Integrated genome-transcriptome analysis unveiled the mechanism of Debaryomyces hansenii-mediated arsenic stress amelioration in rice.

Globally, rice is becoming more vulnerable to arsenic (As) pollution, posing a serious threat to public food safety. Previously Debaryomyces hansenii was found to reduce grain As content of rice. To better understand the underlying mechanism, we performed a genome analysis to identify the key genes in D. hansenii responsible for As tolerance and plant growth promotion. Notably, genes related to As resistance (ARR, Ycf1, and Yap) were observed in the genome of D. hansenii. The presence of auxin pathway and glutathione metabolism-related genes may explain the plant growth-promoting potential and As tolerance mechanism of this novel yeast strain. The genome annotation of D. hansenii indicated that it contains a repertoire of genes encoding antioxidants, well corroborated with the in vitro studies of GST, GR, and glutathione content. In addition, the effect of D. hansenii on gene expression profiling of rice plants under As stress was also examined. The Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed 307 genes, annotated in D. hansenii-treated rice, related to metabolic pathways (184), photosynthesis (12), glutathione (10), tryptophan (4), and biosynthesis of secondary metabolite (117). Higher expression of regulatory elements like AUX/IAA and WRKY transcription factors (TFs), and defense-responsive genes dismutases, catalases, peroxiredoxin, and glutaredoxins during D. hansenii+As exposure was also observed. Combined analysis revealed that D. hansenii genes are contributing to stress mitigation in rice by supporting plant growth and As-tolerance. The study lays the foundation to develop yeast as a beneficial biofertilizer for As-prone areas.

Description of lipase producing novel yeast species Debaryomyces apis f.a., sp. nov. and a modified pH indicator dye-based method for the screening of lipase producing microorganisms.

Four yeast strains were isolated from the gut of stingless bee, collected in Churdhar, Himachal Pradesh, India. Physiological characterization, morphological examination, and sequence analysis of small subunit ribosomal RNA (18S rRNA) genes, internal transcribed spacer (ITS) region, and D1/D2 domain of the large subunit rRNA gene revealed that the four strains isolated from the gut of stingless bee belonged to the Debaryomyces clade. Strain CIG-23HT showed sequence divergence of 7.5% from Debaryomyces nepalensis JCM 2095T, 7.8% from Debaryomyces udenii JCM 7855T, and Debaryomyces coudertii JCM 2387T in the D1/D2 domain. In the ITS region sequences, strain CIG-23HT showed a 15% sequence divergence from Debaryomyces nepalensis JCM 2095T and Debaryomyces coudertii JCM 2387T. In 18S rRNA gene sequence, the strain CIG-23HT showed 1.14% sequence divergence from Debaryomyces nepalensis JCM 2095 and and Debaryomyces coudertii JCM 2387, and 0.83% sequence divergence from Debaryomyces hansenii NRRL Y-7426. Strain CIG-23HT can utilize more carbon sources than closely related species. The findings suggest that strain CIG-23HT is a novel species of the genus Debaryomyces, and we propose to name it as Debaryomyces apis f.a., sp. nov. The holotype is CBS 16297T, and the isotypes are MTCC 12914T and KCTC 37024T. The MycoBank number of Debaryomyces apis f.a., sp. nov. is MB836065. Additionally, a method using cresol red and Bromothymol blue pH indicator dyes was developed to screen for lipase producers, which is more sensitive and efficient than the currently used phenol red and rhodamine B dye-based screening methods, and avoids the problem of less differentiable zone of hydrolysis.

Genomic features, aroma profiles, and probiotic potential of the Debaryomyces hansenii species complex strains isolated from Korean soybean fermented food.

Fermented soybean products are gaining attention in the food industry owing to their nutritive value and health benefits. In this study, we performed genomic analysis and physiological characterization of two Debaryomyces spp. yeast isolates obtained from a Korean traditional fermented soy sauce "ganjang". Both Debaryomyces hansenii ganjang isolates KD2 and C11 showed halotolerance to concentrations of up to 15% NaCl and improved growth in the presence of salt. Ploidy and whole-genome sequencing analyses indicated that the KD2 genome is haploid, whereas the C11 genome is heterozygous diploid with two distinctive subgenomes. Interestingly, phylogenetic analysis using intron sequences indicated that the C11 strain was generated via hybridization between D. hansenii and D. tyrocola ancestor strains. The D. hansenii KD2 and D. hansenii-hybrid C11 produced various volatile flavor compounds associated with butter, caramel, cheese, and fruits, and showed high bioconversion activity from ferulic acid to 4-vinylguaiacol, a characteristic flavor compound of soybean products. Both KD2 and C11 exhibited viability in the presence of bile salts and at low pH and showed immunomodulatory activity to induce high levels of the anti-inflammatory cytokine IL-10. The safety of the yeast isolates was confirmed by analyzing virulence and acute oral toxicity. Together, the D. hansenii ganjang isolates possess physiological properties beneficial for improving the flavor and nutritional value of fermented products.

Debaryomyces hansenii: an old acquaintance for a fresh start in the era of the green biotechnology.

The halophilic yeast Debaryomyces hansenii has been studied for several decades, serving as eukaryotic model for understanding salt and osmotic tolerance. Nevertheless, lack of consensus among different studies is found and, sometimes, contradictory information derived from studies performed in very diverse conditions. These two factors hampered its establishment as the key biotechnological player that was called to be in the past decade. On top of that, very limited (often deficient) engineering tools are available for this yeast. Fortunately Debaryomyces is again gaining momentum and recent advances using highly instrumented lab scale bioreactors, together with advanced -omics and HT-robotics, have revealed a new set of interesting results. Those forecast a very promising future for D. hansenii in the era of the so-called green biotechnology. Moreover, novel genetic tools enabling precise gene editing on this yeast are now available. In this review, we highlight the most recent developments, which include the identification of a novel gene implicated in salt tolerance, a newly proposed survival mechanism for D. hansenii at very high salt and limiting nutrient concentrations, and its utilization as production host in biotechnological processes.

Selection of Debaryomyces hansenii isolates as starters in meat products based on phenotypic virulence factors, tolerance to abiotic stress conditions and aroma generation.

INTRODUCTION: Debaryomyces hansenii is a yeast widely used in meat fermentations as starter for the purpose of improving the aromatic quality of the final product. However, it has not been the subject of an extensive study regarding phenotypic characteristics important for starter selection, such as the capacity to grow at abiotic stress conditions occurring during fermentation, the ability to generate desirable aromas and the absence of virulence traits in yeasts. AIMS: The aim of this study was to screen 60 strains of D. hansenii isolated from assorted foods for their potential application as starters in dry-cured fermented sausages manufacture. METHODS: The abiotic stress factors tested were low aw and pH and high concentration of salt, acetic acid and lactic acid. The phenotypic virulence traits explored were growth at 37°C, pseudohyphal and biofilm generation, invasiveness and enzymatic activities present in virulent yeasts. The generation of desirable meat aromas was tested in models containing aroma precursors applying an olfactory analysis. A quantitative profiling of stress tolerance was used to test the potential performance of selected strains in meat fermentations. RESULTS: The results demonstrated that most strains displayed no virulence trait or were only positive for biofilm production. Moreover, the strains showed large heterogeneity regarding their tolerance to abiotic stress factors, although most of them could grow at intermediate to high levels of the traits. The sensory analysis was the criteria determining the selection of starter strains. CONCLUSIONS: The evaluation of the phenotypic traits demonstrates that D. hansenii is a safe yeast, it is able to tolerate the stress in meat fermentation and it is able to generate desirable aromas. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study confirm the adequacy of selected D. hansenii strains to be applied as starters in meat products.

DebaryOmics: an integrative -omics study to understand the halophilic behaviour of Debaryomyces hansenii.

Debaryomyces hansenii is a non-conventional yeast considered to be a well-suited option for a number of different industrial bioprocesses. It exhibits a set of beneficial traits (halotolerant, oleaginous, xerotolerant, inhibitory compounds resistant) which translates to a number of advantages for industrial fermentation setups when compared to traditional hosts. Although D. hansenii has been highly studied during the last three decades, especially in regards to its salt-tolerant character, the molecular mechanisms underlying this natural tolerance should be further investigated in order to broadly use this yeast in biotechnological processes. In this work, we performed a series of chemostat cultivations in controlled bioreactors where D. hansenii (CBS 767) was grown in the presence of either 1M NaCl or KCl and studied the transcriptomic and (phospho)proteomic profiles. Our results show that sodium and potassium trigger different responses at both expression and regulation of protein activity levels and also complemented previous reports pointing to specific cellular processes as key players in halotolerance, moreover providing novel information about the specific genes involved in each process. The phosphoproteomic analysis, the first of this kind ever reported in D. hansenii, also implicated a novel and yet uncharacterized cation transporter in the response to high sodium concentrations.

Effect of Debaryomyces hansenii and the antifungal PgAFP protein on Alternaria spp. growth, toxin production, and RHO1 gene expression in a tomato-based medium.

Tomato fruit is susceptible to Alternaria spp. spoilage, which poses a health risk due to their mycotoxin production. Biopreservation relies on the use of whole microorganisms or their metabolites to manage spoilage microorganisms including filamentous fungi. However, the use of treatments at fungistatic level might activate intracellular pathways, which can cause an increment in mycotoxin accumulation. The objective of this work was to evaluate the effect of two strains of Debaryomyces hansenii and the antifungal protein PgAFP at 10 and 40 µg/mL. Both growth and production of two of the most common mycotoxins (tenuazonic acid and alternariol monomethyl ether) by Alternaria tenuissima sp.-grp. and Alternaria arborescens sp.-grp. on a tomato-based matrix, were analysed at 12 °C. Additionally, the impact of these biocontrol agents on the stress-related RHO1 gene expression was assessed. All treatments reduced mycotoxin accumulation (from 27 to 92% of inhibition). Their mode of action against Alternaria spp. in tomato seems unrelated to damages to fungal cell wall integrity at the genomic level. Therefore, the two D. hansenii strains (CECT 10352 and CECT 10353) and the antifungal protein PgAFP at 10 µg/mL are suggested as biocontrol strategies in tomato fruit at postharvest stage.

CRISPR/Cas9-Mediated Genome Engineering Reveals the Contribution of the 26S Proteasome to the Extremophilic Nature of the Yeast Debaryomyces hansenii.

The marine yeast Debaryomyces hansenii is of high importance in the food, chemical, and medical industries. D. hansenii is also a popular model for studying molecular mechanisms of halo- and osmotolerance. The absence of genome editing technologies hampers D. hansenii research and limits its biotechnological application. We developed novel and efficient single- and dual-guide CRISPR systems for markerless genome editing of D. hansenii. The single-guide system allows high-efficiency (up to 95%) mutation of genes or regulatory elements. The dual-guide system is applicable for efficient deletion of genomic loci. We used these tools to study transcriptional regulation of the 26S proteasome, an ATP-dependent protease complex whose proper function is vital for all cells and organisms. We developed a genetic approach to control the activity of the 26S proteasome by deregulation of its essential subunits. The mutant strains were sensitive to geno- and proteotoxic stresses as well as high salinity and osmolarity, suggesting a contribution of the proteasome to the extremophilic properties of D. hansenii. The developed CRISPR systems allow efficient D. hansenii genome engineering, providing a genetic way to control proteasome activity, and should advance applications of this yeast.

Debaryomyces hansenii Metabolism of Sulfur Amino Acids As Precursors of Volatile Sulfur Compounds of Interest in Meat Products.

The ability of Debaryomyces hansenii to produce volatile sulfur compounds from sulfur amino acids and the metabolic pathway involved have been studied in seven strains from different food origins. Our results proved that l-methionine is the main precursor for sulfur compound generation. Crucial differences in the sulfur compound profile and amino acid consumption among D. hansenii strains isolated from different food sources were observed. Strains isolated from dry pork sausages displayed the most complex sulfur compound profiles. Sulfur compound production, such as that of methional, could result from chemical reactions or yeast metabolism, while according to this study, thioester methyl thioacetate appeared to be generated by yeast metabolism. No relationship between sulfur compounds production by D. hansenii strains and the expression of genes involved in sulfur amino acid metabolism was found, except for the ATF2 gene in the L1 strain for production of methyl thioacetate. Our results suggest a complex scenario during sulfur compound production by D. hansenii.

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.

Septic shock due to Candida famata (Debaryomyces hansenii) candidemia in an ICU immunocompetent trauma-patient.

Sepsis related to Candida famata (C. famata) fungemia is extremely rare in immunocompetent patients. Moreover, septic shock has not been reported due to this yeast. A previously healthy young multi-trauma male, presented septic shock from C. famata, after he had been admitted in the Intensive Care Unit (ICU) due to haemorrhagic shock. Risk factors for candidemia in ICU patients are the presence of a central venous catheter (CVC), Total Parenteral Nutrition (TPN), use of broad-spectrum antimicrobials, immunosuppression and the length of ICU stay. The presence of CVCs, prior use of antibiotics, prolonged hospitalization, disruption of skin flora and immunocompromised states have been identified as predisposing risk factors for C. famata fungemia. It is worth noting that the present case concerns a non-immunocompromised patient, but long ICU stay and brain injury may indicate a state of immunoparalysis. Identification of the yeast was performed by partial amplification and sequencing of the 26S ribosomal DNA gene [hypervariable region D1/D2; partial sequencing of the act1 gene confirmed the identity of the strain as Debaryomyces hansenii (GenBank submission ID: 1688297)] The patient quickly recovered from sepsis after initiation of amphotericin B and was discharged on the 60th day.

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.

Non-canonical Activities of Hog1 Control Sensitivity of Candida albicans to Killer Toxins From Debaryomyces hansenii.

Certain yeasts secrete peptides known as killer toxins or mycocins with a deleterious effect on sensitive yeasts or filamentous fungi, a common phenomenon in environmental species. In a recent work, different Debaryomyces hansenii (Dh) strains isolated from a wide variety of cheeses were identified as producing killer toxins active against Candida albicans and Candida tropicalis. We have analyzed the killer activity of these toxins in C. albicans mutants defective in MAPK signaling pathways and found that the lack of the MAPK Hog1 (but not Cek1 or Mkc1) renders cells hypersensitive to Dh mycocins while mutants lacking other upstream elements of the pathway behave as the wild type strain. Point mutations in the phosphorylation site (T174A-176F) or in the kinase domain (K52R) of HOG1 gene showed that both activities were relevant for the survival of C. albicans to Dh killer toxins. Moreover, Hog1 phosphorylation was also required to sense and adapt to osmotic and oxidative stress while the kinase activity was somehow dispensable. Although the addition of supernatant from the killer toxin- producing D. hansenii 242 strain (Dh-242) induced a slight intracellular increase in Reactive Oxygen Species (ROS), overexpression of cytosolic catalase did not protect C. albicans against this mycocin. This supernatant induced an increase in intracellular glycerol concentration suggesting that this toxin triggers an osmotic stress. We also provide evidence of a correlation between sensitivity to Dh-242 killer toxin and resistance to Congo red, suggesting cell wall specific alterations in sensitive strains.

Transcriptomic response of Debaryomyces hansenii during mixed culture in a liquid model cheese medium with Yarrowia lipolytica.

Yeasts play a crucial role in cheese ripening. They contribute to the curd deacidification, the establishment of acid-sensitive bacterial communities, and flavour compounds production via proteolysis and catabolism of amino acids (AA). Negative yeast-yeast interaction was observed between the yeast Yarrowia lipolytica 1E07 (YL1E07) and the yeast Debaryomyces hansenii 1L25 (DH1L25) in a model cheese but need elucidation. YL1E07 and DH1L25 were cultivated in mono and co-cultures in a liquid synthetic medium (SM) mimicking the cheese environment and the growth inhibition of DH1L25 in the presence of YL1E07 was reproduced. We carried out microbiological, biochemical (lactose, lactate, AA consumption and ammonia production) and transcriptomic analyses by microarray technology to highlight the interaction mechanisms. We showed that the DH1L25 growth inhibition in the presence of YL1E07 was neither due to the ammonia production nor to the nutritional competition for the medium carbon sources between the two yeasts. The transcriptomic study was the key toward the comprehension of yeast-yeast interaction, and revealed that the inhibition of DH1L25 in co-culture is due to a decrease of the mitochondrial respiratory chain functioning.

Putative 3-nitrotyrosine detoxifying genes identified in the yeast Debaryomyces hansenii: in silico search of regulatory sequences responsive to salt and nitrogen stress

Background: During salt stress, the yeast Debaryomyces hansenii synthesizes tyrosine as a strategy to avoid the oxidation of proteins. Tyrosine reacts with nitrogen radicals to form 3-nitrotyrosine. 3-nitrotyrosine prevents the effects of associated oxidative stress and thus contributes to the high halotolerace of the yeast. However, the mechanism of how D. hansenii counteracts the presence of this toxic compound is unclear. In this work, we evaluated D. hansenii's capacity to assimilate 3-nitrotyrosine as a unique nitrogen source and measured its denitrase activity under salt stress. To identify putative genes related to the assimilation of 3-nitrotyrosine, we performed an in silico search in the promoter regions of D. hansenii genome. Results: We identified 15 genes whose promoters had binding site sequences for transcriptional factors of sodium, nitrogen, and oxidative stress with oxidoreductase and monooxygenase GO annotations. Two of these genes, DEHA2E24178g and DEHA2C00286g, coding for putative denitrases and having GATA sequences, were evaluated by RT-PCR and showed high expression under salt and nitrogen stress. Conclusions: D. hansenii can grow in the presence of 3-nitrotyrosine as the only nitrogen source and has a high specific denitrase activity to degrade 3-nitrotyrosine in 1 and 2 M NaCl stress conditions. The results suggest that given the lack of information on transcriptional factors in D. hansenii, the genes identified in our in silico analysis may help explain 3-nitrotyrosine assimilation mechanisms.

Early gut mycobiota and mother-offspring transfer.

BACKGROUND: The fungi in the gastrointestinal tract, the gut mycobiota, are now recognised as a significant part of the gut microbiota, and they may be important to human health. In contrast to the adult gut mycobiota, the establishment of the early gut mycobiota has never been described, and there is little knowledge about the fungal transfer from mother to offspring. METHODS: In a prospective cohort, we followed 298 pairs of healthy mothers and offspring from 36 weeks of gestation until 2 years of age (1516 samples) and explored the gut mycobiota in maternal and offspring samples. Half of the pregnant mothers were randomised into drinking probiotic milk during and after pregnancy. The probiotic bacteria included Lactobacillus rhamnosus GG (LGG), Bifidobacterium animalis subsp. lactis Bb-12 and Lactobacillus acidophilus La-5. We quantified the fungal abundance of all the samples using qPCR of the fungal internal transcribed spacer (ITS)1 segment, and we sequenced the 18S rRNA gene ITS1 region of 90 high-quantity samples using the MiSeq platform (Illumina). RESULTS: The gut mycobiota was detected in most of the mothers and the majority of the offspring. The offspring showed increased odds of having detectable faecal fungal DNA if the mother had detectable fungal DNA as well (OR = 1.54, p = 0.04). The fungal alpha diversity in the offspring gut increased from its lowest at 10 days after birth, which was the earliest sampling point. The fungal diversity and fungal species showed a succession towards the maternal mycobiota as the child aged, with Debaryomyces hansenii being the most abundant species during breast-feeding and Saccharomyces cerevisiae as the most abundant after weaning. Probiotic consumption increased the gut mycobiota abundance in pregnant mothers (p = 0.01). CONCLUSION: This study provides the first insight into the early fungal establishment and the succession of fungal species in the gut mycobiota. The results support the idea that the fungal host phenotype is transferred from mother to offspring. TRIAL REGISTRATION: Clinicaltrials.gov NCT00159523.

The halotolerant Debaryomyces hansenii, the Cinderella of non-conventional yeasts.

Debaryomyces hansenii is a halotolerant yeast with a high biotechnological potential, particularly in the food industry. However, research in this yeast is limited by its molecular peculiarities. In this review we summarize the state of the art of research in this microorganisms, describing both pros and cons. We discuss (i) its halotolerance, (ii) the molecular factors involved in saline and osmotic stress, (iii) its high gene density and ambiguous CUG decoding, and (iv) its biotechnological and medical interests. We trust that all the bottlenecks in its study will soon be overcome and D. hansenii will become a fundamental organism for food biotechnological processes. Copyright © 2016 John Wiley & Sons, Ltd.

Direct utilization of purple sweet potato by sake yeasts to produce an anthocyanin-rich alcoholic beverage.

OBJECTIVE: To produce an alcoholic beverage containing anthocyanins that can act as antioxidants and have anticarcinogenic activities and antihypertensive effects. RESULTS: High starch-assimilating sake yeast strain of Saccharomyces cerevisiae co-expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis using the double rDNA-integration system was developed. The new strain grew substantially using 5 % (w/v) purple sweet potato flour as the sole carbon source. Its cell yield reached 14.5 mg ml(-1) after 3 days. This value was 2.4-fold higher than that of the parental wild-type strain. It produced 12 % (v/v) ethanol from 20 % (w/v) purple sweet potato flour and consumed 98 % of the starch content in purple sweet potato flour after 5 days of fermentation. CONCLUSION: We have produced a health-promoting alcoholic beverage abundant in anthocyanins from purple sweet potato.

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.

Oxidative phosphorylation in Debaryomyces hansenii: physiological uncoupling at different growth phases.

Physiological uncoupling of mitochondrial oxidative phosphorylation (OxPhos) was studied in Debaryomyces hansenii. In other species, such as Yarrowia lipolytica and Saccharomyces cerevisiae, OxPhos can be uncoupled through differential expression of branched respiratory chain enzymes or by opening of a mitochondrial unspecific channel (ScMUC), respectively. However D. hansenii mitochondria, which contain both a branched respiratory chain and a mitochondrial unspecific channel (DhMUC), selectively uncouple complex I-dependent rate of oxygen consumption in the stationary growth phase. The uncoupled complex I-dependent respiration was only 20% of the original activity. Inhibition was not due to inactivation of complex I, lack of protein expression or to differential expression of alternative oxidoreductases. Furthermore, all other respiratory chain activities were normal. Decrease of complex I-dependent respiration was due to NAD(+) loss from the matrix, probably through an open of DhMUC. When NAD(+) was added back, coupled complex I-activity was recovered. NAD(+) re-uptake was independent of DhMUC opening and seemed to be catalyzed by a NAD(+)-specific transporter, which was sensitive to bathophenanthroline, bromocresol purple or pyridoxal-5'-phosphate as described for S. cerevisiae mitochondrial NAD(+) transporters. Loss of NAD(+) from the matrix through an open MUC is proposed as an additional mechanism to uncouple OxPhos.