pH 3. We suggest a constant equilibrium fractionation factor during incorporation of Mg into ribosomes and ATP as a cause of enrichment of 26Mg in the cells. We suggest too that the proton gradient across the cell wall and cytoplasmic membrane controls Mg2+ transport into the fungal cell. As the strength of this gradient is a function of extracellular solution pH, the pH-dependence on Mg isotope fractionation is thus due to differences in fungal cell mass fluxes. Through a mass balance model we show that Mg uptake into the fungal cell is not associated with a unique Mg isotope fractionation factor. This Mg isotope fractionation dependence on pH might also be observed in any organism with cells that follow similar Mg uptake and metabolic pathways and serves to reveal Mg cycling in ecosystems.

A novel qPCR protocol for the specific detection and quantification of the fuel-deteriorating fungus Hormoconis resinae.

A wide variety of fungi and bacteria are known to contaminate fuels and fuel systems. These microbial contaminants have been linked to fuel system fouling and corrosion. The fungus Hormoconis resinae, a common jet fuel contaminant, is used in this study as a model for developing innovative risk assessment methods. A novel qPCR protocol to detect and quantify H. resinae in, and together with, total fungal contamination of fuel systems is reported. Two primer sets, targeting the markers RPB2 and ITS, were selected for their remarkable specificity and sensitivity. These primers were successfully applied on fungal cultures and diesel samples demonstrating the validity and reliability of the established qPCR protocol. This novel tool allows clarification of the current role of H. resinae in fuel contamination cases, as well as providing a technique to detect fungal outbreaks in fuel systems. This tool can be expanded to other well-known fuel-deteriorating microorganisms.

Flow Chamber System for the Statistical Evaluation of Bacterial Colonization on Materials.

Biofilm formation on materials leads to high costs in industrial processes, as well as in medical applications. This fact has stimulated interest in the development of new materials with improved surfaces to reduce bacterial colonization. Standardized tests relying on statistical evidence are indispensable to evaluate the quality and safety of these new materials. We describe here a flow chamber system for biofilm cultivation under controlled conditions with a total capacity for testing up to 32 samples in parallel. In order to quantify the surface colonization, bacterial cells were DAPI (4`,6-diamidino-2-phenylindole)-stained and examined with epifluorescence microscopy. More than 100 images of each sample were automatically taken and the surface coverage was estimated using the free open source software g'mic, followed by a precise statistical evaluation. Overview images of all gathered pictures were generated to dissect the colonization characteristics of the selected model organism Escherichia coli W3310 on different materials (glass and implant steel). With our approach, differences in bacterial colonization on different materials can be quantified in a statistically validated manner. This reliable test procedure will support the design of improved materials for medical, industrial, and environmental (subaquatic or subaerial) applications.

Genetic transformation of Knufia petricola A95 - a model organism for biofilm-material interactions.

We established a protoplast-based system to transfer DNA to Knufia petricola strain A95, a melanised rock-inhabiting microcolonial fungus that is also a component of a model sub-aerial biofilm (SAB) system. To test whether the desiccation resistant, highly melanised cell walls would hinder protoplast formation, we treated a melanin-minus mutant of A95 as well as the type-strain with a variety of cell-degrading enzymes. Of the different enzymes tested, lysing enzymes from Trichoderma harzianum were most effective in producing protoplasts. This mixture was equally effective on the melanin-minus mutant and the type-strain. Protoplasts produced using lysing enzymes were mixed with polyethyleneglycol (PEG) and plasmid pCB1004 which contains the hygromycin B (HmB) phosphotransferase (hph) gene under the control of the Aspergillus nidulans trpC. Integration and expression of hph into the A95 genome conferred hygromycin resistance upon the transformants. Two weeks after plating out on selective agar containing HmB, the protoplasts developed cell-walls and formed colonies. Transformation frequencies were in the range 36 to 87 transformants per 10 µg of vector DNA and 10(6) protoplasts. Stability of transformation was confirmed by sub-culturing the putative transformants on selective agar containing HmB as well as by PCR-detection of the hph gene in the colonies. The hph gene was stably integrated as shown by five subsequent passages with and without selection pressure.

Geodermatophilus africanus sp. nov., a halotolerant actinomycete isolated from Saharan desert sand.

A novel Gram-strain positive, aerobic, actinobacterial strain, designated CF11/1(T), was isolated from a sand sample obtained in the Sahara Desert, Chad. The black-pigmented isolate was aerobic and exhibited optimal growth from 25 to 35 °C at pH 6.0-8.0 and with 0-8 % (w/v) NaCl, indicating that it is a halotolerant mesophile. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The G+C content in the genome was 74.4 mol%. The peptidoglycan contained meso-diaminopimelic acid as diagnostic diaminoacid. The main phospholipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and a minor fraction of phosphatidylglycerol; MK-9(H4) was the dominant menaquinone, and galactose was detected as a diagnostic sugar. The major cellular fatty acid was branched-chain saturated acid iso-C16:0. Analysis of 16S rRNA gene sequences showed 95.3-98.6 % pairwise sequence identity with the members of the genus Geodermatophilus. Based on phenotypic and chemotaxonomic properties, as well as phylogenetic distinctiveness, the isolate represents a novel species, Geodermatophilus africanus, with the type strain CF11/1(T) (DSM 45422 = CCUG 62969 = MTCC 11556).

Nutritional physiology of a rock-inhabiting, model microcolonial fungus from an ancestral lineage of the Chaetothyriales (Ascomycetes).

Rock-inhabiting black fungi [also microcolonial or meristematic fungi (MCF)] are a phylogenetically diverse group of melanised ascomycetes with distinctive morphological features that confer extensive stress tolerance and permit survival in hostile environments. The MCF strain A95 Knufia petricola (syn. Sarcinomyces petricola) belongs to an ancestral lineage of the order Chaetothyriales (class Eurotiomycetes). K. petricola strain A95 is a rock-inhabiting MCF and its growth requirements were studied using the 96-well plate-based Biolog System under ∼1070 different conditions (osmotic stress, pH growth optima, growth factor requirements and nutrient catabolism). A95 is an osmotolerant, oligotrophic MCF that grows best around pH 5. Remarkably, A95 shows metabolic activity in the absence of added nitrogen, phosphorus or sulphur. Correlations could be drawn between the known nutrient requirements of A95 and what probably is available in sub-aerial systems (rock and other material surfaces). Detailed knowledge of A95's metabolic requirements allowed formulation of a synthetic medium that supports strong fungal growth.

Geodermatophilus normandii sp. nov., isolated from Saharan desert sand.

A novel Gram-reaction-positive actinobacterial strain, designated CF5/3(T), was isolated from a sand sample obtained in the Sahara Desert, Chad. The greenish-black-pigmented isolate was aerobic and exhibited optimal growth from 25-40 °C at pH 6.0-10.0 with 0-1% (w/v) NaCl. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The DNA G+C content of the genome of the novel strain was 75.5 mol%. The peptidoglycan contained meso-diaminopimelic acid as diagnostic diamino acid. The main phospholipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol and a minor fraction of phosphatidylglycerol. MK-9(H4) was the dominant menaquinone, and galactose was detected as a diagnostic sugar. The major cellular fatty acids were branched-chain saturated acids: iso-C(15:0) and iso-C(16:0). Analysis of 16S rRNA gene sequences showed 95.6-98.8% pairwise sequence identity with the members of the genus Geodermatophilus. Based on phenotypic and chemotaxonomic properties, as well as phylogenetic distinctiveness, the isolate represents a novel species, Geodermatophilus normandii, with the type strain CF5/3(T) ( =DSM 45417(T) =CCUG 62814(T) =MTCC 11412(T)).

Geodermatophilus tzadiensis sp. nov., a UV radiation-resistant bacterium isolated from sand of the Saharan desert.

Three novel Gram-positive, aerobic, actinobacterial strains, CF5/2(T), CF5/1 and CF7/1, were isolated in 2007 during environmental screening of arid desert soil in the Sahara desert, Chad. Results from riboprinting, MALDI-TOF protein spectra and 16S rRNA sequence analysis confirmed that all three strains belonged to the same species. Phylogenetic analysis of 16S rRNA sequences with the strains' closest relatives indicated that they represented a distinct species. The three novel strains also shared a number of physiological and biochemical characteristics distinct from previously named Geodermatophilus species. The novel strains' peptidoglycan contained meso-diaminopimelic acid; their main phospholipids were phosphatidylcholine, phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylinositol and a small amount of phosphatidylglycerol; MK-9(H4) was the dominant menaquinone. The major cellular fatty acids were the branched-chain saturated acids iso-C16:0 and iso-C15:0. Galactose was detected as diagnostic sugar. Based on these chemotaxonomic results, 16S rRNA gene sequence analysis and DNA-DNA hybridization between strain CF5/2(T) and the type strains of Geodermatophilus saharensis, Geodermatophilus arenarius, Geodermatophilus nigrescens, Geodermatophilus telluris and Geodermatophilus siccatus, the isolates CF5/2(T), CF5/1 and CF7/1 are proposed to represent a novel species, Geodermatophilus tzadiensis, with type strain CF5/2(T)=DSM 45416=MTCC 11411 and two reference strains, CF5/1 (DSM 45415) and CF7/1 (DSM 45420).

Geodermatophilus siccatus sp. nov., isolated from arid sand of the Saharan desert in Chad.

A novel Gram-positive, aerobic, actinobacterial strain, CF6/1(T), was isolated in 2007 during environmental screening of arid desert soil in the Sahara near to Ourba, Chad. The isolate was found to grow best in a temperature range of 20-37 °C and at pH 6.0-8.5 and showed no NaCl tolerance, forming black-coloured and nearly circular colonies on GYM agar. Chemotaxonomic and molecular characteristics determined for the isolate match those previously described for members of the genus Geodermatophilus. The DNA G + C content of the novel strain was determined to be 74.9 mol %. The peptidoglycan was found to contain meso-diaminopimelic acid as the diagnostic diamino acid. The main phospholipids were determined to be phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine, diphosphatidylglycerol and traces of phosphatidylglycerol; MK-9(H(4)) was identified as the dominant menaquinone and galactose as the diagnostic sugar. The major cellular fatty acids were found to be the branched-chain saturated acids iso-C(16:0) and iso-C(15:0), as well as C(17:1ω8c). The 16S rRNA gene sequence shows 97.5-97.9 % sequence identity with the four validly named or at least effectively published members of the genus: Geodermatophilus obscurus (97.5 %), Geodermatophilus arenarius (97.7 %), Geodermatophilus ruber (97.9 %) and Geodermatophilus nigrescens (97.9 %). Based on the results from this polyphasic taxonomic analysis and DNA-DNA hybridizations with all type strains of the genus, we propose that strain CF6/1(T) represents a novel species, Geodermatophilus siccatus, with the type strain CF6/1(T) = DSM 45419(T) = CCUG 62765(T) = MTCC 11414(T).

Geodermatophilus telluris sp. nov., an actinomycete isolated from Saharan desert sand.

A novel Gram-positive, multiloculated thalli-forming, aerobic, actinobacterial strain, CF9/1/1(T), was isolated in 2007 during environmental screening for xerophilic fungi in arid desert soil from the Sahara desert, Chad. The isolate grew best at a temperature range of 20-35 °C and at pH 6.0-8.5 and with 0-4% (w/v) NaCl, forming black-coloured and irregular colonies on GYM agar. Chemotaxonomic and molecular characteristics of the isolate matched those described for members of the genus Geodermatophilus. The DNA G+C content of the novel strain was 75.4 mol%. The peptidoglycan contained meso-diaminopimelic acid as a diagnostic diamino acid. The main phospholipids were diphosphatidylglycerol, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, a not yet structurally identified aminophospholipid and a small amount of phosphatidylglycerol; MK-9(H4) was identified as the dominant menaquinone and galactose was a diagnostic sugar. The major cellular fatty acids were branched-chain saturated acids: iso-C16:0 and iso-C15:0. The 16S rRNA gene sequence of the isolate showed 94.6-97.0% sequence similarities with those of five members of the genus: Geodermatophilus ruber DSM 45317(T) (94.6%), Geodermatophilus obscurus DSM 43160(T) (94.8%), Geodermatophilus siccatus DSM 45419(T) (96.2%), Geodermatophilus nigrescens DSM 45408(T) (96.7%) and Geodermatophilus arenarius DSM 45418(T) (97.0%). Based on the evidence from this polyphasic taxonomic study, a novel species, Geodermatophilus telluris sp. nov., is proposed; the type strain is CF9/1/1(T) (=DSM 45421(T)=CCUG 62764(T)).