Discovery of a MUC3B gene reconstructs the membrane mucin gene cluster on human chromosome 7

Human tissue surfaces are coated with mucins, a family of macromolecular sugar-laden proteins serving diverse functions from lubrication to formation of selective biochemical barriers against harmful microorganisms and molecules. Membrane mucins are a distinct group of mucins that are attached to epithelial cell surfaces where they create a dense glycocalyx facing the extracellular environment. All mucin proteins carry long stretches of tandemly repeated sequences that undergo extensive O-linked glycosylation to form linear mucin domains. However, the repetitive nature of mucin domains makes them prone to recombination and render their genetic sequences particularly difficult to read with standard sequencing technologies. As a result, human mucin genes suffer from significant sequence gaps that have hampered investigation of gene function in health and disease. Here we leveraged a recent human genome assembly to identify a previously unmapped MUC3B gene located within a cluster of four structurally related membrane mucin genes that we entitle the MUC3 cluster at q22 locus in chromosome 7. We found that MUC3B shares high sequence identity with the known MUC3A gene, and that the two genes are governed by evolutionarily conserved regulatory elements. Furthermore, we show that MUC3A, MUC3B, MUC12 and MUC17 in the human MUC3 cluster are exclusively expressed in intestinal epithelial cells. Our results complete existing genetic gaps in the MUC3 cluster that is a conserved genetic unit during primate evolution. We anticipate our results to be the starting point for detection of new polymorphisms in the MUC3 cluster associated with human diseases. Moreover, our study provides the basis for exploration of intestinal mucin gene function in widely used experimental models such as human intestinal organoids and genetic mouse models.

Discovery of a new species of trichomonasvirus in the human parasite Trichomonas vaginalis using transcriptome mining

Trichomonas vaginalis is the most common nonviral cause of sexually transmitted infections globally, with an estimated quarter of a billion people infected around the world. Infection by the protozoan parasite results in the clinical syndrome trichomoniasis, which manifests as an inflammatory syndrome with acute and chronic consequences. Half or more of these parasites are themselves infected with one or more dsRNA viruses which can exacerbate the inflammatory disease. Four distinct viruses have been found in T. vaginalis to date, Trichomonas vaginalis virus 1 through 4 (or TVVs). Despite the global prevalence of these viruses, few coding-complete genome sequences have been determined. We conducted viral sequence mining in publicly available transcriptomes across 60 RNA-seq datasets representing 13 distinct T. vaginalis isolates. We assembled sequences for 27 new trichomonasvirus strains across all known TVV species, with 17 of these assemblies representing coding-complete genomes. Using a strategy of de novo sequence assembly followed by taxonomic classification, we discovered a fifth species of TVV that we term Trichomonas vaginalis virus 5 (TVV5). Six strains of TVV5 were assembled, including two coding-complete genomes. These TVV5 sequences exhibit high sequence identity to each other, but low identity to any strains of TVV1-4. Phylogenetic analysis corroborates the species-level designation. These results substantially increase the number of coding-complete TVV genome sequences and demonstrate the utility of mining publicly available transcriptomes for the discovery of RNA viruses in a critical human pathogen.

Mechanistic basis for understanding the dual activities of the bifunctional Azotobacter vinelandii mannuronan C-5 epimerase and alginate lyase AlgE7

The structure and functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5 epimerases determine the monomer composition by catalysing the epimerization of β- d -mannuronic acid residues (M) into α- l -guluronic acid residues (G). The molecular weight is affected by alginate lyases, which catalyse a β-elimination mechanism that cleaves alginate chains. The reaction mechanisms for the epimerization and lyase reactions are similar and some enzymes can perform both reactions. These dualistic enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity. The mechanism behind their activity and the amino acid residues responsible for it are still unknown. We investigate mechanistic determinants involved in the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii . Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Our results show that calcium promotes lyase activity whereas NaCl reduces the lyase activity of AlgE7. By using defined poly-M and poly-MG substrates, the preferred cleavage sites of AlgE7 were found to be M|XM and G|XM, where X can be either M or G. From the study of AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study we suggest a unified catalytic reaction mechanism for both epimerase and lyase activity where H154 functions as the catalytic base and Y149 as the catalytic acid. Importance Post-harvest valorisation and upgrading of algal constituents is a promising strategy in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring of the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine, and material industries. By providing a better understanding of the catalytic mechanism and of how the two enzyme actions can be altered by changes in reaction conditions, this study opens for further applications of bacterial epimerases and lyases in enzymatic tailoring of alginate polymers.

Yeast Plasma Membrane Fungal Oligopeptide Transporters Display Distinct Substrate Preferences despite Their High Sequence Identity

Fungal Oligopeptide Transporters (Fot) Fot1, Fot2 and Fot3 have been found in Saccharomyces cerevisiae wine strains, but not in strains from other environments. In the S. cerevisiae wine strain EC1118, Fot1 and Fot2 are responsible for a broader range of oligopeptide utilization in comparison with strains not containing any Fot. This leads to better fermentation efficiency and an increased production of desirable organoleptic compounds in wine. Despite the benefits associated with Fot activity in S. cerevisiae within the wine environment, little is known about this family of transporters in yeast. The presence of Fot1, Fot2 and Fot3 in S. cerevisiae wine strains is due to horizontal gene transfer from the yeast Torulaspora microellipsoides, which harbors Fot2Tm, FotX and FotY proteins. Sequence analyses revealed that Fot family members have a high sequence identity in these yeast species. In this work, we aimed to further characterize the different Fot family members in terms of subcellular localization, gene expression in enological fermentation and substrate specificity. Using CRISPR/Cas9, we constructed S. cerevisiae wine strains containing each different Fot as the sole oligopeptide transporter to analyze their oligopeptide preferences by phenotype microarrays. The results of oligopeptide consumption show that Fot counterparts have different di-/tripeptide specificities, suggesting that punctual sequence divergence between FOT genes can be crucial for substrate recognition, binding and transport activity. FOT gene expression levels in different S. cerevisiae wine strains during enological fermentation, together with predicted binding motifs for transcriptional regulators in nitrogen metabolism, indicate that these transporters may be under the control of the Nitrogen Catabolite Repression (NCR) system. Finally, we demonstrated that Fot1 is located in the yeast plasma membrane. This work contributes to a better understanding of this family of oligopeptide transporters, which have demonstrated a key role in the utilization of oligopeptides by S. cerevisiae in enological fermentation.

Functional implication of heat shock protein 70/90 and tubulin in cold stress of Dermacentor silvarum

Background The tick Dermacentor silvarum Olenev (Acari: Ixodidae) is a vital vector tick species mainly distributed in the north of China and overwinters in the unfed adult stage. The knowledge of the mechanism that underlies its molecular adaptation against cold is limited. In the present study, genes of hsp70 and hsp90 cDNA, named Dshsp70 and Dshsp90, and tubulin were cloned and characterized from D. silvarum, and their functions in cold stress were further evaluated. Methods The genome of the heat shock proteins and tubulin of D. silvarum were sequenced and analyzed using bioinformatics methods. Each group of 20 ticks were injected in triplicate with Dshsp90-, Dshsp70-, and tubulin-derived dsRNA, whereas the control group was injected with GFP dsRNA. Then, the total RNA was extracted and cDNA was synthesized and subjected to RT-qPCR. After the confirmation of knockdown, the ticks were incubated for 24 h and were exposed to − 20 °C lethal temperature (LT50), and then the mortality was calculated. Results Results indicated that Dshsp70 and Dshsp90 contained an open reading frame of 345 and 2190 nucleotides that encoded 114 and 729 amino acid residues, respectively. The transcript Dshsp70 showed 90% similarity with that identified from Dermacentor variabilis, whereas Dshsp90 showed 85% similarity with that identified from Ixodes scapularis. Multiple sequence alignment indicates that the deduced amino acid sequences of D. silvarum Hsp90, Hsp70, and tubulin show very high sequence identity to their corresponding sequences in other species. Hsp90 and Hsp70 display highly conserved and signature amino acid sequences with well-conserved MEEVD motif at the C-terminal in Hsp90 and a variable C-terminal region with a V/IEEVD-motif in Hsp70 that bind to numerous co-chaperones. RNA interference revealed that the mortality of D. silvarum was significantly increased after injection of dsRNA of Dshsp70 (P = 0.0298) and tubulin (P = 0.0448), whereas no significant increases were observed after the interference of Dshsp90 (P = 0.0709). Conclusions The above results suggested that Dshsp70 and tubulin play an essential role in the low-temperature adaptation of ticks. The results of this study can contribute to the understanding of the survival and acclimatization of overwintering ticks. Graphical abstract

First report of root rot on Manglietia decidua caused by Calonectria ilicicola in China

Manglietia decidua (Magnoliaceae) was a class I endangered plant in China. During 2018-2020, a severe root rot (about 10% - 90% disease incidence ) was observed on 2-year-old seedlings in the nursery in Yichun, Jiangxi province (N27°52’20”; E114°27’46”). Symptoms started on leaves showing dehydration and chlorosis, the root of diseased plant became black and rotted, and in severe cases, the plants withered and died. The symptomatic root tissues were cut and dipped in a 3% hydrogen peroxide solution for 5 mins, rinsed thrice with sterile water, and then placed on potato glucose agar medium containing ampicillin (50mg/L). The plates were kept in an incubator at 25-28°C for 2-3 days in the dark. The Calonectria-like fungus was consistently isolated from 100% of tissues and the colonies were feathery, moderate white aerial mycelium, surface pale brown, reverse with white outer margin, and brown inner region. The perithecia produced on carnation leaf agar were solitary, subglobose to ovoid, dark red-brown, and measured 273.8 - 427.2 × 362.6 - 628.9 µm (av. 360.9 × 429.9 µm) (n = 31). Clavate asci contained eight spores and tapered into a long thin stalk. Ascospores were hyaline, guttulate, straight to slightly curved with rounded ends, 36.8- 66.1×4.4-7.3 μm (av. 49.9 × 5.9 μm) (n = 52), 1-septate, constricted at the septum and aggregated in the upper third of the ascus. On PDA，conidia formed on penicillate conidiophores within 10 days were hyaline, 1(-3)-septate, cylindrical, rounded at both ends, straight, 36.5-61.7 × 5.0-7.2 μm (av. 50.7 × 6.2 µm) (n=48). Isolate HML 20 and 27 were used to further confirm species identity by five loci analysis：ITS (MZ389092 and MZ389093), ACT (MZ398252 and MZ398253), HIS3 (MZ398254 and MZ398255), TEF1-α (MZ398256 and MZ398257), and TUB2 (MZ398258 and MZ398259). NCBI BLASTN showed the high sequence identity with Calonectria ilicicola ex-type culture CBS 190.50 (CMW 30998) (Liu et al 2020): 100 % for ITS (MT359727), TUB2 (AY725631), and HIS3 (MT335506), 99.22% for ACT (MT335036), 99.80% for TEF1-α (MT412797). Maximum likelihood (ML) analysis and Bayesian inference (BI) based on the combined ITS, tub2, his3 and tef1 sequence using RAxML v.1.0.0 and MrBayes v. 3.2.1 software revealed that isolate HML 20 and 27 clustered together with C. ilicicola strains in C. kyotensis species complex. Thus, the fungus was identified as C. ilicicola (anamorph: Cylindrocladium parasiticum) based on morpho-molecular criteria (Lombard et al. 2010). Pathogenicity was determined under greenhouse conditions (25-30 ℃). The 2-year-old plants grown in 25-cm pots for 20 days were inoculated. Five 6-mm mycelial plugs from 7-day culture on PDA were buried 5 cm under the soil adjacent to the unwounded taproot of each plant and the plants were watered regularly to keep the soil moisture content at about 15%. After ten days, inoculated plants began to show chlorosis symptoms on leaves and collapsed within 15 to 20 days, while no symptoms were observed on control plants. The same colonial fungus was successfully reisolated. Calonectria ilicicola is an economically important plant pathogen worldwide, which causes diseases on Arachis hypogaea, Cinnamomum kanahirai, Glycine max, Medicago sativa, Sassafras randaiense, and Vaccinium spp. etc. in China (Gai et al 2017, Fei et al 2018, Zhang et al 2020 ). As far as we know, it is first report of C. ilicicola causing root rot on M. decidua. At present, this disease is an important threat to the conservation of M. decidua.

Genomic and physiological characterization of Novosphingobium terrae sp. nov., an alphaproteobacterium isolated from Cerrado soil containing a megasized chromid

A novel bacterial strain, designated GeG2T, was isolated from soils of native Cerrado, a highly biodiverse savanna-like Brazilian biome. 16S rRNA gene sequence analysis of strain GeG2T revealed high sequence identity (100%) to the alphaproteobacterium Novosphingobium rosa, however, comparisons with N. rosa DSM7285T showed several distinctive features, prompting a full characterization of the new strain in terms of growth, morphology, biochemistry and, ultimately, its genome. GeG2T cells were Gram-stain negative bacilli, facultatively anaerobic, motile, positive for catalase and oxidase activities and for starch hydrolysis. Strain GeG2T presented planktonic-sessile dimorphism and cell aggregates surrounded by extracellular matrix and nanometric spherical structures were observed in liquid cultures, suggesting the production of exopolysaccharides (EPS) and outer membrane vesicles (OMVs). Whole genome assembly revealed four circular replicons: a 4.1 Mb chromosome, a 2.7 Mb extrachromosomal megareplicon and two plasmids (212.7 and 68.6 kb). The megareplicon contains few core genes and plasmid-type replication/maintenance systems, consistent with its classification as a chromid. Genome annotation shows a vast repertoire of carbohydrate active enzymes and genes involved in the degradation of aromatic compounds, highlighting the biotechnological potential of the new isolate obtained from Cerrado soils, especially regarding EPS production and biodegradation of recalcitrant compounds. Chemotaxonomic features, including polar lipid and fatty acid profiles, as well as physiological, molecular and whole genome comparisons showed significant differences between strain GeG2T and a N. rosa, clearly indicating that it represents a novel species, for which the name Novosphingobium terrae is proposed. The type strain is GeG2T (=CBMAI 2313T =CBAS 753T ).

Characterization of Shiga Toxin 2a Encoding Bacteriophages Isolated From High-Virulent O145:H25 Shiga Toxin-Producing Escherichia coli

Shiga toxin-producing Escherichia coli (STEC) may cause severe disease mainly due to the ability to produce Shiga toxins (Stx) encoded on bacteriophages. In Norway, more than 30% of the reported cases with STEC O145:H25 develop hemolytic uremic syndrome (HUS), and most cases, with known travel history, acquired the infection domestically. To describe phage characteristics associated with high virulence, we extracted the Stx2a phage sequences from eight clinical Norwegian O145:H25 STEC to conduct in-depth molecular characterization using long and short read sequencing. The Stx2a phages were annotated, characterized, and compared with previously published Stx2a phages isolated from STEC of different serotypes. The Norwegian O145:H25 Stx2a phages showed high sequence identity (>99%) with 100% coverage. The Stx2a phages were located at the integration site yciD, were approximately 45 kbp long, and harbored several virulence-associated genes, in addition to stx2a, such as nanS and nleC. We observed high sequence identity (>98%) and coverage (≥94%) between Norwegian O145:H25 Stx2a phages and publicly available Stx2a phages from O145:H25 and O145:H28 STEC, isolated from HUS cases in the USA and a hemorrhagic diarrhea case from Japan, respectively. However, low similarity was seen when comparing the Norwegian O145:H25 Stx2a phage to Stx2a phages from STEC of other serotypes. In all the Norwegian O145:H25 STEC, we identified a second phage or remnants of a phage (a shadow phage, 61 kbp) inserted at the same integration site as the Stx2a phage. The shadow phage shared similarity with the Stx2a phage, but lacked stx2a and harbored effector genes not present in the Stx2a phage. We identified a conserved Stx2a phage among the Norwegian O145:H25 STEC that shared integration site with a shadow phage in all isolates. Both phage and shadow phage harbored several virulence-associated genes that may contribute to the increased pathogenicity of O145:H25 STEC.

Urotensin receptor in GtoPdb v.2021.3

The urotensin-II (U-II) receptor (UT, nomenclature as agreed by the NC-IUPHAR Subcommittee on the Urotensin receptor [26, 36, 93]) is activated by the endogenous dodecapeptide urotensin-II, originally isolated from the urophysis, the endocrine organ of the caudal neurosecretory system of teleost fish [7, 92]. Several structural forms of U-II exist in fish and amphibians [93]. The goby orthologue was used to identify U-II as the cognate ligand for the predicted receptor encoded by the rat gene gpr14 [2, 20, 63, 69, 72]. Human urotensin-II, an 11-amino-acid peptide [20], retains the cyclohexapeptide sequence of goby U-II that is thought to be important in ligand binding [61, 53, 10]. This sequence is also conserved in the deduced amino-acid sequence of rat urotensin-II (14 amino-acids) and mouse urotensin-II (14 amino-acids), although the N-terminal is more divergent from the human sequence [19]. A second endogenous ligand for the UT has been discovered in rat [86]. This is the urotensin II-related peptide, an octapeptide that is derived from a different gene, but shares the C-terminal sequence (CFWKYCV) common to U-II from other species. Identical sequences to rat urotensin II-related peptide are predicted for the mature mouse and human peptides [32]. UT exhibits relatively high sequence identity with somatostatin, opioid and galanin receptors [93].