Dimorphic Mechanism on cAMP Mediated Signal Pathway in Mucor circinelloides.

Mucor circinelloides is a dimorphic fungus that is a non-pathogen strain belonging to zygomycetes. In this research, a part of hypothetical mechanism on yeast-like cell induction of M. circinelloides in CO2 atmosphere was reported from the viewpoint of gene expression. To explain the relation between the change and the expressions of some genes involved in morphological changes of the strain, these were analyzed on the filamentous and yeast cell by real-time qPCR. The compared genes were Nce103, Ras3, Cyr1, Pde, and Efg1 encoding carbonic anhydrase, GTPase, adenylate cyclase, phosphodiesterase, and elongation factor G1, respectively. In anaerobic grown yeast cell with 70%N2 + 30%CO2, the Nce103 and Ras3 gene expressions decreased to 24 h whereas that of the filamentous cell increased. However, a downstream gene of Cyr1 expression level in the yeast cell was higher than that of filamentous cell. A lower level of Pde in the yeast cell than that of the filamentous cell indicated intracellular cAMP accumulation. The actual cAMP in the yeast cell remained whereas that of the filamentous cell decreased with cultivation. The Efg1 expression level controlling hyphal elongation was suppressed in the yeast cell. The intracellular cAMP accumulation and Efg1 expression regulate hyphal elongation or yeast forming.

Fungal Wound Healing through Instantaneous Protoplasmic Gelation.

Multicellular organisms employ fluid transport networks to overcome the limit of diffusion and promote essential long-distance transport. Connectivity and pressurization render these networks especially vulnerable to wounding. To mitigate this risk, animals, plants, and multicellular fungi independently evolved elaborate clotting and plugging mechanisms. In the septate filamentous fungi, membrane-bound organelles plug septal pores in wounded hyphae. By contrast, vegetative hyphae in the early-diverging Mucoromycota are largely aseptate, and how their hyphae respond to wounding is unknown. Here, we show that wounding in the Mucorales leads to explosive protoplasmic discharge that is rapidly terminated by protoplasmic gelation. We identify Mucoromycota-specific Gellin proteins, whose loss of function leads to uncontrolled wound-induced protoplasmic bleeding. Gellins contain ten related ß-trefoil Gll domains, each of which possesses unique features that impart distinct gelation-related properties: some readily unfold and form high-order sheet-like structures when subjected to mechanical force from flow, while others possess hydrophobic motifs that enable membrane binding. In cell-free reconstitution, sheet-like structures formed by a partial Gellin incorporate membranous organelles. Together, these data define a mechanistic basis for regulated protoplasmic gelation, and provide new design principles for the development of artificial flow-responsive biomaterials.

Mechanism of interaction of an endofungal bacterium Serratia marcescens D1 with its host and non-host fungi.

Association of bacteria with fungi is a major area of research in infection biology, however, very few strains of bacteria have been reported that can invade and reside within fungal hyphae. Here, we report the characterization of an endofungal bacterium Serratia marcescens D1 from Mucor irregularis SS7 hyphae. Upon re-inoculation, colonization of the endobacterium S. marcescens D1 in the hyphae of Mucor irregularis SS7 was demonstrated using stereo microscopy. However, S. marcescens D1 failed to invade into the hyphae of the tested Ascomycetes (except Fusarium oxysporum) and Basidiomycetes. Remarkably, Serratia marcescens D1 could invade and spread over the culture of F. oxysporum that resulted in mycelial death. Prodigiosin, the red pigment produced by the Serratia marcescens D1, helps the bacterium to invade fungal hyphae as revealed by the increasing permeability in fungal cell membrane. On the other hand, genes encoding the type VI secretion system (T6SS) assembly protein TssJ and an outer membrane associated murein lipoprotein also showed significant up-regulation during the interaction process, suggesting the involvement of T6SS in the invasion process.

Dual production of polyunsaturated fatty acids and beta-carotene with Mucor wosnessenskii by the process of solid-state fermentation using agro-industrial waste.

Solid-state fermentation is a technique employing microorganisms grown on a solid substrate in the absence of free water. The substrates used in this process are mostly waste from the agro-industry (brans, spent malt grains, distiller grains, etc.) that improves not only the economy of the process but also has positive effect on waste management problems. Zygomycetous fungi are not only able to grow in such conditions but also enrich fermented materials with various types of bioactive compounds. Mucor sp. strains have been identified as producers of gamma-linolenic acid and beta-carotene in submerged fermentation. The aim of the present study was to identify the best microbial producer of gamma-linolenic acid and beta-carotene among four different Mucor strains and to study the requirements for the dual production of these metabolites. Mucor wosnessenskii was identified as the most suitable producer of both metabolites. After optimization of the fermentation conditions, the highest yields obtained were 10.7 g of gamma-linolenic acid/kg of fermented product and 261.5 mg of beta-carotene/kg of fermented product. This yield of beta-carotene is the highest among the results published so far.

Heterotrimeric G-alpha subunits Gpa11 and Gpa12 define a transduction pathway that control spore size and virulence in Mucor circinelloides.

Mucor circinelloides is one of the causal agents of mucormycosis, an emerging and high mortality rate fungal infection produced by asexual spores (sporangiospores) of fungi that belong to the order Mucorales. M. circinelloides has served as a model genetic system to understand the virulence mechanism of this infection. Although the G-protein signaling cascade plays crucial roles in virulence in many pathogenic fungi, its roles in Mucorales are yet to be elucidated. Previous study found that sporangiospore size and calcineurin are related to the virulence in Mucor, in which larger spores are more virulent in an animal mucormycosis model and loss of a calcineurin A catalytic subunit CnaA results in larger spore production and virulent phenotype. The M. circinelloides genome is known to harbor twelve gpa (gpa1 to gpa12) encoding G-protein alpha subunits and the transcripts of the gpa11 and gpa12 comprise nearly 72% of all twelve gpa genes transcript in spores. In this study we demonstrated that loss of function of Gpa11 and Gpa12 led to larger spore size associated with reduced activation of the calcineurin pathway. Interestingly, we found lower levels of the cnaA mRNAs in sporangiospores from the Δgpa12 and double Δgpa11/Δgpa12 mutant strains compared to wild-type and the ΔcnaA mutant had significantly lower gpa11 and gpa12 mRNA levels compared to wild-type. However, in contrast to the high virulence showed by the large spores of ΔcnaA, the spores from Δgpa11/Δgpa12 were avirulent and produced lower tissue invasion and cellular damage, suggesting that the gpa11 and gpa12 define a signal pathway with two branches. One of the branches controls spore size through regulation of calcineurin pathway, whereas virulences is controlled by an independent pathway. This virulence-related regulatory pathway could control the expression of genes involved in cellular responses important for virulence, since sporangiospores of Δgpa11/Δgpa12 were less resistant to oxidative stress and phagocytosis by macrophages than the ΔcnaA and wild-type strains. The characterization of this pathway could contribute to decipher the signals and mechanism used by Mucorales to produce mucormycosis.

Gastrointestinal microbiota alteration induced by Mucor circinelloides in a murine model.

Mucor circinelloides is a pathogenic fungus and etiologic agent of mucormycosis. In 2013, cases of gastrointestinal illness after yogurt consumption were reported to the US FDA, and the producer found that its products were contaminated with Mucor. A previous study found that the Mucor strain isolated from an open contaminated yogurt exhibited virulence in a murine systemic infection model and showed that this strain is capable of surviving passage through the gastrointestinal tract of mice. In this study, we isolated another Mucor strain from an unopened yogurt that is closely related but distinct from the first Mucor strain and subsequently examined if Mucor alters the gut microbiota in a murine host model. DNA extracted from a ten-day course of stool samples was used to analyze the microbiota in the gastrointestinal tracts of mice exposed via ingestion of Mucor spores. The bacterial 16S rRNA gene and fungal ITS1 sequences obtained were used to identify taxa of each kingdom. Linear regressions revealed that there are changes in bacterial and fungal abundance in the gastrointestinal tracts of mice which ingested Mucor. Furthermore, we found an increased abundance of the bacterial genus Bacteroides and a decreased abundance of the bacteria Akkermansia muciniphila in the gastrointestinal tracts of exposed mice. Measurements of abundances show shifts in relative levels of multiple bacterial and fungal taxa between mouse groups. These findings suggest that exposure of the gastrointestinal tract to Mucor can alter the microbiota and, more importantly, illustrate an interaction between the intestinal mycobiota and bacteriota. In addition, Mucor was able to induce increased permeability in epithelial cell monolayers in vitro, which might be indicative of unstable intestinal barriers. Understanding how the gut microbiota is shaped is important to understand the basis of potential methods of treatment for gastrointestinal illness. How the gut microbiota changes in response to exposure, even by pathogens not considered to be causative agents of food-borne illness, may be important to how commercial food producers prevent and respond to contamination of products aimed at the public. This study provides evidence that the fungal microbiota, though understudied, may play an important role in diseases of the human gut.

Functional complementarity of ancient plant-fungal mutualisms: contrasting nitrogen, phosphorus and carbon exchanges between Mucoromycotina and Glomeromycotina fungal symbionts of liverworts.

Liverworts, which are amongst the earliest divergent plant lineages and important ecosystem pioneers, often form nutritional mutualisms with arbuscular mycorrhiza-forming Glomeromycotina and fine-root endophytic Mucoromycotina fungi, both of which coevolved with early land plants. Some liverworts, in common with many later divergent plants, harbour both fungal groups, suggesting these fungi may complementarily improve plant access to different soil nutrients. We tested this hypothesis by growing liverworts in single and dual fungal partnerships under a modern atmosphere and under 1500 ppm [CO2 ], as experienced by early land plants. Access to soil nutrients via fungal partners was investigated with 15 N-labelled algal necromass and 33 P orthophosphate. Photosynthate allocation to fungi was traced using 14 CO2 . Only Mucoromycotina fungal partners provided liverworts with substantial access to algal 15 N, irrespective of atmospheric CO2 concentration. Both symbionts increased 33 P uptake, but Glomeromycotina were often more effective. Dual partnerships showed complementarity of nutrient pool use and greatest photosynthate allocation to symbiotic fungi. We show there are important functional differences between the plant-fungal symbioses tested, providing new insights into the functional biology of Glomeromycotina and Mucoromycotina fungal groups that form symbioses with plants. This may explain the persistence of the two fungal lineages in symbioses across the evolution of land plants.

A combined effort to avoid strongyle infection in horses in an oceanic climate region: rotational grazing and parasiticidal fungi.

BACKGROUND: An approach to preventing strongyle infection in horses was tested, comprising rotational pasturing and the administration of spores of two parasiticidal fungi, Mucor circinelloides and Duddingtonia flagrans. METHODS: Twenty-two adult Spanish Sport Horses were dewormed with ivermectin (1 mg pour-on/kg body weight) and then randomly divided into three groups. G-1 was maintained with continuous grazing, and G-2 and G-3 were kept on a four-paddock rotation system. Commercial pelleted feed (2.5 kg/horse) was supplied to G-1 and G-2 twice a week; horses in G-3 received pellets containing 2 × 106 spores/kg of each fungus. Fecal samples were analyzed by the flotation method to estimate the reduction in the fecal egg counts (FECR), the percentage of horses shedding eggs (PHR), and the egg reappearance period (ERP). RESULTS: Third-stage larvae were identified in fecal pats as Cyathostomum (sensu lato) types A, C and D, Gyalocephalus capitatus, Triodontophorus serratus, Poteriosthomum spp., Strongylus vulgaris and S. edentatus. Two weeks after treatment, the FECR values were 100% in G-1, 96% in G-2 and 99% in G-3; the PHR values were 100% in G-1, 75% in G-2 and 88% in G-3. A strongyle ERP of 6 weeks was observed in G-1, ERP of 10 weeks was observed in G-2, and ERP of 16 weeks was observed in G-3. The counts of eggs per gram of feces (EPG) were > 300 EPG in G-1 and G-2 but remained below 250 EPG in G-3 throughout the observation period of 12 months. CONCLUSIONS: These results suggest that horse strongyle infection could be decreased by combining rotational pasturing with feeding pellets containing the spores of parasiticidal fungi.

Biochemical mechanism of phytoremediation process of lead and cadmium pollution with Mucor circinelloides and Trichoderma asperellum.

This study focused on the bioremediation mechanisms of lead (0, 100, 500, 1000 mg kg-1) and cadmium (0,10,50,100 mg kg-1) contaminated soil using two indigenous fungi selected from mine tailings as the phytostimulation of Arabidopsis thaliana. The two fungal strains were characterized as Mucor circinelloides (MC) and Trichoderma asperellum (TA) by internal transcribed spacer sequencing at the genetic levels. Our research revealed that Cadmium was more toxic to plant growth than lead and meanwhile, MC and TA can strengthen A. thaliana tolerance to cadmium and lead with 40.19-117.50% higher root length and 58.31-154.14% shoot fresh weight of plant compared to non-inoculation. In this study, TA exhibited a higher potential to the inactivation of cadmium; however, MC was more effective in lead passivation. There was a direct correlation between the type of fungi, heavy metal content, heavy metal type and oxidative damage in plant. Both lead and cadmium induced oxidative damage as indicated by increased superoxide dismutase and catalase activities, while the antioxidant levels were significantly higher in fungal inoculated plants compared with those non-inoculated. The analysis of soil enzyme activity and taxonomic richness uncovered that the dominant structures of soil microbial community were altered by exogenous microbial agents. MC enhanced higher microbial diversity and soil enzyme activity than TA. The two indigenous fungi lessened several limiting factors with respect to phytoremediation technology, such as soil chemistry, contamination level and transformation, and metal solubility.

Does co-inoculation of Lactuca serriola with endophytic and arbuscular mycorrhizal fungi improve plant growth in a polluted environment?

Phytoremediation of polluted sites can be improved by co-inoculation with mycorrhizal and endophytic fungi. In this study, the effects of single- and co-inoculation of Lactuca serriola with an arbuscular mycorrhizal (AM) fungus, Rhizoglomus intraradices, and endophytic fungi, Mucor sp. or Trichoderma asperellum, on plant growth, vitality, toxic metal accumulation, sesquiterpene lactone production and flavonoid concentration in the presence of toxic metals were evaluated. Inoculation with the AM fungus increased biomass yield of the plants grown on non-polluted and polluted substrate. Co-inoculation with the AM fungus and Mucor sp. resulted in increased biomass yield of plants cultivated on the polluted substrate, whereas co-inoculation with T. asperellum and the AM fungus increased plant biomass on the non-polluted substrate. In the presence of Mucor sp., mycorrhizal colonization and arbuscule richness were increased in the non-polluted substrate. Co-inoculation with the AM fungus and Mucor sp. increased Zn concentration in leaves and roots. The concentration of sesquiterpene lactones in plant leaves was decreased by AM fungus inoculation in both substrates. Despite enhanced host plant costs caused by maintaining symbiosis with numerous microorganisms, interaction of wild lettuce with both mycorrhizal and endophytic fungi was more beneficial than that with a single fungus. The study shows the potential of double inoculation in unfavourable environments, including agricultural areas and toxic metal-polluted areas.

A protein complex bearing an oxidase with napthalene dihydrodiol dehydrogenase activity is induced in Mucor circinelloides strain YR-1 during growth on polycyclic aromatic compounds.

Fungi are organisms capable of growing in a myriad of conditions and respond to counteract environmental cues. Several locations in the world are polluted with oil and its derivatives, and some microorganisms tolerant to these compounds have been isolated. Some fungi can grow in the presence of molecules such as polycyclic aromatic hydrocarbons as sole carbon sources. In this report, we further characterized the induced enzymes with phenanthrene from Mucor circinelloides YR-1 strain, isolated from a polluted field near a petrochemical facility in México. We identified a putative oxidase that is induced when growth with phenanthrene as sole carbon source at a pH of 8.5 and is NADP+ dependent. We show that this enzyme bears naphthalene dihydrodiol dehydrogenase activity with substrate preference for the cis-naphthalene over the trans-naphthalene, with an optimal pH in the range of 8-10. Mass spectrometry analysis revealed that the induced enzyme belongs to the NADP+ oxidase family enzymes with the typical Rossmann-fold for NADP+ binding. This enzyme seems to form a high molecular weight structure (~ 541 kDa) and with a monomer of 57 kDa, suggesting that the multimer is constituted of 10 subunits. Our findings contribute to understanding of the roles that dihydrodiol dehydrogenases have in organisms exposed to toxic compounds in the environment and can regulate their expression.

The gut mycobiome composition is linked to carotid atherosclerosis.

The mycobiotic component of the microbiota comprises an integral, yet under-researched, part of the gastrointestinal tract. Here, we present a preliminary study of the possible contribution of gut mycobiota to sub-clinical atherosclerosis in a well-characterised group of obese and non-obese subjects in association with the Framingham Risk Score (FRS) and carotid intima-media thickness (cIMT). From all taxa identified, the relative abundance of the phylum Zygomycota, comprising the family Mucoraceae and genus Mucor, was negatively associated with cIMT and this association remained significant after controlling for false discovery rate. Obese subjects with detectable Mucor spp. had a similar cardiovascular risk profile as non-obese subjects. Interestingly, the relative abundance of Mucor racemosus was negatively associated both with FRS and cIMT. Partial least square discriminant analyses modelling, evaluating the potential relevance of gut mycobiota in patients stratified by mean values of cIMT, showed that even a 1 component model had a high accuracy (0.789), with a high R2 value (0.51). Variable importance in projection scores showed that M. racemosus abundance had the same impact in the model as waist-to-hip ratio, high-density lipoprotein-cholesterol, fasting triglycerides or fasting glucose, suggesting that M. racemosus relative abundance in the gut may be a relevant biomarker for cardiovascular risk.

The influence of the mating type on virulence of Mucor irregularis.

Mucor irregularis is an emerging fungal pathogen that cause cutaneous infection and could cause death. However, little is known about its mechanism of pathogenesis. There is evidence suggesting virulence vary with mating types in fungi, including the Mucorales. Here, we characterized the mating type locus of M. irregularis and the mating type ratio of 17 clinical isolates in China. Genomic data indicated M. irregularis is heterothallic having two mating types - bearing either SexP or SexM allele. Also, we employed a mice model to study the inflammation and pathological effects of different mating types. The comparison of the inflammatory response, cytokine profiles and Th-1, Th-2 and Th-17 cells numbers in each mating type treated mice showed that the severity and disease progress were enhanced in (+) mating type treated mice. One (+/0) mutant strain, with multiple mutations at the mating locus, had defects in sexual mating ability but appeared to be more virulent than the (-) mating type. Although (+) mating type appeared to be more virulent, most of our clinical isolates presented belonged to (-) mating type. Our findings support the involvement of MAT genes in sexual fertility, and the influence of mating type on the severity of cutaneous infection.

Responses of the antioxidative and biotransformation enzymes in the aquatic fungus Mucor hiemalis exposed to cyanotoxins.

OBJECTIVES: To investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications. RESULTS: Catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M. hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100 µg MC-LR l-1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100 µg l-1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. ß-N-methylamino-L-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected. CONCLUSIONS: M. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.

Effect of the Filamentous Fungus Mucor circinelloides On The Development of Eggs of the Rumen Fluke Calicophoron daubneyi (Paramphistomidae).

Ruminants infected by Paramphistomidae flukes shed eggs in the feces, which pass through different stages in the environment until the infective stages (metacercariae) are reached. The activity of the soil fungus Mucor circinelloides on the development of eggs of the rumen fluke Calicophoron daubneyi was presently tested with 3 probes, i.e., in petri plates, feces, and an aqueous environment (tubes). The effect of the fungus was assessed by recording the numbers of undeveloped, nonviable, and embryonated eggs. Nonviable eggs were considered when vacuolization occurred, the inner structures were not clearly observed, the eggshell was broken, or the embryo inside was destroyed. By considering the ability of hyphae of M. circinelloides to develop in the presence of C. daubneyi eggs, attach to their surface, and penetrate and destroy the inner embryo, this ovicidal effect was classified as type 3. After a period of 50 days, the percentage of undeveloped eggs in the feces of infected cattle was 40%; furthermore, 27% of the eggs were nonviable, and 33% were embryonated (1 miracidium inside). The addition of 4 doses of M. circinelloides spores directly onto the feces resulted in 9-31% undeveloped eggs, 38-60% nonviable eggs, and 9-21% embryonated eggs, and no statistical significances were obtained among the different doses. Placing the eggs of C. daubneyi into an aqueous solution containing 107 spores of M. circinelloides/ml for 29 days resulted in 43% undeveloped eggs, 40% nonviable eggs, and 17% embryonated eggs, whereas in the controls, the percentages were 48%, 12%, and 40%, respectively. These data demonstrate the usefulness of the spores of the fungus M. circinelloides in limiting the development of the eggs of the trematode C. daubneyi.

The Effects of Different Carbon Sources on the Antifungal Activity by Lactic Acid Bacteria.

Lactobacillus rhamnosus R-2002 strain isolated from Armenian pickled cheese sample has a fungicide effect, which was carried out by the first-time designed method. The fungicide effect of LAB strain was expressed after 5 days of co-cultivation with Mucor plumbeus and 6 days of co-cultivation with Penicillium aurantioviolaceum. The growth as well as the antifungal activity of L. rhamnosus R-2002 was shown to directly depend on the nature of the medium's carbon source. The antifungal activity of this strain against both moulds was inhibited when the combination of 10 g l-1 glucose and 10 ml l-1 ethanol in the medium was used as a carbon source. The results could be applied in food industry, medicine, and veterinary.

Fungus-associated bacteriome in charge of their host behavior.

Bacterial-fungal interactions are widespread in nature and there is a growing number of studies reporting distinct fungus-associated bacteria. However, little is known so far about how shifts in the fungus-associated bacteriome will affect the fungal host's lifestyle. In the present study, we describe for the first time the bacterial community associated with the saprotrophic fungus Mucor hiemalis, commonly found in soil and rhizosphere. Two broad-spectrum antibiotics that strongly altered the bacterial community associated with the fungus were applied. Our results revealed that the antibiotic treatment did not significantly reduce the amount of bacteria associated to the fungus but rather changed the community composition by shifting from initially dominating Alpha-Proteobacteria to dominance of Gamma-Proteobacteria. A novel approach was applied for the isolation of fungal-associated bacteria which also revealed differences between bacterial isolates obtained from the original and the antibiotic-treated M. hiemalis. The shift in the composition of the fungal-associated bacterial community led to significantly reduced fungal growth, changes in fungal morphology, behavior and secondary-metabolites production. Furthermore, our results showed that the antibiotic-treated isolate was more attractive and susceptible to mycophagous bacteria as compared to the original isolate. Overall, our study highlights the importance of the fungus-associated bacteriome for the host's lifestyle and interactions and indicate that isolation with antibacterials is not sufficient to eradicate the associated bacteria.

Post-translational regulation by structural changes of 4-dihydromethyltrisporate dehydrogenase, a key enzyme in sexual and parasitic communication mediated by the trisporic acid pheromone system, of the fungal fusion parasite Parasitella parasitica.

Sexual communication between complementary mating partners in the fungal group of zygomycetes is mediated by the trisporoid pheromone system. A key enzyme towards biosynthesis of hormonally active trisporoids is 4-dihydromethyltrisporate dehydrogenase (TSP1), an enzyme occurring in all zygomycetous fungi. Trisporic acid and some of its precursor molecules serve as pheromones for recognizing complementary mating partners and for induction of the differentiation program towards sexual spore formation. In the parasitic zygomycete Parasitella parasitica, a biotrophic fusion parasite infecting many other zygomycetes, these substances have an additional function: They are also responsible for host-parasite recognition and the formation of the characteristic infection structures. Parasitic interactions are mating type dependent as well. In the Mucor-related mycoparasite P. parasitica we can study both types of communication in parallel. We were interested in protein structures of TSP1 from P. parasitica, the genome of which was recently sequenced by us, and especially in the mechanisms involved in the switch from sexual to parasitic communication. P. parasitica contains at least six genes coding for TSP1-like proteins. We created models of tertiary structures and performed protein-protein docking with the resulting protein structures to simulate dimerization and to provide support for probable regulatory mechanisms at the protein level. The resulting structure models show differences in putative activity and binding preferences between the different TSP1-like proteins. Two of them seem to be able to form solid binding pockets for substrate and cosubstrate after dimerization. The other four TSP1-like proteins are more likely to represent regulating subunits for the two active isoforms. The ability to form homodimers with enzymatic activity could be the crucial difference between sexual and parasitic communication pathways. TSP1 PARPA_07791 forms enzymatically inactive homodimers. The second TSP1, PARPA_04105, forms active homodimers and could be responsible for the parasitic pathway of communication. Both TSP1 proteins can form more or less active heterodimers with the additional TSP1-like proteins. TSP1 PARPA_07791 mediates the sexual pathway probably as in other zygomycetous fungi like Mucor mucedo. High sequence identities between this TSP1 isomer and TSP1 proteins from other zygomycetes substantiate its function. This bioinformatic study supports previous experimental findings of post-translational regulation of 4-dihydromethyltrisporate dehydrogenases in zygomycetes and, for the first time, provides a substantiated hypothesis of the underlying mechanism.

Proteomic analysis of the adaptative response of Mucor spp. to cheese environment.

In the cheese industry context, Mucor species exhibit an ambivalent behavior as some species are essential "technological" organisms of some cheeses while others can be spoiling agents. Previously, we observed that cheese "technological" species exhibited higher optimal growth rates on cheese related matrices than on synthetic media. This growth pattern combined with morphological differences raise the question of their adaptation to cheese. In this study, using a comparative proteomic approach, we described the metabolic pathways of three Mucor strains considered as "technological" or "contaminant" in the cheese environment (M. lanceolatus UBOCC-A-109153, M. racemosus UBOCC-A-109155, M. circinelloides CBS 277-49) as well as a non-cheese related strain (M. endophyticus CBS 385-95). Overall, 15.8 to 19.0% of the proteomes showed a fold change ≥1.6 in Potato Dextrose Agar (PDA) versus Cheese Agar (CA), a cheese mimicking-medium. The 289 differentially expressed proteins identified by LC MS-MS analysis were mostly assigned to energy and amino-acid metabolisms in PDA whereas a higher diversity of biological processes was observed for cheese related strains in CA. Surprisingly, the vast majority (72.9%) of the over-accumulated proteins were different according to the considered medium and strain. These results strongly suggest that the observed better adaptative response of "technological" strains to cheese environment is mediated by species-specific proteins. BIOLOGICAL SIGNIFICANCE: The Mucor genus consists of a multitude of poorly known species. In the food context, few species are known for their positive role in the production of various food products, including cheese, while others are spoiling agents. The present study focused on the analysis of morphological and proteome differences of various Mucor spp. representative strains known as either positively (hereafter referred as "technological") or negatively (hereafter referred as "contaminant") associated with cheese or non-related to cheese (endophyte) on two different media, a synthetic medium and a cheese-mimicking medium. The main goal was to assess if adaptative traits of "technological" strains to the cheese environment could be identified. This work was based on observations we did in a recently published physiological study (Morin-Sardin et al., 2016). One of the important innovative aspects lies in the use for the first time of an extensive 2-DE approach to compare proteome variations for 4 strains on two different media. Results obtained offered an insight in the metabolic mechanisms associated with growth on a given medium and showed that adaptation to cheese environment is probably supported by species-specific proteins. The obtained data represent an essential step point for more targeted studies at the genomic and transcriptomic levels.

Characterization and control of Mucor circinelloides spoilage in yogurt.

Consumer confidence in the food industry is severely affected by large-scale spoilage incidents. However, relatively little research exists on spoilage potential of members of the fungal subphylum Mucormycotina (e.g. Mucor), which includes dimorphic spoilage organisms that can switch between a yeast-like and hyphal phase depending on environmental conditions. The presence of Mucor circinelloides in yogurt may not cause spoilage, but growth and subsequent changes in quality (e.g. container bloating) can cause spoilage if not controlled. The purpose of this study was to evaluate the effects on M. circinelloides of pasteurization regimen, natamycin concentrations, and storage temperature in yogurt production, as measured by fungal proliferation and carbon dioxide production. A strain of M. circinelloides isolated from commercially spoiled yogurt showed greater yogurt-spoilage potential than clinical isolates and other industrial strains. D-values and z-values were determined for the spoilage isolate in milk as an evaluation of the fungus' ability to survive pasteurization. Natamycin was added to yogurt at 0, 5, 10, 15, and 20ppm (µg/ml) to determine its ability to inhibit M. circinelloides over the course of month-long challenge studies at 4°C, 15°C, and 25°C. Survivors were recovered on acidified PDA and carbon dioxide levels were recorded. The D-values at 54°C, 56°C, and 58°C for hyphae/sporangiospores were (in min) 38.31±0.02, 10.17±0.28, and 1.94±0.53, respectively, which yielded a z-value of 3.09°C. The D-values at 51°C, 53°C, and 55°C for yeast-like cells were (in min) 14.25±0.12, 6.87±1.19, and 2.44±0.35, respectively, which yielded a z-value of 0.34°C. These results indicated that M. circinelloides would not survive fluid milk pasteurization if contamination occurred prior to thermal treatment. CO2 production was only observed when M. circinelloides was incubated under low-oxygen conditions, and occurred only at temperatures above 4°C. Addition of 10ppm and greater of natamycin inhibited the growth and CO2 production of M. circinelloides under moderate temperature abuse when compared to the untreated control. These data suggest that yogurt spoilage (container bloating) caused by anaerobic growth of M. circinelloides is due to post-pasteurization contamination. Temperature abuse facilitated spoilage as CO2 production was observed in yogurt incubated at 15°C and 25°C, but not at 4°C. The addition of at least 10ppm of natamycin prevented M. circinelloides growth in both hyphal and yeast-like phases, as well as CO2 production in temperatures of up to 15°C for 30days.