Probiotic Potential,Antioxidant Activity,and Phytase Production of Indigenous Yeasts Isolated from Indigenous Fermented Foods

While many bacteria have been used as probiotics by industries, only two yeasts, Saccharomyces cerevisiae var. boulardii and Kluyveromyces fragilis (B0399), have been used for this purpose. In the present work, a total of 116 yeasts isolated from Brazilian indigenous fermented food, cocoa fermentation, and kefir were in vitro characterized for probiotic attributes. From 116 isolates, 36 were tolerant to gastrointestinal conditions evaluated by tolerance to pH 2.0, bile salts (0.3% w/v), and 37 °C temperature. From those, 15 isolates showed a similar or higher percentage (P < 0.05) of hydrophobicity, autoaggregation, and coaggregation with E. coli than the reference strain S. boulardii. All these strains showed a high percentage of adhesion to Caco-2 cells (> 63%) and antioxidant activity (ranging from 18 to 62%). Phytate hydrolysis was evaluated for these yeasts and 13 strains showed positive results, which is important for nutrient availability in plant-based foods. These results are important insights for characterization of novel probiotic yeast strains as well as to aggregate functional value to these food products.

     

Identification of a homolog of Arabidopsis DSP4 (SEX4) in chestnut: its induction and accumulation in stem amyloplasts during winter or in response to the cold

Oligosaccharide synthesis is an important cryoprotection strategy used by woody plants during winter dormancy. At the onset of autumn, starch stored in the stem and buds is broken down in response to the shorter days and lower temperatures resulting in the buildup of oligosaccharides. Given that the enzyme DSP4 is necessary for diurnal starch degradation in Arabidopsis leaves, this study was designed to address the role of DSP4 in this seasonal process in Castanea sativa Mill. The expression pattern of the CsDSP4 gene in cells of the chestnut stem was found to parallel starch catabolism. In this organ, DSP4 protein levels started to rise at the start of autumn and elevated levels persisted until the onset of spring. In addition, exposure of chestnut plantlets to 4 °C induced the expression of the CsDSP4 gene. In dormant trees or cold-stressed plantlets, the CsDSP4 protein was immunolocalized both in the amyloplast stroma and nucleus of stem cells, whereas in the conditions of vegetative growth, immunofluorescence was only detected in the nucleus. The studies indicate a potential role for DSP4 in starch degradation and cold acclimation following low temperature exposure during activity-dormancy transition.     

Amyloid fibril formation by circularly permuted and C-terminally deleted mutants

The natural N- and C-termini, i.e., the given order of secondary structure segments, are critical for protein folding and stability, as shown by several studies using circularly permuted proteins, mutants that have their N- and C-termini linked and are then digested at another site to create new termini. A previous work showed that circularly permuted mutants of sperm whale myoglobin (Mb) are functional, have native-like folding and bind heme, but are less stable than the wild-type protein and aggregate. The ability of wild-type myoglobin to form amyloid fibrils has been established recently, and because circularly permuted mutations are destabilizing, we asked whether these permutations would also affect the rate of amyloid fibril formation. Our investigations revealed that, indeed, the circularly permuted mutants formed cytotoxic fibrils at a rate higher than that of the wild-type. To further investigate the role of the C-terminus in the overall stability of the protein, we investigated two C-terminally deleted mutant, Mb(1-123) and Mb(1-99), and found that Mb(1-123) formed cytotoxic fibrils at a higher rate than that of the wild-type while Mb(1-99) formed cytotoxic fibrils at a similar rate than that of the wild-type. Collectively, our findings show that the native position of both the N-and C-termini is important for the precise structural architecture of myoglobin.     

Role of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> peroxiredoxins in mitochondrial bioenergetics

Trypanosoma cruzi cytosolic (TcCPx) and mitochondrial tryparedoxin peroxidase (TcMPx) play a fundamental role in H₂O₂ detoxification. Herein, mitochondrial bioenergetics was evaluated in cells that overexpressed TcCPx (CPx) and TcMPx (MPx) and in pTEX. In MPx, a higher expression was observed for TcCPx, and the same correlation was true for CPx. Differences in H₂O₂ release among the overexpressing cells were detected when the mitochondrial respiratory chain was inhibited using antimycin A or thenoyltrifluoroacetone. MPx had higher O₂ consumption rates than pTEX and CPx, especially in the presence of oligomycin. In all of the cells, the mitochondrial membrane potential and the ATP levels were similar. Because of the mild uncoupling that was observed in MPx, the presence or induction of a proton transporter in the mitochondrial membrane is suggested when TcMPx is expressed at higher levels. Our results show a possible interplay between the cytosolic and mitochondrial antioxidant systems in a trypanosomatid.     

Kinetics of production and characterization of the fucose-containing exopolysaccharide from Enterobacter A47

A fucose-containing exopolysaccharide (EPS) was produced by the bacterium Enterobacter A47 using glycerol byproduct from the biodiesel industry. The analysis of kinetic data suggested a partially growth associated EPS synthesis model. Although the EPS was composed of fucose, galactose and glucose at all cultivation stages, their relative proportion has varied considerably during the run. At the beginning (24 h), glucose was the main component (82.4 wt.%), being fucose and galactose minor components (5.0 wt.% and 10.9 wt.%, respectively), while at the end (96 h) it was composed of 26.0 wt.% fucose, 28.9 wt.% galactose and 43.7 wt.% glucose. The acyl groups content and composition have also changed, reaching their maximum content (19.2 wt.%) at the end of the run. Moreover, the molecular weight has increased linearly during the run (from 8 × 10⁵ to 5 × 10⁶). The changes observed in EPS composition and molecular weight have also had an impact upon the polymer's intrinsic viscosity, as shown by its linear increase from 3.95 to 10.72 dL g⁻¹. The results suggest that the culture might have synthesized at least two distinct EPS, with different sugar composition and average molecular weight, which predominated at different cultivation stages.     

Identification of high utility SNPs for population assignment and traceability purposes in the pig using high-throughput sequencing

The objectives of this study were to develop breed-specific single nucleotide polymorphisms (SNPs) in five pig breeds sequenced with Illumina's Genome Analyzer and to investigate their usefulness for breed assignment purposes. DNA pools were prepared for Duroc, Landrace, Large White, Pietrain and Wild Boar. The total number of animals used for sequencing was 153. SNP discovery was performed by aligning the filtered reads against Build 7 of the pig genome. A total of 313,964 high confidence SNPs were identified and analysed for the presence of breed-specific SNPs (defined in this context as SNPs for which one of the alleles was detected in only one breed). There were 29,146 putative breed-specific SNPs identified, of which 4441 were included in the PorcineSNP60 beadchip. Upon re-examining the genotypes obtained using the beadchip, 193 SNPs were confirmed as being breed specific. These 193 SNPs were subsequently used to assign an additional 490 individuals from the same breeds, using the sequenced individuals as reference populations. In total, four breed assignment tests were performed. Results showed that for all methods tested 99% of the animals were correctly assigned, with an average probability of assignment of at least 99.2%, indicating the high utility of breed-specific markers for breed assignment and traceability. This study provides a blueprint for the way next-generation sequencing technologies can be used for the identification of breed-specific SNPs, as well as evidence that these SNPs may be a powerful tool for breed assignment and traceability of animal products to their breeds of origin.     

Regulation of the cyclopropane synthase cfaB gene in Pseudomonas putida KT2440

In Pseudomonas putida, as in many other eubacteria, cyclopropane fatty acids (CFAs) accumulate in the membrane during the stationary phase of growth. Here, we show that cfaB gene expression in P. putida KT2440 is dependent on the RpoS sigma factor that recognizes the sequence 5'-CTACTCT-3' between -8 and -14. We have carried out a mutational study of the cfa promoter and have determined that positions -9, -12, -13 and -14 are the most critical for maximal activity. In P. putida, the substrates of the CFA synthase, cis-unsaturated fatty acids (cis-UFAs), are also substrates for another stress-related enzyme, the cis-trans isomerase (CTI). Despite using the same substrates, we have found that the activity of the CTI is not limited by the CFA synthase activity and vice versa. For instance, in a cfaB knockout mutant, the amount of trans-UFAs synthesized after a specific stress was no higher than in the parental background despite the fact that there are more cis-UFAs available to be used by the CTI as substrates. In this regard, in a cti-deficient mutant background, the levels of CFAs were similar to those in the parental one under the same conditions.     

Physiologically relevant divalent cations modulate citrate recognition by the McpS chemoreceptor

The McpS chemoreceptor of Pseudomonas putida KT2440 recognizes six different tricarboxylic acid (TCA) cycle intermediates. However, the magnitude of the chemotactic response towards these compounds differs largely, which has led to distinguish between strong attractants (malate, succinate, fumarate, oxaloacetate) and weak attractants (citrate, isocitrate). Citrate is abundantly present in plant tissues and root exudates and can serve as the only carbon source for growth. Citrate is known to form complexes with divalent cations which are also abundantly present in natural habitats of this bacterium. We have used isothermal titration calorimetry to study the formation of citrate-metal ion complexes. In all cases binding was entropy driven but significant differences in affinity were observed ranging from K(D)=157 μM (for Mg(2+)) to 3 μM (for Ni(2+)). Complex formation occurred over a range of pH and ionic strength. The ligand binding domain of McpS (McpS-LBD) was found to bind free citrate, but not complexes with physiologically relevant Mg(2+) and Ca(2+). In contrast, complexes with divalent cations which are present as trace elements (Co(2+), Cd(2+) and Ni(2+)) were recognized by McpS-LBD. This discrimination differs from other citrate sensing proteins. These results are discussed in the context of the three dimensional structure of free citrate and its complex with Mg(2+). Chemotaxis assays using P. putida revealed that taxis towards the strong attractant malate is strongly reduced in the presence of free citrate. However, this reduction is much less important in the presence of citrate-Mg(2+) complexes. The physiological relevance of these findings is discussed.     

Regenerated luminal epithelial cells are derived from preexisting luminal epithelial cells in adult mouse prostate

Determining the source of regenerated luminal epithelial cells in the adult prostate during androgen deprivation and replacement will provide insights into the origin of prostate cancer cells and their fate during androgen deprivation therapy. Prostate stem cells in the epithelial layer have been suggested to give rise to luminal epithelium. However, the extent of stem cell participation to prostate regrowth is not clear. In this report, using prostate-specific antigen-CreER(T2)-based genetic lineage marking/tracing in mice, preexisting luminal epithelial cells were shown to be a source of regenerated luminal epithelial cells in the adult prostate. Prostatic luminal epithelial cells could survive androgen deprivation and were capable of proliferating upon androgen replacement. Prostate cancer cells, typically exhibiting a luminal epithelial phenotype, may retain this intrinsic capability to survive and regenerate in response to changes in androgen signaling, providing part of the mechanism for the ultimate failure of androgen deprivation therapy in prostate cancer.     

Phenazine-1-carboxylic acid promotes bacterial biofilm development via ferrous iron acquisition

The opportunistic pathogen Pseudomonas aeruginosa forms biofilms, which render it more resistant to antimicrobial agents. Levels of iron in excess of what is required for planktonic growth have been shown to promote biofilm formation, and therapies that interfere with ferric iron [Fe(III)] uptake combined with antibiotics may help treat P. aeruginosa infections. However, use of these therapies presumes that iron is in the Fe(III) state in the context of infection. Here we report the ability of phenazine-1-carboxylic acid (PCA), a common phenazine made by all phenazine-producing pseudomonads, to help P. aeruginosa alleviate Fe(III) limitation by reducing Fe(III) to ferrous iron [Fe(II)]. In the presence of PCA, a P. aeruginosa mutant lacking the ability to produce the siderophores pyoverdine and pyochelin can still develop into a biofilm. As has been previously reported (P. K. Singh, M. R. Parsek, E. P. Greenberg, and M. J. Welsh, Nature 417:552-555, 2002), biofilm formation by the wild type is blocked by subinhibitory concentrations of the Fe(III)-binding innate-immunity protein conalbumin, but here we show that this blockage can be rescued by PCA. FeoB, an Fe(II) uptake protein, is required for PCA to enable this rescue. Unlike PCA, the phenazine pyocyanin (PYO) can facilitate biofilm formation via an iron-independent pathway. While siderophore-mediated Fe(III) uptake is undoubtedly important at early stages of infection, these results suggest that at later stages of infection, PCA present in infected tissues may shift the redox equilibrium between Fe(III) and Fe(II), thereby making iron more bioavailable.