Oxalate degradation is one of lactic acid bacteria's desirable activities. It is achieved by two enzymes, formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc). The current study aimed to screen 15 locally isolated lactic acid bacteria to select those with the highest oxalate degradation ability. It also aimed to amplify the genes involved in degradation. MRS broth supplemented with 20 mM sodium oxalate was used to culture the tested isolates for 72 h. This was followed by an enzymatic assay to detect remaining oxalate. All isolates showed oxalate degradation activity to variable degrees. Five isolates demonstrated high oxalate degradation, 78 to 88%. To investigate the oxalate-degradation potential of the selected isolates, they have been further tested for the presence of genes that encode for enzymes involved in oxalate catabolism, formyl coenzyme A transferase (frc) and oxalyl coenzyme A decarboxylase (oxc). Three strains showed bands with the specific OXC and FRC forward and reverse primers designated as (SA-5, 9 and 37). Species-level identification revealed Loigolactobacillus bifermentans, Lacticaseibacillus paracasei, and Lactiplantibacillus plantarum. Preliminary results revealed that the tested probiotic strains harbored both oxc and frc whose products are putatively involved in oxalate catabolism. The probiotic potential of the selected strains was evaluated, and they showed high survival rates to both simulated gastric and intestinal fluids and variable degrees of antagonism against the tested Gram-positive and negative pathogens and were sensitive to clarithromycin but resistant to both metronidazole and ceftazidime. Finally, these strains could be exploited as an innovative approach to establish oxalate homeostasis in humans and prevent kidney stone formation.
- MeSH
- acylkoenzym A * MeSH
- CoA-transferasy genetika metabolismus MeSH
- karboxylyasy * genetika MeSH
- lidé MeSH
- oxaláty metabolismus MeSH
- probiotika * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Auxins are hormones that regulate growth and development in plants. Besides plants, various microorganisms also produce auxins. Here we investigate whether and how the phytopathogenic fungus Leptosphaeria maculans biosynthesizes auxins. We characterized the auxin profile of in vitro grown L. maculans. The culture was further supplied with the auxin biosynthetic-precursors tryptophan and tryptamine and gene expression and phytohormone content was analyzed. L. maculans in vitro produced IAA (indole-3-acetic acid) as the predominant auxin metabolite. IAA production could be further stimulated by supplying precursors. Expression of indole-3-pyruvate decarboxylase LmIPDC2, tryptophan aminotransferase LmTAM1 and nitrilase LmNIT1 genes was mainly upregulated after adding tryptophan and correlated with IAA production, suggesting that these genes are the key components of auxin biosynthesis in L. maculans. Tryptamine acted as a potent inducer of IAA production, though a pathway independent of LmIPDC2/LmTAM1 may be involved. Despite L. maculans being a rich source of bioactive IAA, the auxin metabolic profile of host plant Brassica napus was not altered upon infection. Exogenous IAA inhibited the growth of L. maculans in vitro when supplied in high concentration. Altogether, we showed that L. maculans is capable of IAA production and we have identified biosynthetic genes that were responsive to tryptophan treatment.
- MeSH
- aminohydrolasy genetika MeSH
- biosyntetické dráhy MeSH
- Brassica napus mikrobiologie MeSH
- fylogeneze MeSH
- genetická transkripce MeSH
- houby klasifikace genetika metabolismus MeSH
- karboxylyasy genetika metabolismus MeSH
- kyseliny indoloctové metabolismus farmakologie MeSH
- Leptosphaeria enzymologie genetika růst a vývoj metabolismus MeSH
- regulace genové exprese u hub MeSH
- regulátory růstu rostlin metabolismus MeSH
- tryptaminy metabolismus farmakologie MeSH
- tryptofan metabolismus farmakologie MeSH
- tryptofantransaminasa genetika metabolismus MeSH
- upregulace MeSH
- Publikační typ
- časopisecké články MeSH
To understand the role of phospholipids on Cdr1p (drug exporter)-mediated drug resistance in yeast, the phospholipids biosynthesis genes PSD1, PSD2, CHO2, and OPI3 were deleted in a strain of Saccharomyces cerevisiae already overexpressing Cdr1-GFP of Candida albicans as a heterologous system. The effect of phospholipids biosynthesis gene deletion was analyzed on Cdr1p-GFP-mediated drug resistance as well as its localization. The results indicate that phospholipids biosynthesis disruption makes the cell sensitive to several drugs including fluconazole (FLC), with Δpsd1/Cdr1-GFP being worst affected. Interestingly, unlike sterols and sphingolipids, the localization of Cdr1p was unaffected by phospholipid biosynthesis gene disruption. Concomitantly, phospholipids mutants also showed an increase in reactive oxygen species (ROS) generation, as verified by fluorescence probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) method. In addition, the sensitivity of phospholipid mutants with FLC was found to be synergistic to ROS generation, resulting in further reduction of growth. Thus, this study proposes phospholipid biosynthesis as a novel target for antifungal therapy.
- MeSH
- ABC transportéry genetika MeSH
- antifungální látky farmakologie MeSH
- biosyntetické dráhy MeSH
- Candida albicans účinky léků genetika MeSH
- delece genu MeSH
- fosfatidylethanolamin-N-methyltransferasa genetika MeSH
- fosfolipidy biosyntéza MeSH
- fungální proteiny genetika MeSH
- karboxylyasy genetika MeSH
- membránové transportní proteiny genetika MeSH
- mikrobiální testy citlivosti MeSH
- mitochondriální proteiny genetika MeSH
- mnohočetná fungální léková rezistence genetika MeSH
- Saccharomyces cerevisiae - proteiny genetika MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
We report for the first time an autosomal recessive inborn error of de novo purine synthesis (DNPS)-PAICS deficiency. We investigated two siblings from the Faroe Islands born with multiple malformations resulting in early neonatal death. Genetic analysis of affected individuals revealed a homozygous missense mutation in PAICS (c.158A>G; p.Lys53Arg) that affects the structure of the catalytic site of the bifunctional enzyme phosphoribosylaminoimidazole carboxylase (AIRC, EC 4.1.1.21)/phosphoribosylaminoimidazole succinocarboxamide synthetase (SAICARS, EC 6.3.2.6) (PAICS). The mutation reduced the catalytic activity of PAICS in heterozygous carrier and patient skin fibroblasts to approximately 50 and 10% of control levels, respectively. The catalytic activity of the corresponding recombinant enzyme protein carrying the mutation p.Lys53Arg expressed and purified from E. coli was reduced to approximately 25% of the wild-type enzyme. Similar to other two known DNPS defects-adenylosuccinate lyase deficiency and AICA-ribosiduria-the PAICS mutation prevented purinosome formation in the patient's skin fibroblasts, and this phenotype was corrected by transfection with the wild-type but not the mutated PAICS. Although aminoimidazole ribotide (AIR) and aminoimidazole riboside (AIr), the enzyme substrates that are predicted to accumulate in PAICS deficiency, were not detected in patient's fibroblasts, the cytotoxic effect of AIr on various cell lines was demonstrated. PAICS deficiency is a newly described disease that enhances our understanding of the DNPS pathway and should be considered in the diagnosis of families with recurrent spontaneous abortion or early neonatal death.
- MeSH
- adenylsukcinátlyasa nedostatek MeSH
- autistická porucha MeSH
- fatální výsledek MeSH
- fenotyp MeSH
- karboxylyasy genetika metabolismus MeSH
- lidé MeSH
- mnohočetné abnormality genetika MeSH
- mutace MeSH
- novorozenec MeSH
- peptidsynthasy genetika metabolismus MeSH
- perinatální smrt MeSH
- poruchy metabolismu purinů a pyrimidinů MeSH
- puriny biosyntéza metabolismus MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Dánsko MeSH
OBJECTIVES: To convert α-acetolactate into acetoin by an α-acetolactate decarboxylase (ALDC) to prevent its conversion into diacetyl that gives beer an unfavourable buttery flavour. RESULTS: We constructed a whole Saccharomyces cerevisiae cell catalyst with a truncated active ALDC from Acetobacter aceti ssp xylinum attached to the cell wall using the C-terminal anchoring domain of α-agglutinin. ALDC variants in which 43 and 69 N-terminal residues were absent performed equally well and had significantly decreased amounts of diacetyl during fermentation. With these cells, the highest concentrations of diacetyl observed during fermentation were 30 % less than those in wort fermented with control yeasts displaying only the anchoring domain and, unlike the control, virtually no diacetyl was present in wort after 7 days of fermentation. CONCLUSIONS: Since modification of yeasts with ALDC variants did not affect their fermentation performance, the display of α-acetolactate decarboxylase activity is an effective approach to decrease the formation of diacetyl during beer fermentation.
