The GEN-ERA toolbox: unified and reproducible workflows for research in microbial genomics.

BACKGROUND: Microbial culture collections play a key role in taxonomy by studying the diversity of their strains and providing well-characterized biological material to the scientific community for fundamental and applied research. These microbial resource centers thus need to implement new standards in species delineation, including whole-genome sequencing and phylogenomics. In this context, the genomic needs of the Belgian Coordinated Collections of Microorganisms were studied, resulting in the GEN-ERA toolbox. The latter is a unified cluster of bioinformatic workflows dedicated to both bacteria and small eukaryotes (e.g., yeasts). FINDINGS: This public toolbox allows researchers without a specific training in bioinformatics to perform robust phylogenomic analyses. Hence, it facilitates all steps from genome downloading and quality assessment, including genomic contamination estimation, to tree reconstruction. It also offers workflows for average nucleotide identity comparisons and metabolic modeling. TECHNICAL DETAILS: Nextflow workflows are launched by a single command and are available on the GEN-ERA GitHub repository (https://github.com/Lcornet/GENERA). All the workflows are based on Singularity containers to increase reproducibility. TESTING: The toolbox was developed for a diversity of microorganisms, including bacteria and fungi. It was further tested on an empirical dataset of 18 (meta)genomes of early branching Cyanobacteria, providing the most up-to-date phylogenomic analysis of the Gloeobacterales order, the first group to diverge in the evolutionary tree of Cyanobacteria. CONCLUSION: The GEN-ERA toolbox can be used to infer completely reproducible comparative genomic and metabolic analyses on prokaryotes and small eukaryotes. Although designed for routine bioinformatics of culture collections, it can also be used by all researchers interested in microbial taxonomy, as exemplified by our case study on Gloeobacterales.

Specialisation of Yeast Genera in Different Phases of Bee Bread Maturation.

Pollen stored by bees undergoes a fermentation marked by the presence of lactic acid bacteria and yeasts. It results in bee bread. Past studies have singled out Starmerella (Candida) magnoliae as the most common yeast species in honey bee-stored bee bread. Starmerella species are ecological specialists with potential biotechnological value. The rarity of recent studies on yeasts in honey bees prompted us to generate new information on yeast diversity during the conversion of bee-collected pollen to bee bread. Bees and stored pollen from two apiaries in Belgium were sampled, a yeast isolation protocol was developed, yeast isolates were grouped according to their macro- and micromorphology, and representative isolates were identified using DNA sequences. Most of the 252 identified isolates belonged to the genera Starmerella, Metschnikowia, and Zygosaccharomyces. The high abundance of yeasts in fresh bee bread decreased rapidly with the storage duration. Starmerella species dominated fresh bee bread, while mostly Zygosaccharomyces members were isolated from aged bee bread. Starmerella (Candida) apis, a rarely isolated species, was the most frequent and abundant species in fresh bee bread. Yeasts from the bee's honey stomach and from pollen pellets obtained from bees hind legs were dominated by Metschnikowia species. The distinctive communities from pollen pellets over fresh bee bread to aged bee bread indicate a non-random distribution of these yeasts.

Nuclear Magnetic Resonance Spectroscopy-Based Identification of Yeast.

Rapid and robust high-throughput identification of environmental, industrial, or clinical yeast isolates is important whenever relatively large numbers of samples need to be processed in a cost-efficient way. Nuclear magnetic resonance (NMR) spectroscopy generates complex data based on metabolite profiles, chemical composition and possibly on medium consumption, which can not only be used for the assessment of metabolic pathways but also for accurate identification of yeast down to the subspecies level. Initial results on NMR based yeast identification where comparable with conventional and DNA-based identification. Potential advantages of NMR spectroscopy in mycological laboratories include not only accurate identification but also the potential of automated sample delivery, automated analysis using computer-based methods, rapid turnaround time, high throughput, and low running costs.We describe here the sample preparation, data acquisition and analysis for NMR-based yeast identification. In addition, a roadmap for the development of classification strategies is given that will result in the acquisition of a database and analysis algorithms for yeast identification in different environments.

Yeast culture collections in the twenty-first century: new opportunities and challenges.

The twenty-first century has brought new opportunities and challenges to yeast culture collections, whether they are long-standing or recently established. Basic functions such as archiving, characterizing and distributing yeasts continue, but with expanded responsibilities and emerging opportunities. In addition to a number of well-known, large public repositories, there are dozens of smaller public collections that differ in the range of species and strains preserved, field of emphasis and services offered. Several collections have converted their catalogues to comprehensive databases and synchronize them continuously through public services, making it easier for users worldwide to locate a suitable source for specific yeast strains and the data associated with these yeasts. In-house research such as yeast taxonomy continues to be important at culture collections. Because yeast culture collections preserve a broad diversity of species and strains within a species, they are able to make discoveries in many other areas as well, such as biotechnology, functional, comparative and evolution genomics, bioprocesses and novel products. Due to the implementation of the Convention of Biological Diversity (CBD) and the Nagoya Protocol (NP), there are new requirements for both depositors and users to ensure that yeasts were collected following proper procedures and to guarantee that the country of origin will be considered if benefits arise from a yeast's utilization. Intellectual property rights (IPRs) are extremely relevant to the current access and benefit-sharing (ABS) mechanisms; most research and development involving genetic resources and associated traditional knowledge will be subject to this topic. Copyright © 2016 John Wiley & Sons, Ltd.

On the reclassification of species assigned to Candida and other anamorphic ascomycetous yeast genera based on phylogenetic circumscription.

Multigene phylogenies have been instrumental in revising the classification of ascosporic (teleomorph) yeasts in a natural system based on lines of descent. Although many taxonomic changes have already been implemented for teleomorph taxa, this is not yet the case for the large genus Candida and smaller anascosporic (anamorph) genera. In view of the recently introduced requirement that a fungal species or higher taxon be assigned only a single valid name under the new International Code of Nomenclature for algae, fungi, and plants (Melbourne Code), the current species of Candida and other anamorph yeast genera must undergo revision to make genus membership consistent with phylogenetic affinities. A review of existing data and analyses shows that certain Candida species may be assigned to teleomorph genera with high confidence using multigene phylogenies. Candida species that form well-circumscribed phylogenetic clades without any teleomorph member justify the creation of new genera. However, a considerable number of Candida species sit at the end of isolated and often long branches, and hence cannot be assigned to larger species groups. They should be maintained in Candida sensu lato until studied by multigene analyses in datasets with comprehensive taxon sampling. The principle of name stability has to be honoured to the largest extent compatible with a natural classification of Candida species.

The microbial diversity of traditional spontaneously fermented lambic beer.

Lambic sour beers are the products of a spontaneous fermentation that lasts for one to three years before bottling. The present study determined the microbiota involved in the fermentation of lambic beers by sampling two fermentation batches during two years in the most traditional lambic brewery of Belgium, using culture-dependent and culture-independent methods. From 14 samples per fermentation, over 2000 bacterial and yeast isolates were obtained and identified. Although minor variations in the microbiota between casks and batches and a considerable species diversity were found, a characteristic microbial succession was identified. This succession started with a dominance of Enterobacteriaceae in the first month, which were replaced at 2 months by Pediococcus damnosus and Saccharomyces spp., the latter being replaced by Dekkera bruxellensis at 6 months fermentation duration.

Starmerella neotropicalis f. a., sp. nov., a yeast species found in bees and pollen.

A novel yeast species was found repeatedly and in high cell densities in underground-nesting stingless bees of the species Melipona quinquefasciata and their provisions in northern Minas Gerais (Brazil). One additional strain was isolated from bee-collected pollen in Cuba. Phylogenetic analyses based on rRNA gene sequences (D1/D2 large subunit gene and internal transcribed spacer) indicated that the novel species belongs to the Starmerella clade and is most closely related to Candida (iter. nom. Starmerella) apicola. Growth reactions on carbon and nitrogen sources were typical of those observed in related species of the Starmerella clade. PCR-fingerprinting with mini- and microsatellite specific primers allowed the distinction of the novel species from Candida apicola, Candida bombi and a yet undescribed species represented by strain CBS 4353. On the basis of phylogenetic relationships, the novel species is assigned to the genus Starmerella despite the failure to observe sexual reproduction after extensive mating tests. We propose the name Starmerella neotropicalis f. a., sp. nov. (Mycobank MB 804285) and designate UFMG PST 09(T) ( = MUCL 53320(T) = CBS 12811(T)) as the type strain.

Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus predominate during well-performed Malaysian cocoa bean box fermentations, underlining the importance of these microbial species for a successful cocoa bean fermentation process.

Two spontaneous Malaysian cocoa bean box fermentations (one farm, two plantation plots) were investigated. Physical parameters, microbial community dynamics, yeast and bacterial species diversity [mainly lactic acid bacteria (LAB) and acetic acid bacteria (AAB)], and metabolite kinetics were monitored, and chocolates were produced from the respective fermented dry cocoa beans. Similar microbial growth and metabolite profiles were obtained for the two fermentations. Low concentrations of citric acid were found in the fresh pulp, revealing low acidity of the raw material. The main end-products of the catabolism of the pulp substrates glucose, fructose, and citric acid by yeasts, LAB, and AAB were ethanol, lactic acid, acetic acid, and/or mannitol. Hanseniaspora opuntiae, Lactobacillus fermentum, and Acetobacter pasteurianus were the prevalent species of the two fermentations. Saccharomyces cerevisiae, Lactobacillus plantarum, Lactobacillus pentosus, and Acetobacter ghanensis were also found during the mid-phase of the fermentation processes. Leuconostoc pseudomesenteroides and Acetobacter senegalensis were among the prevailing species during the initial phase of the fermentations. Tatumella saanichensis and Enterobacter sp. were present in the beginning of the fermentations and they could be responsible for the degradation of citric acid and/or the production of gluconic acid and lactic acid, respectively. The presence of facultative heterofermentative LAB during the fermentations caused a high production of lactic acid. Finally, as these fermentations were carried out with high-quality raw material and were characterised by a restricted microbial species diversity, resulting in successfully fermented dry cocoa beans and good chocolates produced thereof, it is likely that the prevailing species H. opuntiae, S. cerevisiae, Lb. fermentum, and A. pasteurianus were responsible for it.

Species diversity, community dynamics, and metabolite kinetics of the microbiota associated with traditional ecuadorian spontaneous cocoa bean fermentations.

Traditional fermentations of the local Ecuadorian cocoa type Nacional, with its fine flavor, are carried out in boxes and on platforms for a short time. A multiphasic approach, encompassing culture-dependent and -independent microbiological analyses of fermenting cocoa pulp-bean samples, metabolite target analyses of both cocoa pulp and beans, and sensory analysis of chocolates produced from the respective fermented dry beans, was applied for the investigation of the influence of these fermentation practices on the yeast and bacterial species diversity and community dynamics during cocoa bean fermentation. A wide microbial species diversity was found during the first 3 days of all fermentations carried out. The prevailing ethanol-producing yeast species were Pichia kudriavzevii and Pichia manshurica, followed by Saccharomyces cerevisiae. Leuconostoc pseudomesenteroides (glucose and fructose fermenting), Fructobacillus tropaeoli-like (fructose fermenting), and Lactobacillus fermentum (citrate converting, mannitol producing) represented the main lactic acid bacterial species in the fermentations studied, resulting in intensive heterolactate metabolism of the pulp substrates. Tatumella saanichensis and Tatumella punctata were among the members of the family Enterobacteriaceae present during the initial phase of the cocoa bean fermentations and could be responsible for the production of gluconic acid in some cases. Also, a potential new yeast species was isolated, namely, Candida sorbosivorans-like. Acetic acid bacteria, whose main representative was Acetobacter pasteurianus, generally appeared later during fermentation and oxidized ethanol to acetic acid. However, acetic acid bacteria were not always present during the main course of the platform fermentations. All of the data taken together indicated that short box and platform fermentation methods caused incomplete fermentation, which had a serious impact on the quality of the fermented dry cocoa beans.

Wickerhamomyces anomalus in the sourdough microbial ecosystem.

We previously found that Wickerhamomyces anomalus (formerly Hansenula anomala, Pichia anomala) was the second most frequently isolated yeast in Belgian artisan bakery sourdoughs and that the yeast dominated laboratory sourdough fermentations. Such findings are of interest in terms of the advantage of W. anomalus over other commonly encountered sourdough yeasts and its potential introduction into the sourdough ecosystem. Here, we provide a brief overview of current knowledge on yeast ecology and diversity in sourdough in the context of the potential natural habitat of W. anomalus. Insight into the population structure of W. anomalus was obtained by comparing internal transcribed spacer rDNA sequences of selected sourdough isolates with publicly available database sequences.

The amsterdam declaration on fungal nomenclature.

The Amsterdam Declaration on Fungal Nomenclature was agreed at an international symposium convened in Amsterdam on 19-20 April 2011 under the auspices of the International Commission on the Taxonomy of Fungi (ICTF). The purpose of the symposium was to address the issue of whether or how the current system of naming pleomorphic fungi should be maintained or changed now that molecular data are routinely available. The issue is urgent as mycologists currently follow different practices, and no consensus was achieved by a Special Committee appointed in 2005 by the International Botanical Congress to advise on the problem. The Declaration recognizes the need for an orderly transitition to a single-name nomenclatural system for all fungi, and to provide mechanisms to protect names that otherwise then become endangered. That is, meaning that priority should be given to the first described name, except where that is a younger name in general use when the first author to select a name of a pleomorphic monophyletic genus is to be followed, and suggests controversial cases are referred to a body, such as the ICTF, which will report to the Committee for Fungi. If appropriate, the ICTF could be mandated to promote the implementation of the Declaration. In addition, but not forming part of the Declaration, are reports of discussions held during the symposium on the governance of the nomenclature of fungi, and the naming of fungi known only from an environmental nucleic acid sequence in particular. Possible amendments to the Draft BioCode (2011) to allow for the needs of mycologists are suggested for further consideration, and a possible example of how a fungus only known from the environment might be described is presented.

Yeast species composition differs between artisan bakery and spontaneous laboratory sourdoughs.

Sourdough fermentations are characterized by the combined activity of lactic acid bacteria and yeasts. An investigation of the microbial composition of 21 artisan sourdoughs from 11 different Belgian bakeries yielded 127 yeast isolates. Also, 12 spontaneous 10-day laboratory sourdough fermentations with daily backslopping were performed with rye, wheat, and spelt flour, resulting in the isolation of 217 yeast colonies. The isolates were grouped according to PCR-fingerprints obtained with the primer M13. Representative isolates of each M13 fingerprint group were identified using the D1/D2 region of the large subunit rRNA gene, internal transcribed spacer sequences, and partial actin gene sequences, leading to the detection of six species. The dominant species in the bakery sourdoughs were Saccharomyces cerevisiae and Wickerhamomyces anomalus (formerly Pichia anomala), while the dominant species in the laboratory sourdough fermentations were W. anomalus and Candida glabrata. The presence of S. cerevisiae in the bakery sourdoughs might be due to contamination of the bakery environment with commercial bakers yeast, while the yeasts in the laboratory sourdoughs, which were carried out under aseptic conditions with flour as the only nonsterile component, could only have come from the flour used.

The role of culture collections as an interface between providers and users: the example of yeasts.

The importance and species diversity of yeasts in food production are described, including a listing of agricultural applications. Two yeast species were selected for case studies on distribution patterns from microbial culture collections: the high representation of Saccharomyces cerevisiae in culture collections enabled global comparison, while Dekkera bruxellensis deposits and distributions were analyzed from the perspective of a single culture collection. In conclusion, culture collections need to cover temporal gaps between deposit and application of strains. The further development of culture collections in countries of high but underexplored species diversity should facilitate the conservation and management of microbial resources.

The trehalose synthesis pathway is an integral part of the virulence composite for Cryptococcus gattii.

The trehalose pathway is essential for stress tolerance and virulence in fungi. We investigated the importance of this pathway for virulence of the pathogenic yeast Cryptococcus gattii using the highly virulent Vancouver Island, Canada, outbreak strain R265. Three genes putatively involved in trehalose biosynthesis, TPS1 (trehalose-6-phosphate [T6P] synthase) and TPS2 (T6P phosphatase), and degradation, NTH1 (neutral trehalose), were deleted in this strain, creating the R265tps1 Delta, R265tps2 Delta, and R265nth1 Delta mutants. As in Cryptococcus neoformans, cellular trehalose was reduced in the R265tps1 Delta and R265tps2 Delta mutants, which could not grow and died, respectively, at 37 degrees C on yeast extract-peptone-dextrose agar, suggesting that T6P accumulation in R265tps2 Delta is directly toxic. Characterizations of the cryptococcal hexokinases and trehalose mutants support their linkage to the control of glycolysis in this species. However, unlike C. neoformans, the C. gattii R265tps1 Delta mutant demonstrated, in addition, defects in melanin and capsule production, supporting an influence of T6P on these virulence pathways. Attenuated virulence of the R265tps1 Delta mutant was not due solely to its 37 degrees C growth defect, as shown in worm studies and confirmed by suppressor mutants. Furthermore, an intact trehalose pathway controls protein secretion, mating, and cell wall integrity in C. gattii. Thus, the trehalose synthesis pathway plays a central role in the virulence composites of C. gattii through multiple mechanisms. Deletion of NTH1 had no effect on virulence, but inactivation of the synthesis genes, TPS1 and TPS2, has profound effects on survival of C. gattii in the invertebrate and mammalian hosts. These results highlight the central importance of this pathway in the virulence composites of both pathogenic cryptococcal species.

Yeast diversity of Ghanaian cocoa bean heap fermentations.

The fermentation of the Theobroma cacao beans, involving yeasts, lactic acid bacteria, and acetic acid bacteria, has a major influence on the quality of the resulting cocoa. An assessment of the microbial community of cocoa bean heap fermentations in Ghana resulted in 91 yeast isolates. These were grouped by PCR-fingerprinting with the primer M13. Representative isolates were identified using the D1/D2 region of the large subunit rRNA gene, internal transcribed spacer sequences and partial actin gene sequences leading to the detection of 15 species. Properties of importance for cocoa bean fermentation, namely sucrose, glucose, and citrate assimilation capacity, pH-, ethanol-, and heat-tolerance, were examined for selected isolates. Pichia kudriavzevii (Issatchenkia orientalis), Saccharomyces cerevisiae, and Hanseniaspora opuntiae formed the major components of the yeast community. Hanseniaspora opuntiae was identified conclusively for the first time from cocoa fermentations. Among the less frequently encountered species, Candida carpophila, Candida orthopsilosis, Kodamaea ohmeri, Meyerozyma (Pichia) caribbica, Pichia manshurica, Saccharomycodes ludwigii, and Yamadazyma (Pichia) mexicana were not yet documented from this substrate. Hanseniaspora opuntiae was preferably growing during the earlier phase of fermentation, reflecting its tolerance to low pH and its citrate-negative phenotype, while no specific temporal distribution was recognized for P. kudriavzevii and S. cerevisiae.

Rapid etiological classification of meningitis by NMR spectroscopy based on metabolite profiles and host response.

Bacterial meningitis is an acute disease with high mortality that is reduced by early treatment. Identification of the causative microorganism by culture is sensitive but slow. Large volumes of cerebrospinal fluid (CSF) are required to maximise sensitivity and establish a provisional diagnosis. We have utilised nuclear magnetic resonance (NMR) spectroscopy to rapidly characterise the biochemical profile of CSF from normal rats and animals with pneumococcal or cryptococcal meningitis. Use of a miniaturised capillary NMR system overcame limitations caused by small CSF volumes and low metabolite concentrations. The analysis of the complex NMR spectroscopic data by a supervised statistical classification strategy included major, minor and unidentified metabolites. Reproducible spectral profiles were generated within less than three minutes, and revealed differences in the relative amounts of glucose, lactate, citrate, amino acid residues, acetate and polyols in the three groups. Contributions from microbial metabolism and inflammatory cells were evident. The computerised statistical classification strategy is based on both major metabolites and minor, partially unidentified metabolites. This data analysis proved highly specific for diagnosis (100% specificity in the final validation set), provided those with visible blood contamination were excluded from analysis; 6-8% of samples were classified as indeterminate. This proof of principle study suggests that a rapid etiologic diagnosis of meningitis is possible without prior culture. The method can be fully automated and avoids delays due to processing and selective identification of specific pathogens that are inherent in DNA-based techniques.

Synonymy of the yeast genera Moniliella and Trichosporonoides and proposal of Moniliella fonsecae sp. nov. and five new species combinations.

Analyses of nucleotide sequences from the D1/D2 domains of the large-subunit rDNA and phenotypic characteristics showed that the genera Moniliella and Trichosporonoides are members of a single, monophyletic clade that would be best represented by a single anamorphic genus. On the basis of taxonomic priority, we propose the transfer of the five species of the genus Trichosporonoides to the genus Moniliella. The description of the genus Moniliella is emended and the following new combinations are proposed: Moniliella madida comb. nov., Moniliella megachiliensis comb. nov., Moniliella nigrescens comb. nov., Moniliella oedocephalis comb. nov. and Moniliella spathulata comb. nov. In addition, ten strains representing a novel yeast species belonging to the Moniliella clade were isolated from flowers in Thailand, Cuba and Brazil. Analysis of the internal transcribed spacer and D1/D2 large-subunit rDNA sequences indicated that the isolates represent a single species that was distinct from other species of the Moniliella clade. The name Moniliella fonsecae sp. nov. is proposed to accommodate these strains. The type strain is BCC 7726(T) (=CBS 10551(T)).

Metschnikowia cubensis sp. nov., a yeast species isolated from flowers in Cuba.

A novel yeast species is described from 19 strains isolated from flowers and insects in three provinces of Cuba. The species is so far known only from Cuba. Characteristic asci and ascospores as well as phylogenetic analysis of the rDNA sequence place the novel species in the genus Metschnikowia. The novel species belongs to the New World subclade of large-spored species of Metschnikowia. Mating tests with other members of the subclade resulted in the formation of sterile asci without ascospores, showing that the Cuban strains represent a distinct biological species. Intraspecies matings lead to the production of fertile asci containing large needle-shaped ascospores. The novel species was further distinguished from its close relatives by rDNA sequences and PCR fingerprinting using primers derived from mini- and microsatellites. We propose the name Metschnikowia cubensis sp. nov. and designate MUCL 45753(T) (=CRGF 279(T) =CBS 10832(T), h(+)) as the type strain and MUCL 45751 (=CRGF 278 =CBS 10833, h(-)) as the allotype.

Re-examining the phylogeny of clinically relevant Candida species and allied genera based on multigene analyses.

Yeasts of the artificial genus Candida include plant endophytes, insect symbionts, and opportunistic human pathogens. Phylogenies based on rRNA gene and actin sequences confirmed that the genus is not monophyletic, and the relationships among Candida species and allied teleomorph genera are not clearly resolved. Protein-coding genes have been useful to resolve taxonomic positions among a broad range of fungi. Over 70 taxa of the genus Candida and its allied sexually reproducing genera were therefore selected, and their phylogenetic relationships were investigated using nuclear sequences of the largest subunit and second largest subunit of RNA polymerase II gene, actin, the second subunit of the mitochondrial cytochrome oxidase gene, and D1/D2 LSU rRNA gene. The DNA sequences were analysed by maximum parsimony and Bayesian inference, resulting in the recognition of six major phylogenetic groups (A-F). Group A contains six facultative pathogenic Candida species, which seem to have derived from nonpathogenic species, while Group B contains species of Clavispora, Metschnikowia, and Pichia guilliermondii. Species of Debaryomyces form an independent group C that is related to groups A and B. Pichia fermentans and other environmental species are concentrated in Group D. Group E, containing Pichia anomala, may be a sibling to group F, which is represented by the Saccharomyces species complex.