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Vaginal infectious diseases caused by viruses and bacteria have been linked to the occurrence of dysbiosis, that is, a reduction in the abundance of the normally dominating vaginal Lactobacillus species. Mucosal infections in the vagina and/or vulva caused by Candida species, usually known as vulvovaginal candidiasis (or VVC), are among the leading causes of diseases in the vaginal tract. The existence of a clear link between the occurrence of dysbiosis and the development of VVC is still unclear, although multiple observations point in that direction. Based on the idea that vaginal health is linked to a microbiota dominated by lactobacilli, several probiotics have been used in management of VVC, either alone or in combination with antifungals, having obtained different degrees of success. In most cases, the undertaken trials resorted to lactobacilli species other than those indigenous to the vaginal tract, although in vitro these vaginal species were shown to reduce growth, viability and virulence of Candida. In this paper we overview the role of lactobacilli and Candida in the vaginal micro- and myco-biomes, while discussing the results obtained in what concerns the establishment of interference mechanisms in vivo and the environmental factors that could determine that. We also overview the molecular mechanisms by which lactobacilli species have been shown to inhibit pathophysiology of Candida , including the description of the genes and pathways determining their ability to thrive in the presence of each other. In a time where concerns are increasing with the emergence of antifungal resistance and the slow pace of discovery of new antifungals, a thorough understanding of the molecular mechanisms underneath the anti- Candida effect prompted by vaginal lactobacilli is of utmost importance to assure a knowledge-based design of what can be a new generation of pharmaceuticals, eventually focusing therapeutic targets other than the usual ones. [ABSTRACT FROM AUTHOR]

Background: Formic acid is an inhibitory compound present in lignocellulosic hydrolysates. Understanding the complex molecular mechanisms underlying Saccharomyces cerevisiae tolerance to this weak acid at the system level is instrumental to guide synthetic pathway engineering for robustness improvement of industrial strains envisaging their use in lignocellulosic biorefineries. Results: This study was performed to identify, at a genome-wide scale, genes whose expression confers protection or susceptibility to formic acid, based on the screening of a haploid deletion mutant collection to search for these phenotypes in the presence of 60, 70 and 80 mM of this acid, at pH 4.5. This chemogenomic analysis allowed the identification of 172 determinants of tolerance and 41 determinants of susceptibility to formic acid. Clustering of genes required for maximal tolerance to this weak acid, based on their biological function, indicates an enrichment of those involved in intracellular trafficking and protein synthesis, cell wall and cytoskeleton organization, carbohydrate metabolism, lipid, amino acid and vitamin metabolism, response to stress, chromatin remodelling, transcription and internal pH homeostasis. Among these genes is HAA1 encoding the main transcriptional regulator of yeast transcriptome reprograming in response to acetic acid and genes of the Haa1-regulon; all demonstrated determinants of acetic acid tolerance. Among the genes that when deleted lead to increased tolerance to formic acid, TRK1, encoding the high-affinity potassium transporter and a determinant of resistance to acetic acid, was surprisingly found. Consistently, genes encoding positive regulators of Trk1 activity were also identified as formic acid susceptibility determinants, while a negative regulator confers protection. At a saturating K+ concentration of 20 mM, the deletion mutant trk1Δ was found to exhibit a much higher tolerance compared with the parental strain. Given that trk1Δ accumulates lower levels of radiolabelled formic acid, compared to the parental strain, it is hypothesized that Trk1 facilitates formic acid uptake into the yeast cell. Conclusions: The list of genes resulting from this study shows a few marked differences from the list of genes conferring protection to acetic acid and provides potentially valuable information to guide improvement programmes for the development of more robust strains against formic acid. [ABSTRACT FROM AUTHOR]

Pulmonary colonization of cystic fibrosis (CF) patients with Burkholderia cenocepacia or other bacteria of the Burkholderia cepacia complex (Bcc) is associated with worse prognosis and increased risk of death. During colonization, the bacteria may evolve under the stressing selection pressures exerted in the CF lung, in particular, those resulting from challenges of the host immune defenses, antimicrobial therapy, nutrient availability and oxygen limitation. Understanding the adaptive mechanisms that promote successful colonization and long-term survival of B. cenocepacia in the CF lung is essential for an improved therapeutic outcome of chronic infections. To get mechanistic insights into these adaptive strategies a transcriptomic analysis, based on DNA microarrays, was explored in this study. The genomic expression levels in two clonal variants isolated during long-term colonization of a CF patient who died from the cepacia syndrome were compared. One of the isolates examined, IST439, is the first B. cenocepacia isolate retrieved from the patient and the other isolate, IST4113, was obtained three years later and is more resistant to different classes of antimicrobials. Approximately 1000 genes were found to be differently expressed in the two clonal variants reflecting a marked reprogramming of genomic expression. The up-regulated genes in IST4113 include those involved in translation, iron uptake (in particular, in ornibactin biosynthesis), efflux of drugs and in adhesion to epithelial lung tissue and to mucin. Alterations related with adaptation to the nutritional environment of the CF lung and to an oxygen-limited environment are also suggested to be a key feature of transcriptional reprogramming occurring during long-term colonization, antibiotic therapy and the progression of the disease. [ABSTRACT FROM AUTHOR]

The success of food and beverage production processes carried out by Saccharomyces cerevisiae and the thriving of food spoilage fungi are dependent on the ability of a cell to cope with the many environmental insults imposed during food production and preservation processes. Post-genomic approaches, especially transcriptomics, proteomics and chemogenomics, applied to S. cerevisiae made possible the unveiling of general and specific genome-wide adaptive response programs against stress induced by weak acids, ethanol, sulfite, heat and cold shock, osmotic pressure and nutrient limitation. These programs and the underlying signaling pathways are overviewed herein, highlighting the recent identification of genes and pathways found to be involved in stress response and tolerance. These are good candidate targets for genetic engineering aiming at the development of improved strains. The extension of the data gathered in S. cerevisiae to food spoilage fungi is considered. The relevance of the different genome-wide approaches in this context is also discussed. [Copyright &y& Elsevier]

Background: Acetic acid is a byproduct of Saccharomyces cerevisiae alcoholic fermentation. Together with high concentrations of ethanol and other toxic metabolites, acetic acid may contribute to fermentation arrest and reduced ethanol productivity. This weak acid is also a present in lignocellulosic hydrolysates, a highly interesting non-feedstock substrate in industrial biotechnology. Therefore, the better understanding of the molecular mechanisms underlying S. cerevisiae tolerance to acetic acid is essential for the rational selection of optimal fermentation conditions and the engineering of more robust industrial strains to be used in processes in which yeast is explored as cell factory. Results: The yeast genes conferring protection against acetic acid were identified in this study at a genome-wide scale, based on the screening of the EUROSCARF haploid mutant collection for susceptibility phenotypes to this weak acid (concentrations in the range 70-110 mM, at pH 4.5). Approximately 650 determinants of tolerance to acetic acid were identified. Clustering of these acetic acid-resistance genes based on their biological function indicated an enrichment of genes involved in transcription, internal pH homeostasis, carbohydrate metabolism, cell wall assembly, biogenesis of mitochondria, ribosome and vacuole, and in the sensing, signalling and uptake of various nutrients in particular iron, potassium, glucose and amino acids. A correlation between increased resistance to acetic acid and the level of potassium in the growth medium was found. The activation of the Snf1p signalling pathway, involved in yeast response to glucose starvation, is demonstrated to occur in response to acetic acid stress but no evidence was obtained supporting the acetic acid-induced inhibition of glucose uptake. Conclusions: Approximately 490 of the 650 determinants of tolerance to acetic acid identified in this work are implicated, for the first time, in tolerance to this weak acid. These are novel candidate genes for genetic engineering to obtain more robust yeast strains against acetic acid toxicity. Among these genes there are number of transcription factors that are documented regulators of a large percentage of the genes found to exert protection against acetic acid thus being considered interesting targets for subsequent genetic engineering. The increase of potassium concentration in the growth medium was found to improve the expression of maximal tolerance to acetic acid, consistent with the idea that the adequate manipulation of nutrient concentration of industrial growth medium can be an interesting strategy to surpass the deleterious effects of this weak acid in yeast cells. [ABSTRACT FROM AUTHOR]

The physiological function of the Saccharomyces cerevisiae RIM101 signaling pathway is extended in this study beyond alkaline pH-induced responses. The transcription factor Rim101p is demonstrated to be required for maximal tolerance to weak acid-induced stress, at pH 4.0, but does not exert protection against low pH itself (range 4.5–2.5), when a strong acid is used as the acidulant. The Rim101p-dependent alterations of the yeast transcriptome following exposure to propionic acid stress (at pH 4.0) include genes of the previously described Rim101p regulon but also new target genes, in particular KNH1, involved in cell wall β-1,6-glucan synthesis and the uncharacterized ORF YIL029c, both required for maximal propionic acid resistance. Clustering of the genes that provide resistance to propionic acid reveals the enrichment of those involved in protein catabolism through the multivesicular body pathway and in the homeostasis of internal pH and vacuolar function. The analysis of the network of interactions established among all the identified propionic acid resistance determinants shows an enrichment of interactions around the RIM101 gene and highlights the role of proteins involved in Rim101p proteolytic processing. RIM101 expression is shown to be required to counteract propionic acid-induced cytosolic acidification and for proper vacuolar acidification and cell wall structure, these having positive implications for a robust adaptive response and resistance to stress promoted by this food preservative. [ABSTRACT FROM AUTHOR]

Hanseniaspora guilliermondii is a well-recognized producer of acetate esters associated with fruity and floral aromas. The molecular mechanisms underneath this production or the environmental factors modulating it remain unknown. Herein, we found that, unlike Saccharomyces cerevisiae, H. guilliermondii over-produces acetate esters and higher alcohols at low carbon-to-assimilable nitrogen (C:N) ratios, with the highest titers being obtained in the amino acid-enriched medium YPD. The evidences gathered support a model in which the strict preference of H. guilliermondii for amino acids as nitrogen sources results in a channeling of keto-acids obtained after transamination to higher alcohols and acetate esters. This higher production was accompanied by higher expression of the four HgAATs, genes, recently proposed to encode alcohol acetyl transferases. In silico analyses of these HgAat's reveal that they harbor conserved AATs motifs, albeit radical substitutions were identified that might result in different kinetic properties. Close homologues of HgAat2, HgAat3, and HgAat4 were only found in members of Hanseniaspora genus and phylogenetic reconstruction shows that these constitute a distinct family of Aat's. These results advance the exploration of H. guilliermondii as a bio-flavoring agent providing important insights to guide future strategies for strain engineering and media manipulation that can enhance production of aromatic volatiles. Acetate esters production by the wine yeastHanseniaspora guilliermondi. [ABSTRACT FROM AUTHOR]

New cyclam-based complexes of formulae [{H2(4-CF3PhCH2)2Cyclam}FeCl2]Cl and [{H2(4-CF3PhCH2)2Cyclam}Cu](CH3COO)2·2H2O were synthesized, in very high yields, by the reaction of H2(4-CF3PhCH2)2Cyclam with the corresponding metal salts in methanol. [{H2(4-CF3PhCH2)2Cyclam}FeCl2]Cl was also prepared, quantitatively, in the solid state by mechanochemistry. The Mössbauer spectra of [{H2(4-CF3PhCH2)2Cyclam}FeCl2]Cl show that the high-spin cis complex is the major product of both reactions. The EPR data of [{H2(4-CF3PhCH2)2Cyclam}Cu](CH3COO)2·2H2O are consistent with two distinct Cu2+ species, whose solid state molecular structures were determined by single crystal X-ray diffraction and correspond to complexes displaying octahedral and square–pyramidal geometries. The screening of the antifungal activity of both metal complexes revealed the higher activity of [{H2(4-CF3PhCH2)2Cyclam}FeCl2]Cl to inhibit the growth of unicellular and multicellular fungal species with described pathogenic potential. [ABSTRACT FROM AUTHOR]

The pathogenic yeast Candida glabrata is intrinsically resilient to azoles and rapidly acquires resistance to these antifungals, in vitro and in vivo. In most cases azole-resistant C. glabrata clinical strains encode hyperactive CgPdr1 variants, however, resistant strains encoding wild-type CgPDR1 alleles have also been isolated, although remaining to be disclosed the underlying resistance mechanism. In this study, we scrutinized the mechanisms underlying resistance to azoles of 8 resistant clinical C. glabrata strains, identified along the course of epidemiological surveys undertaken in Portugal. Seven of the strains were found to encode CgPdr1 gain-of-function variants (I392M, E555K, G558C, and I803T) with the substitutions I392M and I803T being herein characterized as hyper-activating mutations for the first time. While cells expressing the wild-type CgPDR1 allele required the mediator subunit Gal11A to enhance tolerance to fluconazole, this was dispensable for cells expressing the I803T variant indicating that the CgPdr1 interactome is shaped by different gain-of-function substitutions. Genomic and transcriptomic profiling of the sole azole-resistant C. glabrata isolate encoding a wild-type CgPDR1 allele (ISTB218) revealed that under fluconazole stress this strain over-expresses various genes described to provide protection against this antifungal, while also showing reduced expression of genes described to increase sensitivity to these drugs. The overall role in driving the azole-resistance phenotype of the ISTB218 C. glabrata isolate played by these changes in the transcriptome and genome of the ISTB218 isolate are discussed shedding light into mechanisms of resistance that go beyond the CgPdr1-signalling pathway and that may alone, or in combination, pave the way for the acquisition of resistance to azoles in vivo. [ABSTRACT FROM AUTHOR]

The discovery of itaconic acid as a product of citric acid pyrolytic distillation in 1837 opened the possibility of using it as a polymer building block. Itaconic acid, featuring two carboxylic acids and an unsaturated group, can potentially be used as a building block in several chemical syntheses, with a particular emphasis on polymer manufacture. The elucidation of biochemical pathways originating from itaconic acid, first in Aspergillus terreus and, recently, in several species of the Ustilago genus, has intensified and diversified research focused on microbe-based itaconic acid production, including at an industrial scale. These efforts include the engineering of naturally producing species/strains along with the exploration of other species that do not naturally produce itaconic acid but may offer potential benefits. The use of renewable wastes or sugar-enriched residues as substrates to produce itaconic acid, from a circular bioeconomy perspective, is another important aspect of the advancements in microbial itaconic acid production. This review provides an overview of the achievements as well as the challenges concerning the engineering of the producing strains/species, substrate selection, optimisation of bioreactor operation, and downstream itaconic acid purification methods. • Itaconic Acid allows addition and condensation polymerization in the same molecule. • Wider application of Itaconic Acid (IA) requires to decrease its bioproduction costs. • Bioreactor amenable natural or heterologous IA microbial productors are still sought. • The use of renewable cheap substrates is key to improve IA production sustainability. • Optimal streamlined bioprocess and efficient downstream are crucial for IA scale-up. [ABSTRACT FROM AUTHOR]

Motivation: Uncovering mechanisms underlying gene expression control is crucial to understand complex cellular responses. Studies in gene regulation often aim to identify regulatory players involved in a biological process of interest, either transcription factors coregulating a set of target genes or genes eventually controlled by a set of regulators. These are frequently prioritized with respect to a context-specific relevance score. Current approaches rely on relevance measures accounting exclusively for direct transcription factor–target interactions, namely overrepresentation of binding sites or target ratios. Gene regulation has, however, intricate behavior with overlapping, indirect effect that should not be neglected. In addition, the rapid accumulation of regulatory data already enables the prediction of large-scale networks suitable for higher level exploration by methods based on graph theory. A paradigm shift is thus emerging, where isolated and constrained analyses will likely be replaced by whole-network, systemic-aware strategies.Results: We present TFRank, a graph-based framework to prioritize regulatory players involved in transcriptional responses within the regulatory network of an organism, whereby every regulatory path containing genes of interest is explored and incorporated into the analysis. TFRank selected important regulators of yeast adaptation to stress induced by quinine and acetic acid, which were missed by a direct effect approach. Notably, they reportedly confer resistance toward the chemicals. In a preliminary study in human, TFRank unveiled regulators involved in breast tumor growth and metastasis when applied to genes whose expression signatures correlated with short interval to metastasis.Availability: Prototype at http://kdbio.inesc-id.pt/software/tfrank/.Contact: jpg@kdbio.inesc-id.pt; sara.madeira@ist.utl.pt;Supplementary Information: Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

The understanding of the molecular basis of yeast resistance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. In this study, the yeast disruptome was screened for mutants with differential susceptibility to stress induced by high ethanol concentrations in minimal growth medium. Over 250 determinants of resistance to ethanol were identified. The most significant gene ontology terms enriched in this data set are those associated with intracellular organization, biogenesis, and transport, in particular, regarding the vacuole, the peroxisome, the endosome, and the cytoskeleton, and those associated with the transcriptional machinery. Clustering the proteins encoded by the identified determinants of ethanol resistance by their known physical and genetic interactions highlighted the importance of the vacuolar protein sorting machinery, the vacuolar H+-ATPase complex, and the peroxisome protein import machinery. Evidence showing that vacuolar acidification and increased resistance to the cell wall lytic enzyme β-glucanase occur in response to ethanol-induced stress was obtained. Based on the genome-wide results, the particular role of the FPS1 gene, encoding a plasma membrane aquaglyceroporin which mediates controlled glycerol efflux, in ethanol stress resistance was further investigated. FPSI expression contributes to decreased [3H]ethanol accumulation in yeast cells, suggesting that Fps1p may also play a role in maintaining the intracellular ethanol level during active fermentation. The increased expression of FPS1 confirmed the important role of this gene in alcoholic fermentation, leading to increased final ethanol concentration under conditions that lead to high ethanol production. [ABSTRACT FROM AUTHOR]

Background: Saccharomycodes ludwigii belongs to the poorly characterized Saccharomycodeacea family and is known by its ability to spoil wines, a trait mostly attributable to its high tolerance to sulfur dioxide (SO2). To improve knowledge about Saccharomycodeacea our group determined whole-genome sequences of Hanseniaspora guilliermondii (UTAD222) and S. ludwigii (UTAD17), two members of this family. While in the case of H. guilliermondii the genomic information elucidated crucial aspects concerning the physiology of this species in the context of wine fermentation, the draft sequence obtained for S. ludwigii was distributed by more than 1000 contigs complicating extraction of biologically relevant information. In this work we describe the results obtained upon resequencing of S. ludwigii UTAD17 genome using PacBio as well as the insights gathered from the exploration of the annotation performed over the assembled genome. Results: Resequencing of S. ludwigii UTAD17 genome with PacBio resulted in 20 contigs totaling 13 Mb of assembled DNA and corresponding to 95% of the DNA harbored by this strain. Annotation of the assembled UTAD17 genome predicts 4644 protein-encoding genes. Comparative analysis of the predicted S. ludwigii ORFeome with those encoded by other Saccharomycodeacea led to the identification of 213 proteins only found in this species. Among these were six enzymes required for catabolism of N-acetylglucosamine, four cell wall β-mannosyltransferases, several flocculins and three acetoin reductases. Different from its sister Hanseniaspora species, neoglucogenesis, glyoxylate cycle and thiamine biosynthetic pathways are functional in S. ludwigii. Four efflux pumps similar to the Ssu1 sulfite exporter, as well as robust orthologues for 65% of the S. cerevisiae SO2-tolerance genes, were identified in S. ludwigii genome. Conclusions: This work provides the first genome-wide picture of a S. ludwigii strain representing a step forward for a better understanding of the physiology and genetics of this species and of the Saccharomycodeacea family. The release of this genomic sequence and of the information extracted from it can contribute to guide the design of better wine preservation strategies to counteract spoilage prompted by S. ludwigii. It will also accelerate the exploration of this species as a cell factory, specially in production of fermented beverages where the use of Non-Saccharomyces species (including spoilage species) is booming. [ABSTRACT FROM AUTHOR]

Fungal infections and, in particular, those caused by species of the Candida genus, are growing at an alarming rate and have high associated rates of mortality and morbidity. These infections, generally referred as candidiasis, range from common superficial rushes caused by an overgrowth of the yeasts in mucosal surfaces to life-threatening disseminated mycoses. The success of currently used antifungal drugs to treat candidiasis is being endangered by the continuous emergence of resistant strains, specially among non-albicans Candida species. In this review article, the mechanisms of action of currently used antifungals, with emphasis on the mechanisms of resistance reported in clinical isolates, are reviewed. Novel approaches being taken to successfully inhibit growth of pathogenic Candida species, in particular those based on the exploration of natural or synthetic chemicals or on the activity of live probiotics, are also reviewed. It is expected that these novel approaches, either used alone or in combination with traditional antifungals, may contribute to foster the identification of novel anti-Candida therapies. [ABSTRACT FROM AUTHOR]

Background: In this paper, we explore the concept of multi-objective optimization in the field of metabolic engineering when both continuous and integer decision variables are involved in the model. In particular, we propose a multi-objective model that may be used to suggest reaction deletions that maximize and/or minimize several functions simultaneously. The applications may include, among others, the concurrent maximization of a bioproduct and of biomass, or maximization of a bioproduct while minimizing the formation of a given by-product, two common requirements in microbial metabolic engineering. Results: Production of ethanol by the widely used cell factory Saccharomyces cerevisiae was adopted as a case study to demonstrate the usefulness of the proposed approach in identifying genetic manipulations that improve productivity and yield of this economically highly relevant bioproduct. We did an in vivo validation and we could show that some of the predicted deletions exhibit increased ethanol levels in comparison with the wild-type strain. Conclusions: The multi-objective programming framework we developed, called Momo, is open-source and uses PolySCIP (Available at http://polyscip.zib.de/). as underlying multi-objective solver. Momo is available at http://momo-sysbio.gforge.inria.fr [ABSTRACT FROM AUTHOR]

Zinc (Zn) has been proposed as an alternative metallic biodegradable material to support transient wound-healing processes. Once a Zn piece is implanted inside the organism the degradation will depend upon the physiological surrounding environment. This, by modulating the composition of the surface layers formed on Zn devices, will govern the subsequent interactions with the surrounding living cells (e.g. biocompatibility and/or antifungal behaviour). In silico simulation of an implanted Zn piece at bone-muscle interface or inside the bone yielded the preferential precipitation of simonkolleite or zincite, respectively. To study the impact of these surface layers in the in vitro behaviour of Zn biomaterials, simonkolleite and zincite where synthesised. The successful production of simonkolleite or zincite was confirmed by an extensive physicochemical characterization. An in vitro layer formed on the top of these surface layers revealed that simonkolleite was rather inert, while zincite yielded a complex matrix containing hydroxyapatite, an important bone analogue. When analysing the “anti-biofilm” activity simonkolleite stood out for its activity against an important pathogenic fungi involved in implant-device infections, Candida albicans . The possible physiological implications of these findings are discussed. [ABSTRACT FROM AUTHOR]

Medical devices colonized by the commensal microflora or infecting pathogens can have impaired functioning while contributing for infection dissemination. The use of ZnO nanoparticles to coat medical devices is emerging as an interesting strategy considering the antimicrobial potential of these particles. For Candida albicans and C. parapsilosis , two relevant microbial colonizers of medical devices, it was reported that a reduced colonization was made at the expenses of a nanostructured ZnO coating disassembling. To overcome the usefulness of ZnO coatings for longer periods or recurrent infections, a microstructured coating was investigated. When comparing the obtained results with our published data it was clear that similar “anti-biofilm” activities were attained independently of ZnO crystal sizes, questioning the paradigm that reduction to nanoscale guarantees an improved antimicrobial efficacy. Regardless the colonization of the microstructured ZnO coating with Candida cells the observed precipitation of CaP-derived compounds on the surface can render biocompatibility advantageous even when a fungal infection occurs. Moreover, the interaction of fungi with the coating revealed that the disassembly reported for the nanosized-based coating was likewise observed for the microsized-based coating. Although ZnO-based coatings are effective against the biofilm formation of pathogenic fungi new challenges arise in the design of disassembly-resistant coatings. [ABSTRACT FROM AUTHOR]

The present work follows a previous report describing the antibacterial activity of silver camphorimine complexes of general formula [Ag(NO3)L]. The synthesis and demonstration of the antifungal and antibacterial activity of three novel [Ag(NO3)L] complexes (named 1, 2 and 3) is herein demonstrated. This work also shows for the first time that the previously studied complexes (named 4 to 8) also exert antifungal activity. The antibacterial activity of complexes was evaluated against Staphylococcus aureus, Pseudomonas aeruginosa, Burkholderia contaminans and Escherichia coli strains, while antifungal activity was tested against the Candida species C. albicans, C. glabrata, C. parapsilosis and C. tropicalis. The antimicrobial activity of the complexes ranged from very high (complex 4) to moderate (complex 6) or low (complex 8), depending on the structural and electronic characteristics of the camphorimine ligands. Notably, the highest antibacterial and anti-Candida activities do not coincide in the same complex and in some cases they were even opposite, as is the case of complex 4 which exhibits a high anti-bacterial and low antifungal activity. These distinct results suggest that the complexes may have different mechanisms against prokaryotic and eukaryotic cells. The antifungal activity of the Ag(I) camphorimine complexes (in particular of complex 1) was found to be very high (MIC = 2 μg/mL) against C. parapsilosis, being also registered a prominent activity against C. tropicalis and C. glabrata. None of the tested compounds inhibited C. albicans growth, being this attributed to the ability of these yeast cells to mediate the formation of less toxic Ag nanoparticles, as confirmed by Scanning Electron Microscopy images. The high antibacterial and anti-Candida activities of the here studied camphorimine complexes, especially of complexes 1 and 7, suggests a potential therapeutic application for these compounds. [ABSTRACT FROM AUTHOR]

The mechanisms of persistence and virulence associated with Candida glabrata infections are poorly understood, limiting the ability to fight this fungal pathogen. In this study, the multidrug resistance transporters CgTpo1_1 and CgTpo1_2 are shown to play a role in C. glabrata virulence. The survival of the infection model Galleria mellonella, infected with C. glabrata, was found to increase upon the deletion of either CgTPO1_1 or CgTPO1_2. The underlying mechanisms were further explored. In the case of CgTpo1_1, this phenotype was found to be consistent with the observation that it confers resistance to antimicrobial peptides (AMP), such as the human AMP histatin-5. The deletion of CgTPO1_2, on the other hand, was found to limit the survival of C. glabrata cells when exposed to phagocytosis and impair biofilm formation. Interestingly, CgTPO1_2 expression was found to be up-regulated during biofilm formation, but and its deletion leads to a decreased expression of adhesin-encoding genes during biofilm formation, which is consistent with a role in biofilm formation. CgTPO1_2 expression was further seen to decrease plasma membrane potential and affect ergosterol and fatty acid content. Altogether, CgTpo1_1 and CgTpo1_2 appear to play an important role in the virulence of C. glabrata infections, being at the cross-road between multidrug resistance and pathogenesis. [ABSTRACT FROM AUTHOR]

Background: The introduction of yeast starter cultures consisting in a blend of Saccharomyces cerevisiae and non-Saccharomyces yeast strains is emerging for production of wines with improved complexity of flavor. The rational use of this approach is, however, dependent on knowing the impact that co-inoculation has in the physiology of S. cerevisiae. In this work the transcriptome of S. cerevisiae was monitored throughout a wine fermentation, carried out in single culture or in a consortium with Hanseniaspora guilliermondii, this being the first time that this relevant yeast- yeast interaction is examined at a genomic scale. Results: Co-inoculation with H. guilliermondii reduced the overall genome-wide transcriptional response of S. cerevisiae throughout the fermentation, which was attributable to a lower fermentative activity of S. cerevisiae while in the mixed-fermentation. Approximately 350 genes S. cerevisiae genes were found to be differently expressed (FDR < 0.05) in response to the presence of H. guilliermondii in the fermentation medium. Genes involved in biosynthesis of vitamins were enriched among those up-regulated in the mixed-culture fermentation, while genes related with the uptake and biosynthesis of amino acids were enriched among those more expressed in the single-culture. The differences in the aromatic profiles of wines obtained in the single and in the mixed-fermentations correlated with the differential expression of S. cerevisiae genes encoding enzymes required for formation of aroma compounds. Conclusions: By integrating results obtained in the transcriptomic analysis performed with physiological data our study provided, for the first time, an integrated view into the adaptive responses of S. cerevisiae to the challenging environment of mixed culture fermentation. The availability of nutrients, in particular, of nitrogen and vitamins, stands out as a factor that may determine population dynamics, fermentative activity and by-product formation. [ABSTRACT FROM AUTHOR]

Advanced impedance spectroscopy analysis based on the transmission line model (TLM) is explored as a novel QCM acoustic biosensing platform for the detection of the single point mutation effect on the binding of the transcription factors (TFs) to immobilized DNA oligoduplexes and the characterization of the protein–DNA mechanical properties. [ABSTRACT FROM AUTHOR]

Objectives The ability of opportunistic pathogenic Candida species to persist and invade specific niches in the human host depends on their resistance to natural growth inhibitors and antifungal therapy. This work describes the role of the Candida glabrata drug:H+ antiporter CgTpo3 (ORF CAGL0I10384g) in this context. Methods Deletion and cloning of CgTPO3 was achieved using molecular biology tools. C. glabrata strain susceptibility was assayed based on growth in liquid and solid media and through MIC determination. Radiolabelled compound accumulation or HPLC were used for the assessment of the role of CgTpo3 as a drug or polyamine transporter. Quantitative RT–PCR was used for expression analysis. Results CgTpo3 was found to confer resistance to azole drugs in C. glabrata. This protein was found to be localized to the plasma membrane and to decrease the intracellular accumulation of [3H]clotrimazole, playing a direct role in its extrusion from pre-loaded C. glabrata cells. CgTPO3 was further found to confer resistance to spermine, complementing the susceptibility phenotypes exhibited by the deletion of its Saccharomyces cerevisiae homologue, TPO3. In spermine-stressed C. glabrata cells, CgTPO3 is transcriptionally activated in a CgPdr1-dependent manner, contributing to a decrease in the intracellular concentration of this polyamine. Clotrimazole exposure was found to lead to the intracellular accumulation of spermine, and pre-exposure to this polyamine was found consistently to lead to increased clotrimazole resistance. Conclusions Altogether, these results point to a significant role for CgTpo3 in azole drug resistance and in the tolerance to high polyamine concentrations, such as those found in the urogenital tract. [ABSTRACT FROM PUBLISHER]

A novel quartz crystal microbalance (QCM) analytical method is developed based on the transmission line model (TLM) algorithm to analyze the binding of transcription factors (TFs) to immobilized DNA oligoduplexes. The method is used to characterize the mechanical properties of biological films through the estimation of the film dynamic shear moduli, GSTACKABOVE′/ABOVEBELOWfilm/BELOW/STACK and GSTACKABOVE′′/ABOVEBELOWfilm/BELOW/STACK, and the film thickness. Using the Saccharomyces cerevisiae transcription factor Haa1 (Haa1DBD) as a biological model two sensors were prepared by immobilizing DNA oligoduplexes, one containing the Haa1 recognition element (HREwt) and another with a random sequence (HREneg) used as a negative control. The immobilization of DNA oligoduplexes was followed in real time and we show that DNA strands initially adsorb with low or non-tilting, laying flat close to the surface, which then lift-off the surface leading to final film tilting angles of 62.9° and 46.7° for HREwt and HREneg, respectively. Furthermore we show that the binding of Haa1DBD to HREwt leads to a more ordered and compact film, and forces a 31.7° bending of the immobilized HREwt oligoduplex. This work demonstrates the suitability of the QCM to monitor the specific binding of TFs to immobilized DNA sequences and provides an analytical methodology to study protein–DNA biophysics and kinetics. [ABSTRACT FROM AUTHOR]

Background: The understanding of the molecular basis of yeast tolerance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. A set of 21 genes encoding multidrug transporters from the ATP-Binding Cassette (ABC) Superfamily and Major Facilitator Superfamily (MFS) in S. cerevisiae were scrutinized for a role in ethanol stress resistance. Results: A yeast multidrug resistance ABC transporter encoded by the PDR18 gene, proposed to play a role in the incorporation of ergosterol in the yeast plasma membrane, was found to confer resistance to growth inhibitory concentrations of ethanol. PDR18 expression was seen to contribute to decreased 3 H-ethanol intracellular concentrations and decreased plasma membrane permeabilization of yeast cells challenged with inhibitory ethanol concentrations. Given the increased tolerance to ethanol of cells expressing PDR18, the final concentration of ethanol produced during high gravity alcoholic fermentation by yeast cells devoid of PDR18 was lower than the final ethanol concentration produced by the corresponding parental strain. Moreover, an engineered yeast strain in which the PDR18 promoter was replaced in the genome by the stronger PDR5 promoter, leading to increased PDR18 mRNA levels during alcoholic fermentation, was able to attain a 6 % higher ethanol concentration and a 17 % higher ethanol production yield than the parental strain. The improved fermentative performance of yeast cells over-expressing PDR18 was found to correlate with their increased ethanol tolerance and ability to restrain plasma membrane permeabilization induced throughout high gravity fermentation. Conclusions: PDR18 gene over-expression increases yeast ethanol tolerance and fermentation performance leading to the production of highly inhibitory concentrations of ethanol. PDR18 overexpression in industrial yeast strains appears to be a promising approach to improve alcoholic fermentation performance for sustainable bio-ethanol production. [ABSTRACT FROM AUTHOR]

The emergence of widespread multidrug resistance (MDR) is a serious challenge for therapeutics, food-preservation and crop protection. Frequently, MDR is a result of the action of drug-efflux pumps, which are able to catalyze the extrusion of unrelated chemical compounds. This review summarizes the current knowledge on the Saccharomyces cerevisiae drug:H+ antiporters of the major facilitator superfamily (MFS), a group of MDR transporters that is still characterized poorly in eukaryotes. Particular focus is given here to the physiological role and expression regulation of these transporters, while we provide a unified view of new data emerging from functional genomics approaches. Although traditionally described as drug pumps, evidence reviewed here corroborates the hypothesis that several MFS-MDR transporters might have a natural substrate and that drug transport might occur only fortuitously or opportunistically. Their role in MDR might even result from the transport of endogenous metabolites that affect the partition of cytotoxic compounds indirectly. Finally, the extrapolation of the gathered knowledge on the MDR phenomenon in yeast to pathogenic fungi and higher eukaryotes is discussed. [Copyright &y& Elsevier]

Microbially produced carboxylic acids (CAs) are considered key players in the implementation of more sustainable industrial processes due to their potential to replace a set of oil-derived commodity chemicals. Most CAs are intermediates of microbial central carbon metabolism, and therefore, a biochemical production pathway is described and can be transferred to a host of choice to enable/improve production at an industrial scale. However, for some CAs, the implementation of this approach is difficult, either because they do not occur naturally (as is the case for levulinic acid) or because the described production pathway cannot be easily ported (as it is the case for adipic, muconic or glucaric acids). Synthetic biology has been reshaping the range of molecules that can be produced by microbial cells by setting new-to-nature pathways that leverage on enzyme arrangements not observed in vivo, often in association with the use of substrates that are not enzymes' natural ones. In this review, we provide an overview of how the establishment of synthetic pathways, assisted by computational tools for metabolic retrobiosynthesis, has been applied to the field of CA production. The translation of these efforts in bridging the gap between the synthesis of CAs and of their more interesting derivatives, often themselves non-naturally occurring molecules, is also reviewed using as case studies the production of methacrylic, methylmethacrylic and poly-lactic acids. [ABSTRACT FROM AUTHOR]