Quantitative proteomic profiling of ochratoxin A repression in Penicillium nordicum by protective cultures.

Dry-cured meat products are usually contaminated with moulds during ripening. Although fungal development contributes to the desired sensory characteristics, some moulds, such as Penicillium nordicum are able to produce ochratoxin A (OTA) on meat products. Therefore, strategies to prevent OTA contamination in ripened meat products are required. Microorganisms isolated from these meat products can be adequate as biocontrol agents, given that no negative sensory impact is expected. The PgAFP antifungal protein-producer Penicillium chrysogenum (Pc) and Debaryomyces hansenii (Dh) have been shown to successfully inhibit toxigenic moulds. However, scarce information about the mechanism of action of these biocontrol agents on toxigenic mould inhibition is available. Comparative proteomic analysis is a powerful tool to investigate the physiological response of microorganisms to stimuli. Proteomic analysis was carried out on P. nordicum co-cultured with Pc, Dh, PgAFP, and their combinations on a dry-cured ham-based medium. Additionally, OTA production by P. nordicum in the different cultures was measured. The individual inoculation of Pc or Dh repressed OTA production by P. nordicum by 5 and 3.15 fold, respectively. A total of 2844 unique P. nordicum proteins were identified by proteomic analysis. The impact of the biocontrol agents on the proteome of P. nordicum was higher for Pc-containing cultures, followed by Dh-containing treatments. PgAFP alone had minimal impact on the proteome of P. nordicum. Proteomic analyses indicated Pc repressed P. nordicum OTA production through nutrient competition, potentially reducing glucose availability. Data also suggest that Dh and Pc inhibited P. nordicum through cell wall integrity impairment. Both Pc and Dh seem to hamper P. nordicum secondary metabolism (SM) as indicated by lower levels of MAP kinases and SM-associated proteins found in the co-inoculated P. nordicum. This work paves the way to use antifungal agents in the most efficient way to prevent OTA formation in meat products.

Inventory of ABC proteins and their putative role in salt and drug tolerance in Debaryomyces hansenii.

ATP-binding cassette (ABC) is one of the largest superfamily of proteins, which are ubiquitously present, performing variety of cellular functions. These proteins as drug transporters have been enticing substantial consideration because of their clinical importance. The present study focuses on genome wide identification of ABC proteins of an important halotolerant yeast Debaryomyces hansenii and explores their role in salt and drug tolerance. Our bioinformatics analysis identified a total of 30 putative ABC protein-coding genes whose expression at transcript level was confirmed by qRT-PCR. Our comparative phylogenetic analysis of nucleotide binding domains of D. hansenii and topology prediction categorized these proteins into six subfamilies; ABCB/MDR, ABCC/MRP, ABCD/ALDP, ABCF/YEF3, ABCE/RLI, and ABCG/PDR based on the nomenclature adopted by the Human Genome Organization (HUGO). Further, our transmembrane domain (TMD) predictions suggest that out of 30 ABC proteins, only 22 proteins possess either two or one TMD and hence are considered as membrane localized ABC proteins. Notably, our transcriptional dynamics of ABC proteins encoding genes following D. hansenii cells treatment with different salts and drugs concentrations illustrated variable transcriptional response of some of the genes, pointing to their role in salt and drug tolerance. This study first time provides a comprehensive inventory of the ABC proteins of a haploid D. hansenii which will be helpful for exploring their functional relevance.

Hanseniaspora thailandica BC9 ß-Glucosidase for the Production of ß-D-Hexyl Glucoside.

For biotechnological production of high-valued ß-D-hexyl glucoside, the catalytic properties of Hanseniaspora thailandica BC9 ß-glucosidase purified from the periplasmic fraction were studied, and the transglycosylation activity for the production of ß-D-hexyl glucoside was optimized. The constitutive BC9 ß-glucosidase exhibited maximum specific activity at pH 6.0 and 40ºC, and the activity of BC9 ß-glucosidase was not significantly inhibited by various metal ions. BC9 ß-glucosidase did not show a significant activity of cellobiose hydrolysis, but the activity was rather enhanced in the presence of sucrose and medium-chain alcohols. BC9 ß-glucosidase exhibited enhanced production of ß-D-hexyl glucoside in the presence of DMSO, and 62% of ß-D-hexyl glucoside conversion was recorded in 4 h in the presence of 5% 1-hexanol and 15% DMSO.

Molecular characterization of Aspergillus fumigatus TcsC, a characteristic type III hybrid histidine kinase of filamentous fungi harboring six HAMP domains.

The type III hybrid histidine kinase (HHK) TcsC enables the pathogenic mold Aspergillus fumigatus to thrive under hyperosmotic conditions. It is, moreover, of particular interest, since it is the target of certain antifungal agents, such as fludioxonil. This study was aimed at a functional characterization of the domains that constitute the sensing and the kinase module of TcsC. The sensing module consists of six HAMP domains, an architecture that is commonly found in type III HHKs of filamentous fungi. To dissect the functional role of the individual domains, we have analyzed a set of truncated derivatives of TcsC with respect to their impact on fungal growth and their ability to respond to hyperosmotic stress and fludioxonil. Our data demonstrate that the TcsC kinase module per se is constitutively active and under the control of the sensing module. We furthermore found that the sixth HAMP domain alone is sufficient to arrest the kinase module in an inactive state. This effect can be partially lifted by the presence of the fifth HAMP domain. Constructs harboring more than these two HAMP domains are per se inactive and all six HAMP domains are required to enable a response to fludioxonil or hyperosmotic stress. When expressed in an A. fumigatus wild type strain, the construct harboring only the sixth HAMP domain exerts a strong dominant negative effect on the native TcsC. This effect is successively reduced in other constructs harboring increasing numbers of HAMP domains. To our knowledge, this is the first molecular characterization of a type III HHK containing six HAMP domains. Our data strongly suggest that TcsC is a positive regulator of its MAPK SakA and thereby differs fundamentally from the prototypic yeast type III HHK DhNik1 of Debaryomyces hansenii, which harbors only five HAMP domains and acts as a negative regulator of its MAPK.

Identification of key residues in Debaryomyces hansenii carbonyl reductase for highly productive preparation of (S)-aryl halohydrins.

Key residues of Debaryomyces hansenii carbonyl reductase in the determination of the reducing activity towards aryl haloketones were identified through combinatorial mutation of conserved residues. This study provides a green and efficient biocatalyst for the synthesis of (S)-aryl halohydrins.

Sporadic distribution of prion-forming ability of Sup35p from yeasts and fungi.

Sup35p of Saccharomyces cerevisiae can form the [PSI+] prion, an infectious amyloid in which the protein is largely inactive. The part of Sup35p that forms the amyloid is the region normally involved in control of mRNA turnover. The formation of [PSI+] by Sup35p's from other yeasts has been interpreted to imply that the prion-forming ability of Sup35p is conserved in evolution, and thus of survival/fitness/evolutionary value to these organisms. We surveyed a larger number of yeast and fungal species by the same criteria as used previously and find that the Sup35p from many species cannot form prions. [PSI+] could be formed by the Sup35p from Candida albicans, Candida maltosa, Debaromyces hansenii, and Kluyveromyces lactis, but orders of magnitude less often than the S. cerevisiae Sup35p converts to the prion form. The Sup35s from Schizosaccharomyces pombe and Ashbya gossypii clearly do not form [PSI+]. We were also unable to detect [PSI+] formation by the Sup35ps from Aspergillus nidulans, Aspergillus fumigatus, Magnaporthe grisea, Ustilago maydis, or Cryptococcus neoformans. Each of two C. albicans SUP35 alleles can form [PSI+], but transmission from one to the other is partially blocked. These results suggest that the prion-forming ability of Sup35p is not a conserved trait, but is an occasional deleterious side effect of a protein domain conserved for another function.

Differential role of HAMP-like linkers in regulating the functionality of the group III histidine kinase DhNik1p.

Nik1 orthologs are sensor kinases that function upstream of the high osmolarity glycerol/p38 MAPK pathway in fungi. They contain a poly-HAMP module at their N terminus, which plays a pivotal role in osmosensing as well as fungal death upon exposure to fludioxonil. DhNik1p is a typical member of this class that contains five HAMP domains and four HAMP-like linkers. We investigated the contribution of each of the HAMP-like linker regions to the functionality of DhNik1p and found that the HAMP4b linker was essential as its deletion resulted in the complete loss of activity. Replacement of this linker with flexible peptide sequences did not restore DhNik1p activity. Thus, the HAMP-like sequence and possibly structural features of this linker region are indispensable for the kinase activity of DhNik1p. To gain insight into the global shape of the poly-HAMP module in DhNik1p (HAMP1­5), multi-angle laser light and small angle x-ray scattering studies were carried out. Those data demonstrate that the maltose-binding protein-tagged HAMP1­5 protein exist as a dimer in solution with an elongated shape of maximum linear dimension ∼365 Å. Placement of a sequence similarity based model of the HAMP1­5 protein inside experimental data-based models showed how two chains of HAMP1­5 are entwined on each other and the overall structure retained a periodicity. Normal mode analysis of the structural model is consistent with the H4b linker being a key to native-like collective motion in the protein. Overall, our shape-function studies reveal how different elements in the HAMP1­5 structure mediate its function.

Hydrolysis of soybean isoflavones by Debaryomyces hansenii UFV-1 immobilised cells and free ß-glucosidase.

An intracellular ß-glucosidase from Debaryomyceshansenii UFV-1 was produced in an YP medium with cellobiose as the carbon source. This enzyme was purified, characterised and presented a Mr of 65.15kDa. Yeast cells containing the intracellular ß-glucosidase were immobilised in calcium alginate. The free ß-glucosidase and immobilised cells containing the enzyme presented optima values of pH and temperature of 6.0 and 45°C and 5.5 and 50°C, respectively. The free enzyme maintained 62% and 47% of its original activity after 90days at 4°C and after 15days at room temperature, respectively. The immobilisation process resulted in higher enzyme thermostability at 45 and 50°C. Soy molasses treatment with the free enzyme and the immobilised cells containing ß-glucosidase, for 2h at 40°C, promoted efficient hydrolysis of isoflavone glicosides to their aglycon forms. The results suggest that this enzyme could be used in the food industry, in the free or immobilised forms, for a safe and efficient process to hydrolyse isoflavone glycosides in soy molasses.

Molecular cloning, characterization, and engineering of xylitol dehydrogenase from Debaryomyces hansenii.

Because of its natural ability to utilize both xylose and arabinose, the halotolerant and osmotolerant yeast Debaryomyces hansenii is considered as a potential microbial platform for exploiting lignocellulosic biomass. To gain better understanding of the xylose metabolism in D. hansenii, we have cloned and characterized a xylitol dehydrogenase gene (DhXDH). The cloned gene appeared to be essential for xylose metabolism in D. hansenii as the deletion of this gene abolished the growth of the cells on xylose. The expression of DhXDH was strongly upregulated in the presence of xylose. Recombinant DhXdhp was expressed and purified from Escherichia coli. DhXdhp was highly active against xylitol and sorbitol as substrate. Our results showed that DhXdhp was thermo-sensitive, and except this, its biochemical properties were quite comparable with XDH from other yeast species. Furthermore, to make this enzyme suitable for metabolic engineering of D. hansenii, we have improved its thermotolerance and modified cofactor requirement through modelling and mutagenesis approach.

Co-factor binding confers substrate specificity to xylose reductase from Debaryomyces hansenii.

Binding of substrates into the active site, often through complementarity of shapes and charges, is central to the specificity of an enzyme. In many cases, substrate binding induces conformational changes in the active site, promoting specific interactions between them. In contrast, non-substrates either fail to bind or do not induce the requisite conformational changes upon binding and thus no catalysis occurs. In principle, both lock and key and induced-fit binding can provide specific interactions between the substrate and the enzyme. In this study, we present an interesting case where cofactor binding pre-tunes the active site geometry to recognize only the cognate substrates. We illustrate this principle by studying the substrate binding and kinetic properties of Xylose Reductase from Debaryomyces hansenii (DhXR), an AKR family enzyme which catalyzes the reduction of carbonyl substrates using NADPH as co-factor. DhXR reduces D-xylose with increased specificity and shows no activity towards "non-substrate" sugars like L-rhamnose. Interestingly, apo-DhXR binds to D-xylose and L-rhamnose with similar affinity (K(d)∼5.0-10.0 mM). Crystal structure of apo-DhXR-rhamnose complex shows that L-rhamnose is bound to the active site cavity. L-rhamnose does not bind to holo-DhXR complex and thus, it cannot competitively inhibit D-xylose binding and catalysis even at 4-5 fold molar excess. Comparison of K(d) values with K(m) values reveals that increased specificity for D-xylose is achieved at the cost of moderately reduced affinity. The present work reveals a latent regulatory role for cofactor binding which was previously unknown and suggests that cofactor induced conformational changes may increase the complimentarity between D-xylose and active site similar to specificity achieved through induced-fit mechanism.

Yeast water channels: an overview of orthodox aquaporins.

In yeast, the presence of orthodox aquaporins has been first recognized in Saccharomyces cerevisiae, in which two genes (AQY1 and AQY2) were shown to be related to mammal and plant water channels. The present review summarizes the putative orthodox aquaporin protein sequences found in available genomes of yeast and filamentous fungi. Among the 28 yeast genomes sequenced, most species present only one orthodox aquaporin, and no aquaporins were found in eight yeast species. Alignment of amino acid sequences reveals a very diverse group. Similarity values vary from 99% among species within the Saccharomyces genus to 34% between ScAqy1 and the aquaporin from Debaryomyces hansenii. All of the fungal aquaporins possess the known characteristic sequences, and residues involved in the water channel pore are highly conserved. Advances in the establishment of the structure are reviewed in relation to the mechanisms of selectivity, conductance and gating. In particular, the involvement of the protein cytosolic N-terminus as a channel blocker preventing water flow is addressed. Methodologies used in the evaluation of aquaporin activity frequently involve the measurement of fast volume changes. Particular attention is paid to data analysis to obtain accurate membrane water permeability parameters. Although the presence of aquaporins clearly enhances membrane water permeability, the relevance of these ubiquitous water channels in yeast performance remains obscure.

Saccharomyces cerevisiae CCMI 885 secretes peptides that inhibit the growth of some non-Saccharomyces wine-related strains.

The nature of the toxic compounds produced by Saccharomyces cerevisiae CCMI 885 that induce the early death of Hanseniaspora guilliermondii during mixed fermentations, as well as their ability to inhibit the growth of other non-Saccharomyces wine-related strains, was investigated. The killing effect of mixed supernatants towards H. guilliermondii was inactivated by protease treatments, thus revealing the proteinaceous nature of the toxic compounds. Analysis of the protein pattern of mixed supernatants on Tricine SDS-PAGE showed that this S. cerevisiae strain secretes peptides (<10 kDa), which were detected only when death of H. guilliermondii was already established. Death-inducing supernatants were ultrafiltrated by 10 and 2 kDa membranes, respectively, and the inhibitory effect of those permeates were tested in H. guilliermondii cultures. Results indicated that the (2-10) kDa protein fraction of those supernatants seemed to contain antimicrobial peptides active against H. guilliermondii. Thus, the (2-10) kDa protein fraction was concentrated and its inhibitory effect tested against strains of Kluyveromyces marxianus, Kluyveromyces thermotolerans, Torulaspora delbrueckii and H. guilliermondii. Under the growth conditions used for these tests, the (2-10) kDa protein fraction of S. cerevisiae CCMI 885 supernatants exhibited a fungistatic effect against all the strains and a fungicidal effect against K. marxianus.

N1,N12-diacetylspermine oxidase from Debaryomyces hansenii T-42: purification, characterization, molecular cloning and gene expression.

An FAD-dependent N(1),N(12)-diacetylspermine oxidase (DASpmOX), which seems suitable for enzymatic determination of the tumor marker N(1),N(12)-diacetylspermine (DASpm), was isolated from Debaryomyces hansenii T-42. DASpmOX exhibited the most excellent specificity toward DASpm among all polyamine oxidases found to date, and the specificity for DASpm could be raised by adjusting the pH of the buffer and adding TritonX-100. In potassium phosphate (pH 7.0) with 0.3% TritonX-100, this enzyme did not have any detectable activity toward free polyamines, and the reaction rate of N(1),N(8)-diacetylspermidine, N(1)-acetylspermine, N(1)-acetylspermidine, and N(8)-acetylspermidine was only 19%, 7.8%, 7.8%, and 1.0% of that of DASpm, respectively. The gene encoding DASpmOX was cloned and expressed in Escherichia coli. The apparent k(cat) and K(m) values of recombinant enzyme for DASpm were found to be 158 s(-1) and 3.1 x 10(-4) M under the conditions described above, respectively.

Cloning and characterization of two K+ transporters of Debaryomyces hansenii.

Two genes from the halotolerant yeast Debaryomyces hansenii were cloned, DhTRK1 and DhHAK1. These genes encode K(+) transporters with sequence similarities to the TRK and HAK transporters from Debaryomyces occidentalis and Candida albicans. The DhHAK1p transporter was only expressed in K(+)-starved cells, as shown by Northern blot analysis. Both DhTRK1p and DhHAK1p were expressed in a trk1Delta trk2Delta mutant of Saccharomyces cerevisiae, unable to grow at low K(+). This expression resulted in partial recovery of growth and ability to retain K(+) at low concentrations. In liquid media, 0.5 M NaCl affected growth of these S. cerevisiae transformants as it does in D. hansenii, resulting in a much less deleterious effect than in wild-type S. cerevisiae. Kinetics of Rb(+) uptake in the transformants suggest that DhTRK1p and DhHAK1p code for moderate-affinity K(+) transporters exhibiting a sigmoid response against Rb(+) concentration and presenting a deviation from classic Michaelis-Menten kinetics at low substrate concentrations. Rb(+) uptake by the DhTRK1p transporter was stimulated by millimolar concentrations of Na(+) at pH 4.5. The good performance of DhTRK1p in the presence of NaCl may be a key feature in the halotolerance of D. hansenii.

Intracellular Na and K distribution in Debaryomyces hansenii. Cloning and expression in Saccharomyces cerevisiae of DhNHX1.

Debaryomyces hansenii is a salt-tolerant yeast that contains high amounts of internal Na(+). Debaryomyces hansenii kept more sodium than Saccharomyces cerevisiae in both the cytoplasm and vacuole when grown under a variety of NaCl concentrations. These results indicate a higher tolerance of Debaryomyces to high internal Na(+), and, in addition, suggest the existence of a transporter driving Na(+) into the vacuole. Moreover, a gene encoding a Na(+) (K(+))/H(+) antiporter from D. hansenii was cloned and sequenced. The gene, designated DhNHX1, exhibited significant homology with genes of the NHE/NHX family. DhNHX1 expression was induced neither at low pH nor by extracellular NaCl. A mutant of S. cerevisiae lacking its own Na(+) transporters (ena1-4Delta nha1 Delta nhx1 Delta), when transformed with DhNHX1, partially recovered cation tolerance as well as the ability to accumulate Na(+) and K(+) into the vacuole. Our analysis provides evidence that DhNhx1p transports Na(+) (and K(+)) into the vacuole and that it can play an important role in ion homeostasis and salt tolerance.

Regulatory genes controlling fatty acid catabolism and peroxisomal functions in the filamentous fungus Aspergillus nidulans.

The catabolism of fatty acids is important in the lifestyle of many fungi, including plant and animal pathogens. This has been investigated in Aspergillus nidulans, which can grow on acetate and fatty acids as sources of carbon, resulting in the production of acetyl coenzyme A (CoA). Acetyl-CoA is metabolized via the glyoxalate bypass, located in peroxisomes, enabling gluconeogenesis. Acetate induction of enzymes specific for acetate utilization as well as glyoxalate bypass enzymes is via the Zn2-Cys6 binuclear cluster activator FacB. However, enzymes of the glyoxalate bypass as well as fatty acid beta-oxidation and peroxisomal proteins are also inducible by fatty acids. We have isolated mutants that cannot grow on fatty acids. Two of the corresponding genes, farA and farB, encode two highly conserved families of related Zn2-Cys6 binuclear proteins present in filamentous ascomycetes, including plant pathogens. A single ortholog is found in the yeasts Candida albicans, Debaryomyces hansenii, and Yarrowia lipolytica, but not in the Ashbya, Kluyveromyces, Saccharomyces lineage. Northern blot analysis has shown that deletion of the farA gene eliminates induction of a number of genes by both short- and long-chain fatty acids, while deletion of the farB gene eliminates short-chain induction. An identical core 6-bp in vitro binding site for each protein has been identified in genes encoding glyoxalate bypass, beta-oxidation, and peroxisomal functions. This sequence is overrepresented in the 5' region of genes predicted to be fatty acid induced in other filamentous ascomycetes, C. albicans, D. hansenii, and Y. lipolytica, but not in the corresponding genes in Saccharomyces cerevisiae.

Cooperative evolution in protein complexes of yeast from comparative analyses of its interaction network.

A comparative analysis among Saccharomyces cerevisiae and the other four yeasts Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica is presented. The broad evolutionary range spanned by the organisms allows to quantitatively demonstrate novel evolutionary effects in protein complexes. The evolution rates within cliques of interlinked proteins are found to bear strong multipoint correlations, witnessing a cooperative coevolution of complex subunits. The coevolution is found to be largely independent of the tendency of the subunits to have similar abundances.

Copper-zinc superoxide dismutase from the marine yeast Debaryomyces hansenii.

We have isolated the cytosolic form of Cu-Zn superoxide dismutase (SOD) from the marine yeast Debaryomyces hansenii. This enzyme has a subunit mass of 18 kDa. The preparation was found to be heterogeneous by IF electrophoresis with two pI ranges: 5.14-4.0 and 1.6-1.8. The enzyme preparation had a remarkably strong stability at pH 6.0-7.0, surviving boiling for 10 min without losing more than 60% of activity. On Western blots, this enzyme was recognized by antibodies raised in rabbits against D. hansenii extracts, while only a weak cross-reaction could be detected using antibodies generated against either Saccharomyces cerevisiae or bovine erythrocyte Cu-Zn SODs. In sequencing analysis, a peptide obtained by trypsin digestion was found to have 85% identity to the S. cerevisiae Cu-Zn SOD.

Identification of a DNA fragment encoding a putative membrane transporter protein expressed in Sporothrix schenckii.

We have isolated two DNA fragments from Sporothrix schenckii. These fragments were nonspecifically amplified from the whole-cell DNA using polymerase chain reaction (PCR) primers originally designed in the gene encoding heat shock protein 70 of Mycobacterium leprae. Reverse transcription PCR demonstrated that the smaller (sp-2) fragment was expressed, and a database search indicated that the deduced amino acids sequence from the sp-2 fragment contained a region homologous to the conserved sequence of the membrane transporter protein family. This is the first report of partial cloning of the gene encoding the putative membrane transporter in S. schenckii.

Molecular characterization of the Aspergillus nidulans treA gene encoding an acid trehalase required for growth on trehalose.

Aspergillus nidulans conidiospores contain high levels of the non-reducing disaccharide trehalose. We show that upon induction of conidiospore germination, the trehalose pool is rapidly degraded and a glycerol pool is transiently accumulated. A trehalase with an acidic pH optimum was purified from conidiospores. Characterization of the treA gene encoding this trehalase shows that it is homologous to Saccharomyces cerevisiae vacuolar acid trehalase, the product of the ATH1 gene, and to two related proteins of unknown function identified in Mycobacterium tuberculosis and Mycobacterium leprae. A. nidulans mutants that lack acid trehalase activity were constructed by gene replacement at the treA locus. Analysis of these mutants suggests that the treA gene product is localized in the conidiospore wall, is required for growth on trehalose as a carbon source, and is not involved in the mobilization of the intracellular pool of trehalose. Therefore, it is proposed that a cytoplasmic regulatory trehalase is controlling this latter process.