High-Throughput, Sequence-Based Analysis of the Microbiota of Greek Kefir Grains from Two Geographic Regions

Pozadina istraživanja. Kefir je prirodni probiotički napitak koji se tradicionalno dobiva fermentacijom mlijeka s kefirnim zrncima. Kefirna zrnca sadržavaju združenu populaciju bakterija i kvasaca uklopljenih u proteinsko-polisaharidni matriks. Geografsko porijeklo kefirnih zrnaca može bitno utjecati na njihov mikrobni sastav, a time i na svojstva dobivenog napitka. Iako u literaturi nalazimo detaljne podatke o sastavu kefirnih zrnaca iz različitih geografskih područja, nema dostupnih informacija o mikrobiomu grčkog kefira. Stoga je svrha ovoga rada bila ispitati strukturu i raznolikost bakterijske zajednice grčkih kefirnih zrnaca. Eksperimentalni pristup. Metodom sekvenciranja visoke propusnosti ispitani su struktura i raznolikost sastava kefirnih zrnaca iz dvaju geografski udaljenih područja u Grčkoj, radi boljeg razumijevanja raznolikosti bakterijskih populacija u zrncima. Ovaj pristup ispitivanju metagenoma, koji ne ovisi o kulturi, za sekvenciranje koristi gen 16S rRNA sa segmentima oko V4 varijabilne regije. Rezultati i zaključci. Koljeno Firmicutes (kojem pripadaju i bakterije mliječno-kiselog vrenja), s 99 % sekvenci u oba uzorka kefirnih zrnaca, izrazito je dominiralo među identificiranim bakterijama. Na razini porodice, sekvence Lactobacillaceae bile su zastupljene s više od 98 % jedinica (engl. operational taxonomic units, OTU), zatim slijede: Ruminococcaceae, Lahnospiraceae, Bacteroidaceae, te ostale, manje zastupljene porodice. Relativno je mali broj vrsta bakterija dominirao, od kojih je nazastupljenija bila bakterija Lac¬tobacillus kefiranofaciens, i to s 95,0 % jedinica u kefiru A i 96,3 % jedinica u kefiru B. Međutim, pronađena je vrlo raznolika subdominantna populacija u oba uzorka zrnaca, uključujući i vrste bakterija koje su dosad bile povezivane s probavnim traktom ljudi i životinja. a za neke od njih se smatra da imaju probiotička svojstva (Faecalibacterium spp., Bacteroides spp. i Blautia spp.). Razlike u profilima bakterijskih vrsta između dvaju uzoraka kefirnih zrnaca su vrlo male, što potvrđuje izrazitu homogenost uzoraka usprkos njihovoj velikoj geografskoj udaljenosti. Novina i znanstveni doprinos. Ovaj rad po prvi put detaljno istražuje raznolikost bakterijskih vrsta i njihovo bogatstvo u grčkom kefiru.Research background. Kefir is a natural probiotic drink traditionally produced by milk fermentation using kefir grains. Kefir grains are composed of a complex population of bacteria and yeasts embedded in a polysaccharide-protein matrix. The geographic origin of kefir grains may largely influence their microbial composition and the associated kefir drink properties. Although the detailed bacterial composition of kefir grains from several geographic regions has been reported, to date, analogous data about the microbiome of Greek kefir are lacking. Hence, the aim of this study is to investigate the structure and the diversity of the bacterial community of Greek kefir grains. Experimental approach. The bacterial community structure and diversity of two different kefir grains from distant geographic regions in Greece were examined via high-throughput sequencing analysis, a culture-independent metagenomic approach, targeting the 16S rRNA V4 variable region, in order to gain a deeper understanding of their bacterial population diversities. Results and conclusions. Firmicutes (a phylum that includes lactic acid bacteria) was strikingly dominant amongst the identified bacterial phyla, with over 99 % of the sequences from both kefir grains classified to this phylum. At the family level, Lactobacillaceae sequences accounted for more than 98 % of the operational taxonomic units (OTUs), followed by Ruminococcaceae, Lahnospiraceae, Bacteroidaceae and other bacterial families of lesser abundance. Α relatively small number of bacterial genera dominated, with Lactobacillus kefiranofaciens being the most abundant in both kefir grains (95.0 % of OTUs in kefir A and 96.3 % of OTUs in kefir B). However, a quite variable subdominant population was also present in both grains, including bacterial genera that have been previously associated with the gastrointestinal tract of humans and animals, some of which are believed to possess probiotic properties (Faecalibacterium spp., Bacteroides spp., Blautia spp.). Differences among the bacterial profiles of the two grains were very small indicating a high homogeneity despite the distant geographic origin. Novelty and scientific contribution. This is the first study to deeply explore and report on the bacterial diversity and species richness of Greek kefir

Over-expression of a tomato N-acetyl-L-glutamate synthase gene (SlNAGS1) in Arabidopsis thaliana results in high ornithine levels and increased tolerance in salt and drought stresses

A single copy of the N-acetyl-L-glutamate synthase gene (SlNAGS1) has been isolated from tomato. The deduced amino acid sequence consists of 604 amino acids and shows a high level of similarity to the predicted Arabidopsis NAGS1 and NAGS2 proteins. Furthermore, the N-terminus ArgB domain and the C-terminus ArgA domain found in SlNAGS1 are similar to the structural arrangements that have been reported for other predicted NAGS proteins. SlNAGS1 was expressed at high levels in all aerial organs, and at basic levels in seeds, whereas it was not detected at all in roots. SlNAGS1 transcript accumulation was noticed transiently in tomato fruit at the red-fruit stage. In addition, an increase of SlNAGS1 transcripts was detected in mature green tomato fruit within the first hour of exposure to low oxygen concentrations. Transgenic Arabidopsis plants have been generated expressing the SlNAGS1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Three homozygous transgenic lines expressing the transgene (lines 1-7, 3-8, and 6-5) were evaluated further. All three transgenic lines showed a significant accumulation of ornithine in the leaves with line 3-8 exhibiting the highest concentration. The same lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to 250 mM NaCl. Similarly, mature plants of all three transgenic lines displayed a higher tolerance to salt and drought stress compared to WT plants. Under most experimental conditions, transgenic line 3-8 performed best, while the responses obtained from lines 1-7 and 6-5 depended on the applied stimulus. To our knowledge, this is the first plant NAGS gene to be isolated, characterized, and genetically modified

Expression profiling of ascorbic acid-related genes during tomato fruit development and ripening and in response to stress conditions

L-Ascorbate (the reduced form of vitamin C) participates in diverse biological processes including pathogen defence mechanisms, and the modulation of plant growth and morphology, and also acts as an enzyme cofactor and redox status indicator. One of its chief biological functions is as an antioxidant. L-Ascorbate intake has been implicated in the prevention/alleviation of varied human ailments and diseases including cancer. To study the regulation of accumulation of this important nutraceutical in fruit, the expression of 24 tomato (Solanum lycopersicon) genes involved in the biosynthesis, oxidation, and recycling of L-ascorbate during the development and ripening of fruit have been characterized. Taken together with L-ascorbate abundance data, the results show distinct changes in the expression profiles for these genes, implicating them in nodal regulatory roles during the process of L-ascorbate accumulation in tomato fruit. The expression of these genes was further studied in the context of abiotic and post-harvest stress, including the effects of heat, cold, wounding, oxygen supply, and ethylene. Important aspects of the hypoxic and post-anoxic response in tomato fruit are discussed. The data suggest that L-galactose-1-phosphate phosphatase could play an important role in regulating ascorbic acid accumulation during tomato fruit development and ripening.status: publishe

Journal of Experimental Botany, Page 1 of 13

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