Heritable variation in resistance to pathogens has been reported in many fish species, but little is known about its genetic architecture. To extend understanding, an investigation was made of the association of resistance to proliferative kidney disease (PKD) in 4 second filial generation (F2) families of Atlantic salmon with molecular markers from different genetic linkage groups in the species' genome, following a natural disease outbreak. PKD causes serious mortality in cultured salmonids. In addition to mortality, associations with growth-related traits were also examined, as immune responses are energetically costly and have been observed to reduce growth. Associations were investigated for 34 microsatellite markers and 5 restriction fragment length polymorphism (RFLP) loci from 3 regions of the growth hormone 1 gene (GH1). The phenotypic and genotypic character of survivors was compared with unexposed fish derived from the same families. Mortality was not size-selective, but growth in the survivors was reduced, and fish had a lower condition factor than unexposed fish, suggesting an energetic cost to resistance. Five markers showed significant allele frequency differences between survivors and unexposed fish, albeit in single families. Prior to correction for multiple tests, 2 of these markers were also linked to variation in growth-related traits among survivors, along with a further 7 markers. Though sample sizes constrained the power of the analysis, the study points to regions of the salmon genome that may contain quantitative trait loci related to PKD resistance, on which further work on the genetic architecture of PKD resistance in this species could focus.

In the plant pathogen Streptomyces scabies, the gene bglC encodes a GH1 family cellobiose beta-glucosidase that is both required for primary metabolism and for inducing virulence of the bacterium. Deletion of bglC (strain ΔbglC) surprisingly resulted in the augmentation of the global beta-glucosidase activity of S. scabies. This paradoxical phenotype is highly robust as it has been observed in all bglC deletion mutants independently generated, thereby highlighting a phenomenon of genetic compensation. Comparative proteomics allowed to identify two glycosyl hydrolases - named BcpE1 and BcpE2 - of which peptide levels were significantly increased in strain ΔbglC. Quantitative RT-PCR revealed that the higher abundance of BcpE1 and BcpE2 is triggered at the transcriptional level, the expression of their respective gene being 100 and 15 times upregulated. Enzymatic studies with pure BcpE proteins showed that they both possess beta-glucosidase activity thereby explaining the genotypic-phenotypic discrepancy of the bglC deletion mutant. The GH1 family BcpE1 could hydrolyze cellobiose and generate glucose similarly to BglC itself thereby mainly contributing to the survival of strain ΔbglC when cellobiose is provided as sole nutrient source. The low affinity of BcpE2 for cellobiose suggests that this GH3 family beta-glucosidase would instead primarily target another and yet unknown glucose-beta-1,4-linked substrate. These results make S. scabies a new model system to study genetic compensation. Discovering how, either the bglC DNA locus, its mRNA, the BglC protein, or either its enzymatic activity controls bcpE genes' expression, will unveil new mechanisms directing transcriptional repression.

Keywords: Beta-glucosidase; Enzyme expression control; Genetic compensation; paradoxical phenotype; transcription awakening.

A homozygous mutation in growth hormone 1 (GH1) was recently identified in an individual with growth failure. This mutation, c.705G>C causes the replacement of cysteine at position 53 of the 191 amino acid sequence of 22 kDa human GH (hGH) with serine (p.Cys53Ser). This hGH molecule (further referred to as GH-C53S) lacks the disulfide bond between p.Cys53 and p.Cys165, which is highly conserved among species. It has been previously reported that monomeric GH-C53S has reduced bioactivity compared with wild-type GH (GH-wt) because of its decreased ability to bind and activate the GH receptor in vitro. In this study, we discovered that the substitution of p.Cys53 in hGH significantly increased formation of hGH-dimers in pituitary cells. We expressed his-tagged hGH variants in the cytoplasm of genetically modified Rosetta gami B DE3 Escherichia coli cells, facilitating high yield production. We observed that the bioactivity of monomeric GH-C53S is 25.2% of that of wild-type GH and that dimeric GH-C53S-his has no significant bioactivity in cell proliferation assays. We also found that the expression of GH-C53S in pituitary cells deviates from that of GH-wt. GH-C53S was exclusively stained in the Golgi apparatus, and no secretory granules formed for this variant, impairing its stimulated release. In summary, the unpaired cysteine C165 in GH-C53S forms a disulfide bond linking two hGH molecules in pituitary cells. We conclude that the GH-C53S dimer is inactive and responsible for the growth failure in the affected individual.

Salt bridges are the strongest electrostatic interactions in proteins. They substantially contribute to a protein's structural stability. Thus, mutations of salt bridges are typically selected against. Here, we report on the evolutionary loss of a highly conserved salt bridge in the GH1 family glycosyl hydrolase BglM-G1. BglM-G1's gene was found in the bacterial metagenome of a temperate, seasonally cold marine habitat. In BglM-G1, arginine 75 is replaced by a histidine. While fully retaining β-glucosidase activity, BglM-G1 is less heat stable than an H75R variant, in which the salt bridge was artificially re-introduced. However, the K m toward its substrates was lower in wild type, leading to an overall higher catalytic efficiency. Our results indicate that this loss of the salt bridge leads to higher flexibility in BglM-G1's active site, trading structural stability at high temperatures, a trait not needed in a temperate, seasonally cold habitat, for a more effective catalytic activity.

The influence of chemical modification of His residues in Mb on the rate of redox reaction in system MbO2--Cyt c has been studied at different ionic strengths and pH medium. The products of alkylation of all available His by bromacetate and iodacetamide, CM-Mb and CA-Mb, respectively, and myoglobin, modified by spin label 2,2', 6,6'-tetramethyl-4-bromoacetoxypiperidine-1-oxyl (SL) at His residue A10--Sl (His-A10)--Mb have been studied. It has been shown, that the character of the ionic strength dependence of reaction SL(His-A10)--MbO2 with Cyt c at pH 6.0 ann 7.0 is basically analogues to that, observed for intact protein. It means that only His-GH1 of two His residues, His-A10 and His-GH1, situated in the region of "active contact" of Mg with Cyt c molecule, participates in the interactions, essential for electron transfer. The interaction of the charge of this His with the negatively charged group of Cyt c is necessary, probably for the proper arrangement of other interactions in the active complex, because the deprotonation of His-GHl in the studied pH interval decreases the rate of the process by more than one order of magnitude. The rate of oxidation of MC-MbO2 and CA-MbO2 by ferricytochrome c, in contrast to intact protein, shows a weak dependence on the ionic strength and does not depend on the pH medium, throughout the range of ionic strengths from 0.005 to 1.0. The cause of the radical change in the ionic strength dependence is, probably, nearly entire disturbance of electrostatic interactions in the active complex due to chemical modification of His residues in the site of "active contact", and first of all, the His-CHl residue. The fact, that during alkylation of all available His in Mb the electron transfer persists in the system, points to that in the process of electron transfer to cytochrome c, uncharged group, most probably "inner" His-B5, participates. Based on the data on spatial structure and the obtained results, the positions of the charged groups in the site of "active contact" of Mb with Cyt c molecule are presented.

The β-glucosidase TnBgl1A catalyses hydrolysis of O-linked terminal β-glycosidic bonds at the nonreducing end of glycosides/oligosaccharides. Enzymes with this specificity have potential in lignocellulose conversion (degrading cellobiose to glucose) and conversion of bioactive flavonoids (modification of glycosylation results in modulation of bioavailability). Previous work has shown TnBgl1A to hydrolyse 3, 4' and 7 glucosylation in flavonoids, and although conversion of 3-glucosylated substrate to aglycone was low, it was improved by mutagenesis of residue N220. To further explore structure-function relationships, the crystal structure of the nucleophile mutant TnBgl1A-E349G was determined at 1.9 Å resolution, and docking studies of flavonoid substrates were made to reveal substrate interacting residues. A series of single amino acid changes were introduced in the aglycone binding region [N220(S/F), N221(S/F), F224(I), F310(L/E), and W322(A)] of the wild type. Activity screening was made on eight glucosylated flavonoids, and kinetic parameters were monitored for the flavonoid quercetin-3-glucoside (Q3), as well as for the model substrate para-nitrophenyl-β-d-glucopyranoside (pNPGlc). Substitution by Ser at N220 or N221 increased the catalytic efficiency on both pNPGlc and Q3. Residue W322 was proven important for substrate accomodation, as mutagenesis to W322A resulted in a large reduction of hydrolytic activity on 3-glucosylated flavonoids. Flavonoid glucoside hydrolysis was unaffected by mutations at positions 224 and 310. The mutations did not significantly affect thermal stability, and the variants kept an apparent unfolding temperature of 101°C. This work pinpoints positions in the aglycone region of TnBgl1A of importance for specificity on flavonoid-3-glucosides, improving the molecular understanding of activity in GH1 enzymes. Proteins 2017; 85:872-884. © 2016 Wiley Periodicals, Inc.

Incubation of GH1 cells with cholera toxin for 24 h inhibits [32P]ADP-ribose incorporation into histones and non-histone nuclear proteins by more than 50%. The toxin produces a generalized decrease of incorporation into all protein acceptors and into the poly(ADP-ribosyl)ated components excised from chromatin after micrococcal nuclease digestion. The cellular levels of NAD were also decreased (40 to 80%) after treatment with cholera toxin. The inhibition of poly(ADP-ribosyl)ation is preceded by an increase of [32P]ADP-ribose incorporation, since incubation with the toxin for 3 h caused an increase instead of a decrease of incorporation. Incubation with dibutyryl cyclic AMP for 24 h also inhibited nuclear poly(ADP-ribosyl)ation, thus showing that the effect of cholera toxin might be mediated by cyclic AMP.

BACKGROUND

Congenital isolated growth hormone deficiency (IGHD) is a rare endocrine disorder that presents with severe proportionate growth failure. Dominant (type II) IGHD is usually caused by heterozygous mutations of GH1. The presentation of newly affected family members in 3 families with dominant IGHD in whom previous genetic testing had not demonstrated a GH1 mutation or had not been performed, prompted us to identify the underlying genetic cause.

METHODS

GH1 was sequenced in 3 Caucasian families with a clinical autosomal dominant IGHD.

RESULTS

All affected family members had severe growth hormone (GH) deficiency that became apparent in the first 2 years of life. GH treatment led to a marked increase in height SDS. So far, no other pituitary dysfunctions have become apparent. In the first family a novel splice site mutation in GH1 was identified (c.172-1G>C, IVS2-1G>C). In two other families a previously reported splice site mutation (c.291+1G>A, IVS3+1G>A) was found.

CONCLUSIONS

These data show that several years after negative genetic testing it was now possible to make a genetic diagnosis in these families with a well-defined, clearly heritable, autosomal dominant IGHD. This underscores the importance of clinical and genetic follow-up in a multidisciplinary setting. It also shows that even without a positive family history, genetic testing should be considered if the phenotype is strongly suggestive for a genetic syndrome. Identification of pathogenic mutations, like these GH1 mutations, has important clinical implications for the surveillance and genetic counseling of patients and expands our knowledge on the genotype-phenotype correlation.

Specific catalytic oxidation of sperm whale oxymyoglobin by small amounts of potassium ferri- and ferrocyanide, from 1 to 20% in relation to the protein concentration, was studied. The mechanism of catalysis was shown to involve specific binding of the ferrocyanide anion to the protein. The influence of pH and ionic strength of the medium, the [Fe(CN)6]4- concentration and of chemical modification of Mb histidines by bromoacetate, as well as the effect of the Mb complexing with redox-inactive zinc ion on the rate of reaction was examined. The zinc ion forms a stable complex with His 119(GH1) on the Mb surface at the equimolar Zn2+ concentration. The kinetic scheme of the reaction was analyzed, and the equilibrium and kinetic parameters were obtained. It was first shown that the strong oxidant such as potassium ferricyanide is able to react with the same protein by two distinct mechanisms: (i) a simple outer sphere electron transfer over the heme edge and (ii) electron transfer after the specific binding of [Fe(CN)6]4- to oxyMb in the His 119(GH1) region, thus catalyzing the protein oxidation.

The pH-dependence of the reduction rate of ferricytochrome C by intact and chemically modified oxymyoglobins has been studied. The modification was performed with respect to histidine residues and alpha-aminogroup of N-terminal valine. Two histidine residues of myoglobin, His A10 and His GH1, are shown to take part in the realization of the "active" contact between the molecules in the course of the reaction. The deprotonation of the first residue contributes to the acceleration and that of the second to the reduction of the reaction. The found orientation of the Mb molecules in the "active complex" implies that at any orientation of cytochrome C the distance between the haemes of the both molecules should be more than 30 A. This makes highly probable that a structure-dependent mechanism of electron transfer in the system under study can be proposed.

Experiments from a number of laboratories, employing clonal lines of rat pituitary tumor cells (GH cells), have shown that the cellular levels of the mRNAs for prolactin and growth hormone are subject to regulation by a variety of hormones and other factors. To provide a framework for further studies of the regulation of the expression of these two genes, we have studied their chromosomal locations in the rat, employing our recently developed technique for identifying a unique gene by direct hybridization in situ with a 125I-labeled cDNA plasmid. A rat prolactin cDNA plasmid ( pPRL -1) yielded a signal significantly above that expected on a random basis over chromosomes 9 and 15 from rat FRE cells. Both chromosomes exhibited a subchromosomal clustering of silver grains near the telomere of chromosome 9 and on the short arm of chromosome 15. Control experiments with plasmid pBR322 also yielded a significant signal clustered over the short arm of chromosome 15. Hence, we conclude that chromosome 15 contains sequences homologous to pBR322 and that the prolactin gene is located near the telomere of chromosome 9 in the rat. A rat growth hormone cDNA plasmid (pBR322-GH1) yielded a signal above that expected on a random basis over FRE cell chromosomes 7, 13, and 15, although the labeling was poorer than with prolactin. The signal over chromosome 15 is probably due to sequences homologous to plasmid pBR322. The signals over chromosomes 7 and 13 showed a clear subchromosomal clustering near the centromere on chromosome 7 and near the telomere on the long arm of chromosome 13.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth hormone (GH) was elevated in young growing, intact female Wistar-Furth rats bearing growth hormone (GH1) or growth hormone and prolactin (GH3) secreting tumors. Animals were injected with GH1 or GH3 cells at 1 wk of age. Total feed intake was measured for the 8-wk period from weaning until killed at 11 wk of age. Animals were fed a commercial chow diet throughout the trial. Body composition and composition of the liver and tibialis anterior muscle were determined. Tumor-bearing rats were about 65% heavier than control rats at 11 wk of age: most of the difference in body weight gain was obtained during the last 4 wk of the trial. Total feed intake during the 8 wk after weaning was increased in both GH1 and GH3 tumor-bearing rats when compared with controls. Overall feed efficiency (grams feed consumed/gram body weight gain) was improved in tumor-bearing animals when compared with controls. The GH1 tumor-bearing rats were slightly hyperphagic during wk 8, 9 and 10 (grams feed consumed/gram body weight) when compared with controls. The total amount of body dry matter, protein and ash was increased in tumor-bearing rats when compared with controls. There was no effect on total body fat. Tumor-bearing rats had increased liver weight and increased fat, protein, RNA, DNA and dry matter content when compared with controls. Tumor induction increased the weight, total RNA and total fat content of the tibialis anterior muscle when compared with controls. There was no effect on muscle protein content.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel β-glucosidase, BglD1 with high β-galactosidase and transglycosidation activities, was screened and cloned from the deep-sea bacterium Bacillus sp. D1. BglD1 exhibited the maximal β-glucosidase and β-galactosidase activities at 55-60 °C and pH 5.5-6.0. The enzyme maintained approximately 50% of its original activity at 35 °C and pH 6.0 after 120-h incubation. When applied to synthesize galacto-oligosaccharides (GOS), BglD1 generated 118.3 g/L GOS (33.8% (w/w)) from 350 g/L lactose, with trisaccharide Gal-β(1 → 3)-Lac and disaccharide Gal-β(1 → 4)-Gal as the main components. Furthermore, BglD1 could hydrolyze lactose in milk and produce GOS simultaneously. Using milk as the substrate, BglD1 hydrolyzed 88.5% lactose and produced 3.3 g/L GOS after incubation at 30 °C for 1 h. To improve the transglycosidation activity, a mutant BglD1:E224T was generated based on the semi-rational design. The GOS yield of BglD1:E224T was 11.5% higher than that of BglD1 when using lactose solution as the substrate. Thus, BglD1 and the mutant could be used as beneficial alternatives of the existing β-galactosidases for the production of GOS.

Biocellulose, named "the biomaterial of the future", is a natural and ecologically friendly polymer, produced by selected acetic acid bacteria strains. Biocellulose impregnated with antimicrobial agents can be used as a novel, safe, and biodegradable food packaging material, helping extend the shelf life of some products and may also have the chance to replace typical plastic packaging, which is a big environmental problem these days. This study aimed to evaluate if cellulose impregned with natural oregano essential oil could show antibacterial activity against Cronobacter strains, which can occur in food, causing diseases and food poisoning. Bacterial cellulose was obtained from two acetic bacteria strains, Gluconacetobacter hansenii ATCC 23769 and Komagataeibacter sp. GH1. Antibacterial activity was studied by the disc-diffusion method against chosen Cronobacter strains, isolated from the plant matrix. Oregano essential oil has been shown to penetrate into the structure of bacterial cellulose, and after applying cellulose to the solid medium, it showed the ability to migrate. Biopolymer from the strain K. sp. GH1 was able to better absorb and retain essential oregano oil (OEO) compared to bacterial cellulose (BC) produced by the G. hansenii ATCC 23769. Bacterial cellulose with oregano essential oil from strain Komagataeibacter GH1 showed generally greater inhibitory properties for the growth of tested strains than its equivalent obtained from G. hansenii. This was probably due to the arrangement of the polymer fibers and its final thickness. The largest zone of inhibition of strain growth was observed in relation to C. condimenti s37 (32.75 mm ± 2.8). At the same time, the control sample using filter paper showed an inhibition zone of 36.0 mm ± 0.7. A similar inhibition zone (28.33 mm ± 2.6) was observed for the C. malonaticus lv31 strain, while the zone in the control sample was 27.1 mm ± 0.7. Based on this study, it was concluded that bacterial cellulose impregnated with oregano essential oil has strong and moderate antimicrobial activity against all presented strains of the genus Cronobacter isolated from plant matrix. Obtained results give a strong impulse to use this biopolymer as ecological food packaging in the near future.

Keywords: Cronobacter; antibacterial activity; biocellulose; food packaging; oregano essential oil.

Among female rats, mating enhances neurosteroid formation in the midbrain ventral tegmental area (VTA; independent of peripheral steroid-secreting glands, ovaries, and adrenals). The sources/targets for these actions are not well understood. In Experiment 1, proestrous rats engaged in a mating paradigm, or did not, and the midbrains had been assessed via the Affymetrix rat genome microarrays. In Experiment 2, the influence of gonadal and adrenal glands on the expression of these genes was assessed in rats that were proestrous, ovariectomized (OVX), or OVX and adrenalectomized (ADX). The microarrays revealed 53 target genes that were significantly up-regulated (>2.0-fold change) in response to mating. Mating significantly enhanced the midbrain mRNA expression of genes involved in hormonal and trophic actions: Gh1, S100g, and Klk1b3 in proestrous, but not OVX and/or ADX, rats; Fshb in all but OVX/ADX rats; and lutenizing hormone β and thyroid-stimulating hormone (TSH) β in all rats. Thus, mating enhances midbrain gene expression independent and dependent of peripheral glands.

Growth hormone (GH) production by GH1 rat pituitary tumor cells in iron restricted serum-free defined medium requires apotransferrin (apoTf) and triiodothyronine (T3). As measured by radioimmunoassay, apoTf plus T3 induced GH levels 2 to 4-fold above controls. Deletion of either apoTf or T3 arrested GH secretion. ApoTf/T3 defined medium regulated GH production as effectively as whole serum. Because glucocorticoids enhance GH secretion in serum containing cultures, the effects of dexamethasone were evaluated in apoTf/T3 defined medium. The steroid hormone showed no enhancing effects unless the cells were exposed to serum prior to incubation in apoTf/T3 defined medium. Even under these conditions, the response to dexamethasone remained T3 dependent. These observations indicate that a yet to be characterized serum factor(s), other than apoTf, regulates the response to the steroid hormone. This is the first report of thyroid hormone regulation of GH secretion by rat pituitary tumor cells under completely serum-free chemically defined conditions.

The effect of zink ions, which according to the X-ray data are bound to the His GH1 residue of myoglobin, has been investigated. It is shown that the electron transfer in the system is almost completely inhibited at the equimolar Zn2+ concentration in the pH range 5 to 8. Unlike the reaction between the intact MbO2 and Cyt c, the electron transfer rate in this case does not depend on pH and ionic strength of the solution. Further increase of Zn2+ concentration up to the 20-fold molar excess has no significant effect on the rate of the process. Since the thermodynamic characteristics of the redox reaction between MbO2 and Cyt c are not altered in the presence of Zn2+, the findings obtained can be interpreted as indicating the important role of His GH1 in the formation of productive electron transfer complex.

In the genome of most vertebrates growth-hormone gene is presented in a single copy, while in salmonids after one of the duplication events many genes were multiplied, including growth hormone gene. In salmonids, the growth-hormone gene exists as two independently inherited functional paralogues, gh1 and gh2. In this study, we performed a comparative analysis of gh1 and gh2 growth-hormone genes and their adjacent sequences in Levanidov's charr Salvelinus levanidovi to determine their functionality and define the potential differences. We found that both genes have the same gene structure and are composed of six exons (I-VI) and five introns (A, B, C, D, E). However, the respective gene sequences differ in length. A comparison of exons showed that the size of each exon is identical in both paralogues. The overall length of genes differs due to the varying lengths of introns. Coding sequence of both genes contains an open reading frame for 210 amino acids. We identified regulatory elements in the promoter region of both genes: TATA box, A/T-rich regions that contain binding sites for pituitary-specific transcriptional activator Pit-1, and regions responsible for interaction with other transcriptional activators and initiators, in particular hormone receptors. The obtained data indicate that both genes are functional.

BACKGROUND

Efficient biomass bioconversion is a promising solution to alternative energy resources and environmental issues associated with lignocellulosic wastes. The Trichoderma species of cellulolytic fungi have strong cellulose-degrading capability, and their cellulase systems have been extensively studied. Currently, a major limitation of Trichoderma strains is their low production of β-glucosidases.

RESULTS

We isolated two Trichoderma hamatum strains YYH13 and YYH16 with drastically different cellulose degrading efficiencies. YYH13 has higher cellobiose-hydrolyzing efficiency. To understand mechanisms underlying such differences, we sequenced the genomes of YYH13 and YYH16, which are essentially identical (38.93 and 38.92 Mb, respectively) and are similar to that of the T. hamatum strain GD12. Using GeneMark-ES, we annotated 11,316 and 11,755 protein-coding genes in YYH13 and YYH16, respectively. Comparative analysis identified 13 functionally important genes in YYH13 under positive selection. Through examining orthologous relationships, we identified 172,655, and 320 genome-specific genes in YYH13, YYH16, and GD12, respectively. We found 15 protease families that show differences between YYH13 and YYH16. Enzymatic tests showed that exoglucanase, endoglucanase, and β-glucosidase activities were higher in YYH13 than YYH16. Additionally, YYH13 contains 10 families of carbohydrate-active enzymes, including <em>GH1</em>, GH3, <em>GH1</em>8, GH35, and GH55 families of chitinases, glucosidases, galactosidases, and glucanases, which are subject to stronger positive selection pressure. Furthermore, we found that the β-glucosidase gene (YYH1311079) and pGEX-KG/YYH1311079 bacterial expression vector may provide valuable insight for designing β-glucosidase with higher cellobiose-hydrolyzing efficiencies.

CONCLUSIONS

This study suggests that the YYH13 strain of T. hamatum has the potential to serve as a model organism for producing cellulase because of its strong ability to efficiently degrade cellulosic biomass. The genome sequences of YYH13 and YYH16 represents a valuable resource for studying efficient production of biofuels.

Exopolysaccharide (EPS)-producing lactic acid bacteria have been widely used in dairy products, but how calcium, the main metal ion component in milk, regulates the EPS biosynthesis in lactic acid bacteria is not clear. In this study, the effect of Ca²⁺ on the biosynthesis of EPS in the probiotic Lactobacillus plantarum K25 was studied. The results showed that addition of CaCl₂ at 20 mg/L in a semi-defined medium did not affect the growth of strain K25, but it increased the EPS yield and changed the microstructure of the polymer. The presence of Ca²⁺ also changed the monosaccharide composition of the EPS with decreased high molecular weight components and more content of rhamnose, though the functional groups of the polymer were not altered as revealed by Fourier transform infrared spectral analysis. These were further confirmed by analysis of the mRNA expression of cps genes, 9 of which were upregulated by Ca²⁺, including cps4F and rfbD associated with EPS biosynthesis with rhamnose. Proteomics analysis showed that Ca²⁺ upregulated most of the proteins related to carbon transport and metabolism, fatty acid synthesis, amino acid synthesis, ion transport, UMP synthesis. Specially, the increased expression of MelB, PtlIIBC, EIIABC, PtlIIC, PtlIID, Bgl, GH1, MalFGK, DhaK, and FBPase provided substrates for the EPS synthesis. Meanwhile, metabolomics analysis revealed significant change of the small molecular metabolites in tricarboxylic acid cycle, glucose metabolism and propionic acid metabolism. Among them the content of active small molecules such as polygalitol, lyxose, and 5-phosphate ribose increased, facilitating the EPS biosynthesis. Furthermore, Ca²⁺ activated HipB signaling pathway to inhibit the expression of manipulator repressor such as ArsR, LytR/AlgR, IscR, and RafR, and activated the expression of GntR to regulate the EPS synthesis genes. This study provides a basis for understanding the overall change of metabolic pathways related to the EPS biosynthesis in L. plantarum K25 in response to Ca²⁺, facilitating exploitation of its EPS-producing potential for application in probiotic dairy products.

Keywords: Lactobacillus plantarum; biosynthesis gene; calcium; exopolysaccharides.

Background: Ellagitannase (Ellagitannin acyl hydrolase) is an inducible enzyme with great potential use in food industry since allows the ellagic acid release from ellagitannins.

Objective: In this work, ellagitannase was produced by the fungus Aspergillus niger GH1 in solid state fermentation using polyurethane foam as solid support and pomegranate husk ellagitannins as sole carbon source and ellagitannase inducer and an initial approach to the enzymatic reaction conditions was reached.

Materials and methods: Ellagitannase was produced by Aspergillus niger GH1 in solid state fermentation and the ideal reaction conditions for ellagitannase activity based on ellagic acid quantification as ellagitannins biotransformation product by high performance liquid chromatographic are reported.

Results: The enzyme ideal reaction conditions were substrate concentration of 1 mg.mL^-1, 60 °C and pH 5.0, during 10 min of reaction. The kinetic enzyme constants (V _max = 30.34 mM.mL^-1.min^-1 and K _m = 1.48 x 10³ mM) using punicalagin assubstrate were determined.

Conclusion: The assay was completed in a short time and may find application in future studies of ellagic acid production.

Keywords: Ellagitannase assay; Ellagitannin acyl hydrolase; HPLC; Pomegranate ellagitannins; Aspergillus niger GH1.

Domesticated dogs show unparalleled diversity in body size across breeds, but within breeds variation is limited by selective breeding. Many heritable diseases of dogs are found among breeds of similar sizes, suggesting that as in humans, alleles governing growth have pleiotropic effects. Here, we conducted independent genome-wide association studies in the small Shetland Sheepdog breed and discovered a locus on chromosome 9 that is associated with a dental abnormality called maxillary canine-tooth mesioversion (MCM) (P = 1.53 × 10^-7) as well as two body size traits: height (P = 1.67 × 10^-5) and weight (P = 1.16 × 10^-7). Using whole-genome resequencing data, we identified variants in two proximal genes: FTSJ3, encoding an RNA methyltransferase, and GH1, encoding growth hormone. A substitution in FTSJ3 and a splice donor insertion in GH1 are strongly associated with MCM and reduced body size in Shetland Sheepdogs. We demonstrated in vitro that the GH1 variant leads to exon 3 skipping, predicting a mutant protein known to cause human pituitary dwarfism. Statistical modeling, however, indicates that the FTSJ3 variant is the stronger predictor of MCM and that each derived allele reduces body size by about 1 inch and 5 pounds. In a survey of 224 breeds, both FTSJ3 and GH1 variants are frequent among very small "toy" breeds and absent from larger breeds. Our findings indicate that a chromosome 9 locus harboring tightly linked variants in FTSJ3 and GH1 reduces growth in the Shetland Sheepdog and toy breed dogs and confers risk for MCM through vertical pleiotropy.

Keywords: FTSJ3; GH1; IGF1; height; weight.

Strains of the Gram-positive, thermophilic bacterium Geobacillus stearothermophilus possess elaborate systems for the utilization of hemicellulolytic polysaccharides, including xylan, arabinan, and galactan. These systems have been studied extensively in strains T-1 and T-6, representing microbial models for the utilization of soil polysaccharides, and many of their components have been characterized both biochemically and structurally. Here, we characterized routes by which G. stearothermophilus utilizes mono- and disaccharides such as galactose, cellobiose, lactose, and galactosyl-glycerol. The G. stearothermophilus genome encodes a phosphoenolpyruvate carbohydrate phospho-transferase system (PTS) for cellobiose. We found that the cellobiose-PTS system is induced by cellobiose and characterized the corresponding GH1 6-phospho-β-glucosidase, Cel1A. The bacterium also possesses two transport systems for galactose, a galactose-PTS system and an ABC galactose transporter. The ABC galactose transport system is regulated by a three-component sensing system. We observed that both systems, the sensor and the transporter, utilize galactose-binding proteins that also bind glucose with the same affinity. We hypothesize that this allows the cell to control the flux of galactose into the cell in the presence of glucose. Unexpectedly, we discovered that G. stearothermophilus T-1 can also utilize lactose and galactosyl-glycerol via the cellobiose-PTS system together with a bifunctional 6-phospho-β-galactosidase/glucosidase, Gan1D. Growth curves of strain T-1 growing in the presence of cellobiose, with either lactose or galactosyl-glycerol, revealed initially logarithmic growth on cellobiose and then linear growth supported by the additional sugars. We conclude that Gan1D allows the cell to utilize residual galactose-containing disaccharides, taking advantage of the promiscuity of the cellobiose-PTS system.

Keywords: 6-phospho-β-galactosidase; ABC transporter; G. stearothermophilus; PTS system; bacteria; bacterial metabolism; cellobiose; galactose; galactosyl-glycerol; glycoside hydrolase; thermophile.