Phenotypic and molecular genetics study of Geotrichum candidumLink (1809) and Geotrichum silvicola Pimenta (2005) cultivated on mitis salivarius agar.

BACKGROUND: Geotrichum is a genus of fungi found in different habitats throughout the world. Although Geotrichum and its related species have been extensively reclassified and taxonomically revised, it is still the target for many researches. METHODS AND RESULTS: In this study, phenotypic and molecular genetics comparisons were performed between Geotrichum candidum and Geotrichum silvicola. Mitis Salivarius Agar was used as the growing medium for the phenotypic comparison study, which was carried out at two temperatures (20-25 and 37 °C). For genotypic comparison, we compared the 18 S, ITS, and 28 S sequences of universal DNA barcode regions of both species. Important findings on the new culture media for fungal isolation were revealed by the results. The phenotypic variation between the two species' colonies, including their shapes, sizes, textures and growth rates, were strikingly different. DNA sequences of both species showed that pairwise identities of the species were 99.9% for 18 S, 100% for ITS and 99.6% for 28 S regions. CONCLUSIONS: Contrary to what is commonly seen, the results showed that 18 S, ITS and 28 S failed to discriminate the species. The first investigation into the performance of Mitis Salivarius Agar as a fungus culture medium is reported in this work, and proved its efficiency. Additionally, this is the first study to compare G. candidum with G. silvicola by means of both phenotypic and genotypic analysis.

Genome sequencing and transcriptome analysis of Geotrichum citri-aurantii on citrus reveal the potential pathogenic- and guazatine-resistance related genes.

Sour rot, caused by Geotrichum citri-aurantii, is a major postharvest disease of citrus，and it causes serious economic losses. In this study, a high-quality genome sequence of G. citri-aurantii was obtained by Single Molecule Real-Time Sequencing (SMRT). Approximately 5.43 Gb of clean data were obtained and a total of 27.94-Mb genomic sequence was mapped to 10 chromosome groups after high-through chromosome conformation capture (Hi-C) assembly. In addition, three polygalacturonase genes which were related to pathogenicity in G. citri-aurantii genome were discovered. And transcriptome data of guazatine-resistance had been analyzed, the results showed that the guazatine-resistance of G. citri-aurantii was related to two ATP-binding cassette (ABC) transporter family genes, six major facilitator superfamily (MFS) transporter family genes and two multidrug and toxic compound extrusion (MATE) transporter family genes. In summary, our research may provide novel insights into the effective control of this pathogen.

Geotrichum candidum gene expression and metabolite accumulation inside the cells reflect the strain oxidative stress sensitivity and ability to produce flavour compounds.

Geotrichum candidum is a fungus-like yeast widely used as a starter culture for cheese ripening for its proteolytic and lipolytic activities and its contribution to the cheese flavours. The sequenced strain G. candidum CLIB 918 was isolated from cheese Pont-L'Evêque. This strain's ability to produce volatile compounds was compared to the ability of a known strong sulphur compound producer G. candidum strain (Gc203). The aminotransferase-coding genes BAT2 and ARO8 were identified to be involved in methionine catabolism. The production of volatile compounds indicated that the sequenced strain was a moderate producer compared to the strong producer strain. The major volatile compounds were produced from sulphur amino acid, branched-chain amino acid and fatty acid metabolisms. Metabolite content of the cells showed that the ability of the strain to produce volatile compounds was inversely proportional to its ability to store amino acids inside the cells. Reduced glutathione, hypotaurine and taurine intracellular concentrations and volatile fatty aldehyde production indicated the role of oxidative stress sensitivity in flavour production. The increase in expression of several genes in a Reblochon-type cheese at the end of ripening confirmed that oxygen and iron were key factors regulating cheese flavour production.

Reversible control of enantioselectivity by the length of ketone substituent in biocatalytic reduction.

Enzyme engineering has been widely employed to tailor the substrate specificity and enantioselectivity of enzymes. In this study, we mutated Trp288, an unconserved residue in the small binding pocket of an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD). Trp288 mutants showed substrate specificity expansion towards bulky-bulky ketones and enantioselectivity alteration which was highly dependent on the substrate substituent length. In aliphatic ketone reduction, enantioselectivity inverted from (S) to (R) when one of the substituents to the carbonyl carbon was elongated from propyl to butyl or pentyl. The best (R)-selective mutant, Trp288Val, achieved the reduction of 3-heptanone to its corresponding (R)-alcohol with 97% ee. Our docking simulation suggested that when enantioselectivity inverted to (R), only pro-R binding poses were productive. Gly94 played an important role to stabilize the butyl or pentyl group for their productive pro-R poses. Interestingly, when the substituent was further elongated, the enantioselectivity inverted back to the (S) form.

Sequencing of FKS Hot Spot 1 from Saprochaete capitata To Search for a Relationship to Reduced Echinocandin Susceptibility.

Saprochaete capitata, formerly known as Geotrichum capitatum, is an emerging fungal pathogen with low susceptibility to echinocandins. Here, we report the nucleotide sequence of the S. capitata hot spot 1 region of the FKS gene (FKS HS1), which codifies for the catalytic subunit of ß-1,3-d-glucan synthase, the target of echinocandins. For that purpose, we first designed degenerated oligonucleotide primers derived from conserved flanking regions of the FKS1 HS1 segment of 12 different fungal species. Interestingly, analysis of the translated FKS HS1 sequences of 12 isolates of S. capitata revealed that all of them exhibited the same F-to-L substitution in a position that is highly related to reduced echinocandin susceptibility.

Characterization and Synergistic Effect of Antifungal Volatile Organic Compounds Emitted by the Geotrichum candidum PF005, an Endophytic Fungus from the Eggplant.

Plant-associated endophytes are recognized as sources of novel bioactive molecules having diverse applications. In this study, an endophytic yeast-like fungal strain was isolated from the fruit of eggplant (Solanum melongena) and identified as Geotrichum candidum through phenotypic and genotypic characterizations. This endophytic G. candidum isolate PF005 was found to emit fruity scented volatiles. The compositional profiling of volatile organic compounds (VOCs) revealed the presence of 3-methyl-1-butanol, ethyl 3-methylbutanoate, 2-phenylethanol, isopentyl acetate, naphthalene, and isobutyl acetate in significant proportion when analyzed on a time-course basis. The VOCs from G. candidum exhibited significant mycelial growth inhibition (54%) of phytopathogen Rhizoctonia solani, besides having mild antifungal activity against a few other fungi. The source of carbon as a nutrient was found to be an important factor for the enhanced biosynthesis of antifungal VOCs. The antifungal activity against phytopathogen R. solani was improved up to 91% by feeding the G. candidum with selective precursors of alcohol and ester volatiles. Furthermore, the antifungal activity of VOCs was enhanced synergistically up to 92% upon the exogenous addition of naphthalene (1.0 mg/plate). This is the first report of G. candidum as an endophyte emitting antifungal VOCs, wherein 2-penylethanol, isopentyl acetate, and naphthalene were identified as important contributors to its antifungal activity. Possible utilization of G. candidum PF005 as a mycofumigant has been discussed based upon its antifungal activity and the qualified presumption of safety status.

Specific populations of the yeast Geotrichum candidum revealed by molecular typing.

Geotrichum candidum is a ubiquitous yeast and an essential component in the production of many soft cheeses. We developed a multilocus sequence typing (MLST) scheme with five retained loci (NUP116, URA1, URA3, SAPT4 and PLB3) which were sufficiently divergent to distinguish 40 sequence types (STs) among the 67 G. candidum strains tested. Phylogenetic analyses defined five main clades; one clade was restricted to environmental isolates, three other clades included distinct environmental isolates and dairy strains, while the fifth clade comprised 34 strains (13 STs), among which all but two were isolated from milk, cheese or the dairy environment. These findings suggest an adaptation to the dairy ecosystems by a group of specialized European G. candidum strains. In addition, we developed a polymerase chain reaction inter-long terminal repeat scheme, a fast and reproducible random amplification of polymorphic DNA-like method for G. candidum, to type the closely related dairy strains, which could not be distinguished by MLST. Overall, our findings distinguished two types of dairy strains, one forming a homogeneous group with little genetic diversity, and the other more closely related to environmental isolates. Neither regional nor cheese specificity was observed in the dairy G. candidum strains analysed. This present study sheds light on the genetic diversity of both dairy and environmental strains of G. candidum and thus extends previous characterizations that have focused on the cheese isolates of this species. Copyright © 2016 John Wiley & Sons, Ltd.

Immobilized enzymes from Geotrichum spp. improve wine quality.

Higher alcohols are the byproducts of yeasts in alcohol fermentation and are harmful to human health at high concentrations. In this study, immobilized crude enzymes extracted from Geotrichum spp. strains S12 and S13 were separately employed to treat red wine, then GC and GC-MS analyses were used to determine the profiles of volatile compounds in untreated and treated wine samples. Immobilized enzymes from S13 (SA-S13E) were more active in decreasing higher alcohols than enzymes from S12. Conditions for preparing SA-S13E were optimized, and best results were obtained at a sodium alginate concentration of 35 g/L, calcium chloride of 20 g/L, and crude enzyme dosage of 3 mL. Treatment with SA-S13E significantly increased the ester content and sensory quality of wine. After being reused three times, SA-S13E still exhibited approximately 80% activity towards 1-propanol, isobutanol, and hexanol and had certain activity even after 3 months storage at -20 °C, indicating high stability in application and storage and thus showing potential in wine processing.

Invasive infections due to Saprochaete and Geotrichum species: Report of 23 cases from the FungiScope Registry.

Saprochaete and Geotrichum spp. are rare emerging fungi causing invasive fungal diseases in immunosuppressed patients and scarce evidence is available for treatment decisions. Among 505 cases of rare IFD from the FungiScope™ registry, we identified 23 cases of invasive infections caused by these fungi reported from 10 countries over a 12-year period. All cases were adults and previous chemotherapy with associated neutropenia was the most common co-morbidity. Fungaemia was confirmed in 14 (61%) cases and deep organ involvement included lungs, liver, spleen, central nervous system and kidneys. Fungi were S. capitata (n=14), S. clavata (n=5), G. candidum (n=2) and Geotrichum spp. (n=2). Susceptibility was tested in 16 (70%) isolates. All S. capitata and S. clavata isolates with the exception of one S. capitata (MIC 4 mg/L) isolate had MICs>32 mg/L for caspofungin. For micafungin and anidulafungin, MICs varied between 0.25 and >32 mg/L. One case was diagnosed postmortem, 22 patients received targeted treatment, with voriconazole as the most frequent first line drug. Overall mortality was 65% (n=15). Initial echinocandin treatment was associated with worse outcome at day 30 when compared to treatment with other antifungals (amphotericin B ± flucytosine, voriconazole, fluconazole and itraconazole) (P=.036). Echinocandins are not an option for these infections.

Metatranscriptome analysis of fungal strains Penicillium camemberti and Geotrichum candidum reveal cheese matrix breakdown and potential development of sensory properties of ripened Camembert-type cheese.

BACKGROUND: Camembert-type cheese ripening is driven mainly by fungal microflora including Geotrichum candidum and Penicillium camemberti. These species are major contributors to the texture and flavour of typical bloomy rind cheeses. Biochemical studies showed that G. candidum reduces bitterness, enhances sulphur flavors through amino acid catabolism and has an impact on rind texture, firmness and thickness, while P. camemberti is responsible for the white and bloomy aspect of the rind, and produces enzymes involved in proteolysis and lipolysis activities. However, very little is known about the genetic determinants that code for these activities and their expression profile over time during the ripening process. RESULTS: The metatranscriptome of an industrial Canadian Camembert-type cheese was studied at seven different sampling days over 77 days of ripening. A database called CamemBank01 was generated, containing a total of 1,060,019 sequence tags (reads) assembled in 7916 contigs. Sequence analysis revealed that 57% of the contigs could be affiliated to molds, 16% originated from yeasts, and 27% could not be identified. According to the functional annotation performed, the predominant processes during Camembert ripening include gene expression, energy-, carbohydrate-, organic acid-, lipid- and protein- metabolic processes, cell growth, and response to different stresses. Relative expression data showed that these functions occurred mostly in the first two weeks of the ripening period. CONCLUSIONS: These data provide further advances in our knowledge about the biological activities of the dominant ripening microflora of Camembert cheese and will help select biological markers to improve cheese quality assessment.

Invasive Geotrichum clavatum fungal infection in an acute myeloid leukaemia patient: a case report and review.

Invasive Geotrichum clavatum fungal infections are extremely rare and unusual, occurring nearly exclusively in patients experiencing prolonged neutropenia during the treatment for acute myeloid leukaemia. Several groups of cases of fatal G. clavatum infection were reported in France between 2011 and 2012, but the ecological niche has not yet been identified. We report a case of a 32-year-old patient with acute myeloid leukaemia who developed G. clavatum sepsis with primary peritonitis, hepatic nodular lesions, and multivisceral failure during aplasia after induction followed by salvage chemotherapy. He was treated with voriconazole and is still alive 1 year after with controlled disease. We then discuss the epidemiological, clinical, and therapeutic features of these serious fungal infections compared to the published data.

Structure of Acidothermus cellulolyticus family 74 glycoside hydrolase at 1.82 Å resolution.

Here, a 1.82 Šresolution X-ray structure of a glycoside hydrolase family 74 (GH74) enzyme from Acidothermus cellulolyticus is reported. The resulting structure was refined to an R factor of 0.150 and an Rfree of 0.196. Structural analysis shows that five related structures have been reported with a secondary-structure similarity of between 75 and 89%. The five similar structures were all either Clostridium thermocellum or Geotrichum sp. M128 GH74 xyloglucanases. Structural analysis indicates that the A. cellulolyticus GH74 enzyme is an endoxyloglucanase, as it lacks a characteristic loop that blocks one end of the active site in exoxyloglucanases. Superimposition with the C. thermocellum GH74 shows that Asp451 and Asp38 are the catalytic residues.

Genetic diversity of dairy Geotrichum candidum strains revealed by multilocus sequence typing.

The introduction of multilocus sequence typing (MLST) for strain characterization provided the first sequence-based approach for genotyping many fungi, leading to reproducible, reliable, and exchangeable data. A MLST scheme based on the analysis of six housekeeping genes was developed for genotyping Geotrichum candidum. The scheme was first developed using 18 isolates for which the complete sequences of the alanyl-tRNA synthetase (ALA1), pyruvate kinase (CDC19), acetyl-coA acetyltransferase (ERG10), glutaminyl-tRNA synthase (GLN4), phosphoglucoisomerase (PGI1), and phosphoglucomutase (PGM2) housekeeping genes were determined. Multiple sequence alignments of these genes were used to define a set of loci showing, as closely as possible, the same phylogenetic resolution level as complete gene sequences. This scheme was subsequently validated with 22 additional isolates from dairy and non-dairy sources. Overall, 58 polymorphic sites were indexed among 3,009 nucleotides analyzed. Depending on the loci, four to eight alleles were detected, generating 17 different sequence types, of which ten were represented by a single strain. MLST analysis suggested a predominantly clonal population for the 40 G. candidum isolates. Phylogenetic analysis of the concatenated sequences revealed a distantly related group of four isolates. Interestingly, this group diverged with respect to internal transcribed spacers 1 (ITS1), 5.8S, and ITS2 analysis. The reproducibility of the MLST approach was compared to random amplification of microsatellites by PCR (RAM-PCR), a gel profiling method previously proposed for G. candidum strain typing. Our results found MLST differentiation to be more efficient than RAM-PCR, and MLST also offered a non-ambiguous, unique language, permitting data exchange and evolutionary inference.

Acetophenone reductase with extreme stability against a high concentration of organic compounds or an elevated temperature.

The gene encoding acetophenone reductase (APRD), a useful biocatalyst for producing optically pure alcohols, was cloned from the cDNA of Geotrichum candidum NBRC 4597. The gene contained an open reading frame that consisted of 1,029 nucleotides corresponding to 342 amino acid residues. The subunit molecular weight was calculated to be 36.7 kDa. The predicted amino acid sequence did not have significant similarity to those of the acetophenone reductase reported previously. The gene was inserted into the pET-21b(+) expression vector and expressed in Escherichia coli Rosetta™(DE3)pLysS by induction with 1 mM of isopropyl-ß-D-thiogalactopyranoside. E. coli cell-free extract gave 21.9 U/mg APRD activity, which was 81 times that of the G. candidum cell-free extract. The enzyme was purified with a HisTrap FF crude column. The enzyme exhibited the highest activity at 60 °C, and optimum reducing and oxidizing activity were observed in a pH range around 7.0-8.0 and 8.5, respectively. The enzyme was most stable at 60 °C and pH 6.5-7.5. The Vmax and the apparent Km value of the reductase were 67.6 µmol/min per milligram of protein and 0.146 mM for acetophenone, respectively. From 4 % (v/v) 4-phenyl-2-butanone, (S)-4-phenyl-2-butanol was obtained with a yield >80 % and an enantiomeric excess >99 % in a 20 h reaction recycling NADH with 15 % (v/v) 2-propanol.

Molecular study of Geotrichum strains isolated from Armada cheese.

The aim of this work was the genetic characterization at the strain level of 39 presumed Geotrichum candidum isolates isolated throughout the artisanal manufacturing and ripening of Armada cheese and tentatively identified at genus and/or species level by phenotypic characteristics. The molecular identification of the strains included among others the amplification and sequencing of the D1/D2 domains of the 26S rRNA gene. A restriction fragment length polymorphism (RFLP) analysis with the ITS1-5.8S-ITS2 PCR amplicons and a randomly amplified polymorphic DNA (RAPD) analysis with five different primers were carried out. The bands pattern profile obtained through RFLP by enzymatic restriction with HinfI was the same for all the strains studied, which confirmed the classification of the strains at species level. A RAPD-PCR analysis with three different primers was applied to assess the intraspecific diversity, in this way 16 band profiles were obtained for the 39 strains studied by the combined use of primers Ari1 and Omt1. This study contributes to know the occurrence and genotypic biodiversity of G. candidum in Armada cheese.

Use of PCR-restriction fragment length polymorphism analysis for identification of yeast species isolated from bovine intramammary infection.

This study reports a rapid PCR-based technique using a one-enzyme RFLP for discrimination of yeasts isolated from bovine clinical and subclinical mastitis milk samples. We analyzed a total of 1,486 milk samples collected over 1 yr in south Sardinia and northern Italy, and 142 yeast strains were preliminarily grouped based on their cultural morphology and physiological characteristics. Assimilation tests were conducted using the identification kit API ID 32C and APILAB Plus software (bioMérieux, Marcy l'Etoile, France). For PCR-RFLP analysis, the 18S-ITS1-5.8S ribosomal(r)DNA region was amplified and then digested with HaeIII, and dendrogram analysis of RFLP fragments was carried out. Furthermore, within each of the groups identified by the API or PCR-RFLP methods, the identification of isolates was confirmed by sequencing of the D1/D2 region using an ABI Prism 310 automatic sequencer (Applied Biosystems, Foster City, CA). The combined phenotypic and molecular approach enabled the identification of 17 yeast species belonging to the genera Candida (47.9%), Cryptococcus (21.1%), Trichosporon (19.7%), Geotrichum (7.1%), and Rhodotorula (4.2%). All Candida species were correctly identified by the API test and their identification confirmed by sequencing. All strains identified with the API system as Geotrichum candidum, Cryptococcus uniguttulatus, and Rhodotorula glutinis also produced characteristic restriction patterns and were confirmed as Galactomyces geotrichum (a teleomorph of G. candidum), Filobasidium uniguttulatum (teleomorph of Crypt. uniguttulatus), and R. glutinis, respectively, by D1/D2 rDNA sequencing. With regard to the genus Trichosporon, preliminary identification by API was problematic, whereas the RFLP technique used in this study gave characteristic restriction profiles for each species. Moreover, sequencing of the D1/D2 region allowed not only successful identification of Trichosporon gracile where API could not, but also correct identification of misidentified isolates. In conclusion, the 18S-ITS1-5.8S region appears to be useful in detecting genetic variability among yeast species, which is valuable for taxonomic purposes and for species identification. We have established an RFLP database for yeast species identified in milk samples using the software GelCompar II and the RFLP database constitutes an initial method for veterinary yeast identification.

Species reassignment of Geotrichum bryndzae, Geotrichum phurueaensis, Geotrichum silvicola and Geotrichum vulgare based on phylogenetic analyses and mating compatibility.

The present classification of Galactomyces and its anamorph, Geotrichum, is based on various studies that used morphology, ecology, biochemistry, DNA-DNA reassociation comparisons and gene sequencing. In this study, the identities of strains of the Centraalbureau voor Schimmelcultures yeast culture collection, as well as seven strains from South Africa, were examined by analyses of the nucleotide divergence in the internal transcribed spacer regions of the nuclear rRNA gene (nrRNA) operon, the D1/D2 domains of the 26S rRNA gene and partial actin gene sequences as well as compatibility studies. The South African strains were assigned to species in the genus Galactomyces. The phylogenetic analyses and mating studies revealed that Geotrichum silvicola and Geotrichum bryndzae are synonyms of Galactomyces candidus and that Geotrichum vulgare is a synonym of Galactomyces pseudocandidus.

Differences in hydrolytic abilities of two crude lipases from Geotrichum candidum 4013.

The fungus Geotrichum candidum 4013 produces two types of lipases (extracellular and cell-bound). Both enzymes were tested for their hydrolytic ability to p-nitrophenyl esters and compounds having a structure similar to the original substrate (triacylglycerols). Higher lipolytic activity of extracellular lipase was observed when triacylglycerols of medium- (C12) and long- (C18) chain fatty acids were used as substrates. Cell-bound lipase preferentially hydrolysed trimyristate (C14). The differences in the abilities of these two enzymes to hydrolyse p-nitrophenyl esters were observed as well. The order of extracellular lipase hydrolysis relation velocity was as follows: p-nitrophenyl decanoate > p-nitrophenyl caprylate > p-nitrophenyl laurate > p-nitrophenyl palmitate > p-nitrophenyl stearate. The cell-bound lipase indicates preference for p-nitrophenyl palmitate. The most striking differences in the ratios between the activity of both lipases (extracellular : cell-bound) towards different fatty acid methyl esters were 2.2 towards methyl hexanoate and 0.46 towards methyl stearate (C18). The Michaelis constant (K(m) ) and maximum reaction rate (V(max) ) for p-nitrophenyl palmitate hydrolysis of cell-bound lipase were significantly higher (K(m) 2.462 mM and V(max) 0.210 U/g/min) than those of extracellular lipase (K(m) 0.406 mM and V(max) 0.006 U/g/min).

Geotrichum siamensis sp. nov. and Geotrichum phurueaensis sp. nov., two asexual arthroconidial yeast species isolated in Thailand.

Two asexual arthroconidial yeast strains, TM3-44(T) and LYSM5(T), were isolated, respectively, from estuarine water in a mangrove forest and soil in a terrestrial forest in Thailand. Analysis of the D1/D2 domains of the large-subunit rRNA gene sequences revealed that strain TM3-44(T) differed from the closest species in terms of pairwise sequence similarity, Dipodascus albidus, by 11.7% nucleotide substitutions, while strain LYSM5(T) was closest to Galactomyces geotrichum with only 2.9% nucleotide substitutions. The phylogenetic tree further demonstrated that strain TM3-44(T) was at a distant position from the closest species, D. albidus, and other related species in the Dipodascus clade, while strain LYSM5(T) clustered with G. geotrichum, it closest relative in the Galactomyces clade. The phenotypic characteristics of the two strains were typical of the genus Geotrichum. On the basis of the above findings, strain TM3-44(T) was assigned as a novel species of Geotrichum, for which the name Geotrichum siamensis sp. nov. is proposed. The type strain is TM3-44(T) (BCC 29903(T)=NBRC 104880(T)=CBS 10929(T)). Strain LYSM5(T) represented another novel species of Geotrichum, which was named Geotrichum phurueaensis sp. nov. The type strain is LYSM5(T) (BCC 34756(T)=NBRC 105674(T)=CBS 11418(T)).