The Shine-Dalgarno (SD) sequence is a key element directing the translation to initiate at the authentic start codons and also enabling translation initiation to proceed in 5' untranslated mRNA ...regions (5'-UTRs) containing moderately strong secondary structures. Bioinformatic analysis of almost forty genomes from the major bacterial phylum Bacteroidetes revealed, however, a general absence of SD sequence, drop in GC content and consequently reduced tendency to form secondary structures in 5'-UTRs. The experiments using the Prevotella bryantii TC1-1 expression system were in agreement with these findings: neither addition nor omission of SD sequence in the unstructured 5'-UTR affected the level of the reporter protein, non-specific nuclease NucB. Further, NucB level in P. bryantii TC1-1, contrary to hMGFP level in Escherichia coli, was five times lower when SD sequence formed part of the secondary structure with a folding energy -5,2 kcal/mol. Also, the extended SD sequences did not affect protein levels as in E. coli. It seems therefore that a functional SD interaction does not take place during the translation initiation in P. bryanttii TC1-1 and possibly other members of phylum Bacteroidetes although the anti SD sequence is present in 16S rRNA genes of their genomes. We thus propose that in the absence of the SD sequence interaction, the selection of genuine start codons in Bacteroidetes is accomplished by binding of ribosomal protein S1 to unstructured 5'-UTR as opposed to coding region which is inaccessible due to mRNA secondary structure. Additionally, we found that sequence logos of region preceding the start codons may be used as taxonomical markers. Depending on whether complete sequence logo or only part of it, such as information content and base proportion at specific positions, is used, bacterial genera or families and in some cases even bacterial phyla can be distinguished.
The effect of standard agricultural management on the genetic heterogeneity of nitrous oxide reductase (nosZ) fragments from denitrifying prokaryotes in native and cultivated soil was explored. ...Thirty-six soil cores were composited from each of the two soil management conditions. nosZ gene fragments were amplified from triplicate samples, and PCR products were cloned and screened by restriction fragment length polymorphism (RFLP). The total nosZ RFLP profiles increased in similarity with soil sample size until triplicate 3-g samples produced visually identical RFLP profiles for each treatment. Large differences in total nosZ profiles were observed between the native and cultivated soils. The fragments representing major groups of clones encountered at least twice and four randomly selected clones with unique RFLP patterns were sequenced to verify nosZ identity. The sequence diversity of nosZ clones from the cultivated field was higher, and only eight patterns were found in clone libraries from both soils among the 182 distinct nosZ RFLP patterns identified from the two soils. A group of clones that comprised 32% of all clones dominated the gene library of native soil, whereas many minor groups were observed in the gene library of cultivated soil. The 95% confidence intervals of the Chao1 nonparametric richness estimator for nosZ RFLP data did not overlap, indicating that the levels of species richness are significantly different in the two soils, the cultivated soil having higher diversity. Phylogenetic analysis of deduced amino acid sequences grouped the majority of nosZ clones into an interleaved Michigan soil cluster whose cultured members are alpha-Proteobacteria. Only four nosZ sequences from cultivated soil and one from the native soil were related to sequences found in gamma-Proteobacteria. Sequences from the native field formed a distinct, closely related cluster (D(mea)n = 0.16) containing 91.6% of the native clones. Clones from the cultivated field were more distantly related to each other (D(mean) = 0.26), and 65% were found outside of the cluster from the native soil, further indicating a difference in the two communities. Overall, there appears to be a relationship between use and richness, diversity, and the phylogenetic position of nosZ sequences, indicating that agricultural use of soil caused a shift to a more diverse denitrifying community.
Five strictly anaerobic Gram-negative bacterial strains, P4-65, P4-76ᵀ, P5-60, P5-119, and P5-125, presumably belonging to the genus Prevotella were isolated from pig fecal samples. Strains were ...tested for various phenotypic traits and nearcomplete genome sequences were obtained and analyzed. Phylogenetic analysis based on 16S rRNA gene sequences and multilocus sequence analysis based on five conserved genes confirmed that the strains belong to the genus Prevotella, revealing that they represent a novel and discrete lineage distinct from other known species of this genus. The size of the genome of the isolated strains is 3–3.3 Mbp, and the DNA G+C content is 47.5–48.1 mol%. The isolates are strictly anaerobic, rod-shaped with rounded ends, non-motile and non-spore-forming. The main fermentation products are succinate and acetate, with minor concentrations of isovalerate, propionate and isobutyrate. Hydrogen is also produced. Major cellular fatty acids consist of anteiso-C₁₅:₀ and iso-C₁₅:₀, and a number of additional acids are present in lower concentrations. A substantial portion of genes involved in carbohydrate utilization is devoted to pectin degradation and utilization, while those supporting growth on xylan in ruminal Prevotella could not have been revealed. On the basis of the presented results, a novel species, Prevotella pectinovora sp. nov. is proposed. The type strain is P4-76ᵀ (=DSM 29996ᵀ =ZIM B1020ᵀ).
Acetic acid bacteria are involved in many biotechnological processes such as vitamin C, gluconic acid, miglitol or acetic acid production, and others. For a technologist trying to control the ...industrial process, the ability to follow the microbiological development of the process is thus of importance. During the past few years hybridization in a combination with flow cytometry has often been used for this purpose. Since vinegar is a liquid, it is an ideal matrix for flow cytometry analysis. In this work we have constructed a specific probe for highly acetic acid-resistant species of the acetic acid bacteria and a protocol for
hybridization, which in combination with flow cytometry enables direct monitoring of bacteria producing vinegar with >10% of acetic acid. The approach was successfully applied for monitoring microbiota during industrial vinegar production.
Although the Prevotella are commonly observed in high shares in the mammalian hindgut and rumen studies using NGS approach, the knowledge on their actual role, though postulated to lie in soluble ...fibre degradation, is scarce. Here we analyse in total 23, more than threefold of hitherto known rumen and hindgut Prevotella species and show that rumen/hindgut Prevotella generally possess extensive repertoires of polysaccharide utilization loci (PULs) and carbohydrate active enzymes targeting various plant polysaccharides. These PUL repertoires separate analysed Prevotella into generalists and specialists yet a finer diversity among generalists is evident too, both in range of substrates targeted and in PUL combinations targeting the same broad substrate classes. Upon evaluation of the shares of species analysed in this study in rumen metagenomes we found firstly, that they contributed significantly to total Prevotella abundance though much of rumen Prevotella diversity may still be unknown. Secondly, the hindgut Prevotella species originally isolated in pigs and humans occasionally dominated among the Prevotella with surprisingly high metagenome read shares and were consistently found in rumen metagenome samples from sites as apart as New Zealand and Scotland. This may indicate frequent passage between different hosts and relatively low barriers to their successful establishment in rumen versus the hindgut.
Metabolic syndrome is a complex disease that is exponentially increasing in the western world, and diet is one of the possible ways to improve the metabolic status of patients. Barley beta glucans ...are dietary fibres that show promise for improvement cholesterol levels and postprandial glucose response, but they have been rarely investigated in human trials with concurrent focus on gut microbiota.
A double-blind, placebo-controlled, randomised clinical trial was conducted with 43 volunteers with high risk for metabolic syndrome development or with diagnosed metabolic syndrome. During a four-week intervention study, participants consumed experimental bread containing 6 g of barley beta glucans or equal bread but without beta glucans. After dietary intervention, total plasma cholesterol decreased in the test group (−0.26 ± 0.54, p = 0.019), but not in the control group. Short chain fatty acids (SCFA) composition in faeces significantly changed with increase of propionic acid in test group (43.2%, p = 0.045) and with decrease of acetic acid in control group (41.8%, p = 0.011). The microbiome analysis based on Illumina paired end sequencing of 16S rRNA genes showed a decrease in microbial diversity and richness in the test group. The pre-intervention gut microbiota composition showed higher abundance of health associated Bifidobacterium spp. and Akkermansia municiphila within cholesterol-responsive group, showing that diet-induced metabolic response is possibly dependent on individual gut microbiota composition.
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•After beta glucans dietary intervention total plasma cholesterol levels decreased.•Short chain fatty acids significantly changed with increase of propionic acid.•After dietary intervention significant changes in microbiota composition occurred.•Pre-intervention gut microbiota appears to be a factor influencing the metabolic response.
Species now affiliated to genus Prevotella have been known for decades as an integral part of human oral cavity microbiota. They were frequently isolated from patients with periodontitis or from ...dental root canals but also from healthy subjects. With the exception of Prevotella intermedia, they were considered opportunistic pathogens, as they were isolated also from various bacterial abscesses from the head, neck, breast, skin and various other body sites. Consequently, Prevotella were not in the focus of research activities. On the other hand, the four species found in the rumen never caused any disease and seemed early on to be numerous and important part of the rumen ecosystem indicating this genus harbored bacteria with enormously diverse habitats and lifestyles. The purpose of this review is to illustrate the main research themes performed in Prevotella on a path from less noted oral bacteria and from hard to cultivate and study rumen organisms to important mutualistic bacteria in guts of various mammals warranting major research efforts.
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•The Prevotella is a large and diverse genus found in anaerobic habitats mainly in animals.•The research of Prevotella is currently gaining momentum due to their newly found importance in the human gut.•The genetic analysis tools are underdeveloped.
The results of metagenomic studies have clearly established that bacteria of the genus Prevotella represent one of the important groups found in the oral cavity and large intestine of man, and they ...also dominate the rumen. They belong to the Bacteroidetes, a phylum well-known for its polysaccharide degrading potential that stems from the outer membrane-localized enzyme/binding protein complexes encoded in polysaccharide utilization loci (PULs). Dozens of Prevotella species have been described, primarily from the oral cavity, and many of them occur simultaneously at the same sites, but research on their ecological adaptation has been neglected. Therefore, in this study, the repertoires of PULs and carbohydrate acting enzymes (CAZYmes) found in Prevotella genomes were analyzed and it was concluded that the Prevotella species were widely heterogeneous in this respect and displayed several distinct adaptations with regard to the number, source and nature of the substrates apparently preferred for growth.
PURPOSE: The purpose of this study was to discover differences in the human fecal microbiota composition driven by long-term omnivore versus vegan/lacto-vegetarian dietary pattern. In addition, the ...possible association of demographic characteristics and dietary habits such as consumption of particular foods with the fecal microbiota was examined. METHODS: This study was conducted on a Slovenian population comprising 31 vegetarian participants (11 lacto-vegetarians and 20 vegans) and 29 omnivore participants. Bacterial DNA was extracted from the frozen fecal samples by Maxwell 16 Tissue DNA Purification Kit (Promega). Relative quantification of selected bacterial groups was performed by real-time PCR. Differences in fecal microbiota composition were evaluated by PCR–DGGE fingerprinting of the V3 16S rRNA region. Participants’ demographic characteristics, dietary habits and health status information were collected through a questionnaire. RESULTS: Vegetarian diet was associated with higher ratio (% of group-specific DNA in relation to all bacterial DNA) of Bacteroides–Prevotella, Bacteroides thetaiotaomicron, Clostridium clostridioforme and Faecalibacterium prausnitzii, but with lower ratio (%) of Clostridium cluster XIVa. Real-time PCR also showed a higher concentration and ratio of Enterobacteriaceae (16S rDNA copies/g and %) in female participants (p < 0.05 and p < 0.01) and decrease in Bifidobacterium with age (p < 0.01). DGGE analysis of the 16S rRNA V3 region showed that relative quantity of DGGE bands from certain bacterial groups was lower (Bifidobacterium, Streptococus, Collinsella and Lachnospiraceae) or higher (Subdoligranulum) among vegetarians, indicating the association of dietary type with bacterial community composition. Sequencing of selected DGGE bands revealed the presence of common representatives of fecal microbiota: Bacteroides, Eubacterium, Faecalibacterium, Ruminococcaceae, Bifidobacterium and Lachnospiraceae. Up to 4 % of variance in microbial community analyzed by DGGE could be explained by the vegetarian type of diet. CONCLUSIONS: Long-term vegetarian diet contributed to quantity and associated bacterial community shifts in fecal microbiota composition. Consumption of foods of animal origin (eggs, red meat, white meat, milk, yoghurt, other dairy products, fish and seafood) and vegetarian type of diet explained the largest share of variance in microbial community structure. Fecal microbiota composition was also associated with participants’ age, gender and body mass.
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