Defining the Influence of the A12.2 Subunit on Transcription Elongation and Termination by RNA Polymerase I In Vivo

Saccharomyces cerevisiae has approximately 200 copies of the 35S rDNA gene, arranged tandemly on chromosome XII. This gene is transcribed by RNA polymerase I (Pol I) and the 35S rRNA transcript is processed to produce three of the four rRNAs required for ribosome biogenesis. An intergenic spacer (IGS) separates each copy of the 35S gene and contains the 5S rDNA gene, the origin of DNA replication, and the promoter for the adjacent 35S gene. Pol I is a 14-subunit enzyme responsible for the majority of rRNA synthesis, thereby sustaining normal cellular function and growth. The A12.2 subunit of Pol I plays a crucial role in cleavage, termination, and nucleotide addition during transcription. Deletion of this subunit causes alteration of nucleotide addition kinetics and read-through of transcription termination sites. To interrogate both of these phenomena, we performed native elongating transcript sequencing (NET-seq) with an rpa12Δ strain of S. cerevisiae and evaluated the resultant change in Pol I occupancy across the 35S gene and the IGS. Compared to wild-type (WT), we observed template sequence-specific changes in Pol I occupancy throughout the 35S gene. We also observed rpa12Δ Pol I occupancy downstream of both termination sites and throughout most of the IGS, including the 5S gene. Relative occupancy of rpa12Δ Pol I increased upstream of the promoter-proximal Reb1 binding site and dropped significantly downstream, implicating this site as a third terminator for Pol I transcription. Collectively, these high-resolution results indicate that the A12.2 subunit of Pol I plays an important role in transcription elongation and termination.

Two new species and one new record of Hyalopsora (Pucciniastraceae) on ferns in China

Three rust fungi belonging to the genus Hyalopsora occurring on ferns were found in China. Hyalopsora minispora and H. tibetica are described as new species in this paper. Hyalopsora minispora can be distinguished from known species by its relatively small urediniospores (19.5–23.0 × 12.0–19.0 μm) with 4–6 scattered germ pores and amphispores in a common pustule. Hyalopsora tibetica is mainly characterized by urediniospores with 2–6 scattered germ pores and amphispores. Hyalopsora neocheilanthis is described and reported as a new record for China. The phylogenetic relationships of Hyalopsora species and related taxa were examined by comparing the sequences of their internal transcribed spacer 2 (ITS2) barcode and 28S rDNA gene segments. Hyalopsora is supported with strong bootstrap support and it differs from the two other fern-infecting genera viz. Milesina and Uredinopsis.

Fecal microbiota profiling in irritable bowel syndrome and inflammatory bowel disease patients with irritable bowel syndrome-type symptoms

Background The intestinal microbiota is thought to be involved in the occurrence of inflammatory bowel disease in remission with irritable bowel syndrome (IBS)-type symptoms, but the specific distinct profile of these bacteria remains unclear. This cross-sectional study aims to investigate the fecal microbiota profiling in patients with these diseases. Methods Fecal samples from 97 subjects, including Crohn’s disease patients in remission with IBS-type symptoms (CDR-IBS+) or without IBS-type symptoms (CDR-IBS−), ulcerative colitis patients in remission with IBS-type symptoms (UCR-IBS+) or without IBS-type symptoms (UCR-IBS−), IBS patients and healthy controls, were collected and applied 16S ribosomal DNA (rDNA) gene sequencing. The V4 hypervariable regions of 16S rDNA gene were amplified and sequenced by the Illumina MiSeq platform. The differences in the sample diversity index in groups were analyzed with R software. Results The richness of the intestinal microbiota in the CDR-IBS group was markedly lower than those in the control and IBS groups based on the analysis of observed species and the Chao index (P < 0.05). The observed species index in the CDR-IBS+ group was higher than that in the CDR-IBS− group (median index: 254.8 vs 203, P = 0.036). No difference was found in alpha diversity between UCR patients with IBS-type symptoms and those without related symptoms. At the genus level, the number of Faecalibacterium in CDR patients with IBS-type symptoms increased significantly, while Fusobacterium decreased versus those without such symptoms (mean relative abundance of Faecalibacterium: 20.35% vs 5.18%, P < 0.05; Fusobacterium: 1.51% vs 5.2%, P < 0.05). However, compared with the UCR-IBS− group, the number of Faecalibacterium in the UCR-IBS+ group decreased, while the number of Streptococcus increased, but there was no significant difference in the genus structure. The abundance and composition of the microbiota of IBS patients were not distinct from those of healthy controls. Conclusions The IBS-type symptoms in CD patients in remission may be related to an increase in Faecalibacterium and a decrease in Fusobacterium. The IBS-type symptoms in UC patients in remission cannot be explained by changes in the abundance and structure of the intestinal microbiota.

Antioxidant Potential of Pediococcus Pentosaceus Strains from the Sow Milk Bacterial Collection in Weaned Piglets

Background: In modern animal husbandry, breeders pay increasing attention to improving sow nutrition during pregnancy and lactation to favor the health of neonates. Sow milk is a main food for piglets during their first three weeks of life, which is not only a rich repository of essential nutrients and a broad range of bioactive compounds, but also an indispensable source of commensal bacteria. Maternal milk microorganisms are important sources of commensal bacteria for neonatal gut. Bacteria from maternal milk may serve as an additive to confer a health benefit on the composition of the indigenous microbiota of piglets. Methods: The sow milk microbiota was collected using the culturomics methods of Continuous Culture and Interval Sampling, following by the identification of 16S rDNA gene sequences. To screen potential probiotics, the functional evaluation was conducted to assess their antagonistic activity against pathogens in vitro and evaluate their resistance against oxidative stress in damaged Drosophila induced by paraquat. In piglet feeding trial, a total of 54 newborn suckling piglets were chosen from nine sows and randomly assigned to three treatments with different concentration of a candidate strain. Multiple approaches were carried out to verify its antioxidant function including western blotting, enzyme activity analysis, metabolomics and 16S rDNA sequencing. Results: The 1240 isolates were screened out from the sow milk microbiota and grouped into 271 bacterial taxa based on a nonredundant set of 16S rDNA gene sequences. Among 80 Pediococcus isolates, a new Pediococcus pentosaceus strain (SMM914) showed best performance in inhibition ability against swine pathogens and in Drosophila model challenged by paraquat. Pretreatment of piglets with SMM914 induces the Nrf2-Keap1 antioxidant signaling pathway and greatly affected the pathways of amino acid metabolism and lipid metabolism in plasma. In colon, Lactobacillus was significantly increased in the high dose of SMM914 group compared with the control group. Conclusion: SMM914 functions as a promising probiotic conferring antioxidant capacity by activating the Nrf2-Keap1 antioxidant signaling pathway in piglets. Our study provided useful resources for deeply understanding the relationships between the maternal microbiota and offspring.

High variability in SSU rDNA gene copy number among planktonic foraminifera revealed by single-cell qPCR

Metabarcoding has become the workhorse of community ecology. Sequencing a taxonomically informative DNA fragment from environmental samples gives fast access to community composition across taxonomic groups, but it relies on the assumption that the number of sequences for each taxon correlates with its abundance in the sampled community. However, gene copy number varies among and within taxa, and the extent of this variability must therefore be considered when interpreting community composition data derived from environmental sequencing. Here we measured with single-cell qPCR the SSU rDNA gene copy number of 139 specimens of five species of planktonic foraminifera. We found that the average gene copy number varied between of ~4000 to ~50,000 gene copies between species, and individuals of the same species can carry between ~300 to more than 350,000 gene copies. This variability cannot be explained by differences in cell size and considering all plausible sources of bias, we conclude that this variability likely reflects dynamic genomic processes acting during the life cycle. We used the observed variability to model its impact on metabarcoding and found that the application of a correcting factor at species level may correct the derived relative abundances, provided sufficiently large populations have been sampled.

Intra-genomic rDNA gene variability of Nassellaria and Spumellaria (Rhizaria, Radiolaria) assessed by Sanger, MinION and Illumina sequencing

Ribosomal DNA (rDNA) genes are known to be valuable markers for the barcoding of eukaryotic life and its phylogenetic classification at various taxonomic levels. The large scale exploration of environmental microbial diversity through metabarcoding approaches have been focused mainly on the hypervariable regions V4 and V9 of the 18S rDNA gene. Yet, the accurate interpretation of such environmental surveys is hampered by technical (e.g., PCR and sequencing errors) and biological biases (e.g., intra-genomic variability). Here we explored the intra-genomic diversity of Nassellaria and Spumellaria specimens (Radiolaria) by comparing Sanger sequencing with two different high-throughput sequencing platforms: Illumina and Oxford Nanopore Technologies (MinION). Our analysis determined that intra-genomic variability of Nassellaria and Spumellaria is generally low, yet in some Spumellaria specimens we found two different copies of the V4 with a similarity lower than 97%. From the different sequencing methods, Illumina showed the highest number of contaminations (i.e., environmental DNA, cross-contamination, tag-jumping), revealed by its high sequencing depth; and Minion showed the highest sequencing rate error (~14%). Yet the long reads produced by MinION (~2900 bp) allowed accurate phylogenetic reconstruction studies. These results, highlight the requirement for a careful interpretation of Illumina based metabarcoding studies, in particular regarding low abundant amplicons, and open future perspectives towards full environmental rDNA metabarcoding surveys.

A new species of the sun coral genus Tubastraea (Scleractinia: Dendrophylliidae) from Hong Kong

Tubastraea, commonly known as sun coral, is a genus of brightly coloured azooxanthellate corals in the family Dendrophylliidae. The diversity of this genus is low, with only seven recognized species. Herein, we describe Tubastraea megacorallita sp. nov. from Hong Kong based on morphological and molecular analyses. This new species exhibits several characteristics of the genus including being colonial, having a rough texture of corallum and no epitheca. It can be distinguished from its congenerics by bigger corallites, and the Pourtalès plan arrangement of its septa. The rDNA gene sequences (consisting of ITS1, 5.8S, ITS2, 18S and 28S) showed 2.45–5.18% divergence from those of its closest relatives, T. coccinea and T. micranthus.

The effect of sodium carboxymethyl starch with high degree of substitution on defecation

Sodium carboxymethyl starch (CMS-Na), a kind of food additive with high degree of substitution, is also known as a prebiotic. The aim of this study was to determine the effect of CMS-Na on defecation. Constipated mouse model was prepared by loperamide. Normal rats were also used in the study. Short-chain fatty acids in rat feces were detected by gas chromatography. The bacterial communities in rat feces were identified by 16S rDNA gene sequencing. 5-hydroxytryptamine (5-HT) and tryptophan hydroxylase 1 (Tph1) were measured by ELISA. The results showed that CMS-Na increased the fecal granule counts and intestinal propulsion rate in constipated mice. The contents of water, acetic acid, propionic acid and n-butyrate in feces, Tph1 in colon and 5-HT in serum of rats were increased. In addition, CMS-Na shortened the colonic transport time in rats. The 16S rDNA gene sequencing results indicated that CMS-Na increased the relative abundance of Alloprevotella and decreased the proportion of Lactobacillus. However, the biodiversity of the normal intestinal flora was not altered. In conclusion, CMS-Na can promote defecation in constipated mice. The mechanism may be related to the regulation of Alloprevotella and Lactobacillus in colon, the increase of short-chain fatty acids, and the promotion of the synthesis of Tph1 and 5-HT.

Molecular genetics and phylogeny of Ephippigera species group of genus Saga Charpentier, 1825 (Orthoptera: Ensifera: Saginae) in Anatolia

The genus Saga, one of the largest predatory bush crickets and insects in the Palearctic region, is represented by nine species in Anatolia. According to the morphological and bioacoustics evaluations of the Ephippigera species group belonging to the Saga genus, it consists of three species: S. ephippigera, S. syriaca, and S. hakkarica. To the present, however, there is no molecular genetic research on this species group. In our study, by using the 16S rDNA gene sequences, the molecular phylogeny of this species group is revealed and the morphological species hypotheses has been tested. In the study, specimens belonging to S. ephippigera, S. syriaca, and S. hakkarica were collected between the years of 2018 and 2020 with sweep netting; the DNA isolation, polymerase chain reactions, purifications and double-stranded sequencing were performed to obtain 16S rDNA gene sequences. Sequences were evaluated phylogenetically by performing alignment, phylogenetic analysis (Maximum Likelihood and Bayesian Inference), and dating of cladogenetic events, respectively. The monophyletic and species hypotheses of all three species have been supported by molecular data. It has been determined that the S. ephippigera species is the origin lineages of the Ephippigera species group which diverged approximately 0.4 million years ago; it has been also revealed that the climatic changes in the glacial and inter-glacial periods in the Middle Pleistocene period affected greatly the evolution of the species group.