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A novel, strictly anaerobic, gram-negative, segmented filamentous bacterium strain AGMB03513T, was isolated from the faeces of a 5-month-old pig. Phylogenetic analysis based on the 16S rRNA gene indicated that the isolate was a member of the family Lachnospiraceae, and the closest strain was Anaerostipes butyraticus. Strain AGMB03513T formed a lineage within the genus Anaerostipes and was closely related to A. butyraticus DSM 22094 T (= KCTC 15125 T, 95.8%), Anaerostipes hadrus DSM 3319 T (= KCTC 15606 T, 95.5%), Anaerostipes caccae DSM 14662 T (= KCTC 15019 T, 94.0%), and Anaerostipes rhamnosivorans DSM 26241 T (= KCTC 15316 T, 93.4%). Strain AGMB03513T grew at temperatures between 30 and 45 °C, within a pH range of 7.0–9.0, and in medium containing up to 1.5% NaCl. Cells were found to utilise d-glucose, d-mannitol, d-lactose, d-saccharose, d-maltose, d-xylose, l-arabinose, d-mannose, and d-sorbitol, and acetate was identified as the major end product of metabolism. The major components of the cellular fatty acids were C12:0, C16:0, and C18:0. In addition, the bacterium contained meso-diaminopimelic acid in the cell wall. According to the comparative analysis of the whole genome sequence, the DNA G + C content of strain AGMB03513 was 37.0 mol%. In addition, Average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridisation (dDDH) values were obtained in comparisons of strain AGMB03513T with reference strains of species in the genus Anaerostipes. ANI values were found to be between 71.0 and 75.7%, AAI values between 66.6 and 73.2%, and dDDH values between 19.5 and 21.4%. All the data were below the threshold range for species determination. Based on phenotypic, phylogenetic, biochemical, chemotaxonomic, and genomic characteristics, we considered it reasonable to assign a novel species status to strain AGMB03513T, for which we propose the name Anaerostipes faecalis sp. nov. The type strain is AGMB03513T (= KCTC 25020 T = NBRC 114896 T).

A novel actinobacterial strain, Gram-positive, anaerobic, non-motile, and rod-shaped, designated KGMB02528T, was isolated from healthy human feces. Cells of strain KGMB02528T grew optimally at pH 7.0 and 37 °C and in the presence of 0% (w/v) NaCl. Based on 16S rRNA gene sequence similarity, strain KGMB04489T belonged to the genus Collinsella and was most closely related to Collinsella aerofaciens DSM 17552T (95.8%). The DNA G + C content was 58.0 mol%. The major cellular fatty acids (> 10%) were C16:0 DMA, C16:0 ALDE, C14:0 DMA, and C12:0. The predominant end product of fermentation was acetic acid. The cell wall peptidoglycan of strain KGMB02528T contained alanine, glutamic acid, and lysine, while diaminopimelic acid was not detected. The polar lipids were composed of two unidentified phospholipids and unidentified nine glycolipids. Based on the phenotypic, chemotaxonomic, and phylogenetic properties, strain KGMB02528T represents a novel species of the genus Collinsella, for which the name Collinsella acetigenes sp. nov. is proposed. The type strain is Collinsella acetigenes KGMB02528T (= KCTC 15847T = CCUG 73987T). The description of the genus Collinsella is emended to accommodate the new species. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Collinsella acetigenes KGMB02528T is MT117838. The whole-genome shotgun BioProject number is PRJNA623694 with the accession number JABBCP000000000.

The genus Izhakiella was established and designated as a member of the family Enterobacteriaceae in 2016. Although the taxonomical classification of most members in this family has been relatively resolved after two reclassifications in 2016 and 2017, the classification of the genus Izhakiella remains ambiguous. In this study, a polyphasic approach was used to provide evidence supporting the fact that the genus Izhakiella should no longer be considered a member of Enterobacteriaceae and proposes its reclassification into the family Erwiniaceae . The phylogenetic tree of type species in the families Enterobacteriaceae and Erwiniaceae based on the sequences of the 16S rRNA gene, rpoB housekeeping gene, and the whole-genome comprising the 92 core genes revealed that the genus Izhakiella forms a phylogenetic lineage within the family Erwiniaceae . The average nucleotide identity (ANI) value of the type species with genus Izhakiella was found to be higher for the family Erwiniaceae than that for the family Enterobacteriaceae . Notably, 12 conserved signature indels (CSIs) that are exclusively shared among the Erwiniaceae clade members were found in the type strains of the genus Izhakiella . Based on these analyses, this study suggests the reclassification of I. capsodis and I. australiensis into the family Erwiniaceae .

An obligately anaerobic, Gram-stain-positive, non-motile and coccoid- or oval-shaped bacterium, designated strain KGMB01111T, was isolated from faeces from a healthy Korean. Comparative analysis of 16S rRNA gene sequences indicated that KGMB01111T was closely related to Ruminococcus gauveauii CCRI-16110T (93.9 %) and Blautia stercoris GAM6-1T (93.7 %), followed by Clostridium nexile DSM 1787T (93.5 %), Blautia producta ATCC 27340T (93.4 %), Blautia hydrogenotrophica DSM 10507T (93.1 %) and Blautia coccoides ATCC 29236T (93.1 %) within the family Lachnospiraceae ( Clostridium rRNA cluster XIVa). Phylogenetic analysis based on the 16S rRNA gene sequences indicated that KGMB01111T formed a separate branch with species in the genus Blautia . The major cellular fatty acids (>10.0 %) were C16 : 0 and C18 : 1 cis 9 dimethyl acetal (DMA), and the major polar lipids were aminophospholipids and lipids. KGMB01111T contained meso-diaminopimelic acid in cell-wall peptidoglycan. The predominant end product of fermentation produced by KGMB01111T was acetic acid. Based on the whole-genome sequence, the DNA G+C content of the isolate was 44.7 mol%. On the basis of the phenotypic, chemotaxonomic and phylogenetic characteristics, KGMB01111T represents a novel species within the genus Blautia for which the name Blautia faecicola sp. nov. is proposed. The type strain is KGMB01111T (=KCTC 15706T=DSM 107827T).

A novel actinobacterial strain, designated KGMB04484T, was isolated from healthy human faeces sampled in the Republic of Korea. Cells of strain KGMB04484T were strictly anaerobic, Gram-stain-positive, catalase-positive, oxidase-negative, non-motile coccobacilli and formed tiny colonies on Columbia agar with 5 % horse blood. On the basis of 16S rRNA gene sequence similarity, strain KGMB04484T was affiliated with the genus Senegalimassilia in the family Coriobacteriaceae and its closest relative was Senegalimassilia anaerobia JC110T (96.28 % sequence similarity). The DNA G+C content of strain KGMB04484T was 61.2 mol%. The polar lipids contained diphosphatidylglycerol, phosphatidylglycerol, an unidentified phospholipid, an unidentified aminolipid and three unidentified glycolipids. The predominant cellular fatty acids (>10 %) of strain KGMB04484T were C14 : 0, C16 : 0 and C16 : 0 dimethyl acetal. Based on its phylogenetic, physiological and chemotaxonomic characteristics, strain KGMB04484T is considered to represent a novel species within the genus Senegalimassilia , for which the name Senegalimassilia faecalis sp. nov. is proposed. The type strain is KGMB04484T (=KCTC 15721T=CCUG 72347T).

A novel Gram-stain-negative and strictly anaerobic bacterial strain, designated KGMB02408T, was isolated from faeces of a healthy human in the Republic of Korea. The isolate was characterized as non-motile, non-spore-forming and rod-shaped (variable in length). The results of phylogenetic analysis based on 16S rRNA gene sequences revealed that strain KGMB02408T belonged to the genus Bacteroides and was most closely related to Bacteroides faecichinchillae JCM 17102T (=KCTC 15666T; 96.5 %). Based on its whole-genome sequence, the DNA G+C content of the isolate was 39.5 mol%. The average nucleotide identity value between strain KGMB02408T and related species, B. faecichinchillae JCM 17102T, was 93.8 %. The major cellular fatty acids (>10 %) of the isolate were anteiso-C15 : 0, iso-C17 : 0-OH, summed feature 11 (iso-C17 : 0-OH and/or C18 : 2 DMA) and C16 : 0. Menaquinone-8 (28.6 %) and menaquinone-10 (47.1 %) were detected as the major respiratory quinones in the isolate. The major end products of glucose fermentation produced by strain KGMB02408T were lactic acid, acetic acid and formic acid. Based on its phylogenetic, phenotypic and chemotaxonomic characteristics, strain KGMB02408T represents a novel species of the genus Bacteroides in the family Bacteroidaceae . The type strain is KGMB02408T (=KCTC 15687T=DSM 107828T).

The gut microbiota (GM) has been shown to be closely associated with the development of colorectal cancer (CRC). However, the involvement of GM is CRC has mainly been demonstrated by metagenomic profiling studies showing the compositional difference between the GM of healthy individuals and that of CRC patients and not by directly studying isolated gut microbes. Thus, to discover novel gut microbes involved in CRC, we isolated the GM from the feces of healthy individuals and evaluated its anti-CRC activity in vitro and in vivo. After GM isolation, cell-free supernatants (CFSs) were prepared from the isolated gut microorganisms to efficiently screen a large amount of the GM for anti-proliferative ability in vitro. Our results showed that the CFSs of 21 GM isolates had anti-proliferative activity against human colon cancer HCT 116 cells. Of these 21 GM isolates, GM07 was chosen for additional study because it had the highest anti-cancer activity against mouse colon cancer CT 26 cells in vitro and was further evaluated in a CT 26 allograft mouse model in vivo. GM07 was identified as Odoribacter splanchnicus through phylogenetic analysis based on 16S rRNA gene sequencing. Further investigation determined that the CFS of O. splanchnicus (OsCFS) induced anti-proliferative activity via apoptosis, but not cell cycle arrest. Moreover, GC/MS analysis suggested that the putative active molecule in OsCFS is malic acid. Finally, in the CRC mouse model, peri-tumoral injection of OsCFS significantly decreased CRC formation, compared to the control group. Altogether, these findings will provide valuable information for the discovery of potential probiotic candidates that inhibit CRC.

A novel endophytic bacterial strain, designated GR-TSA-9T, was isolated from surface-sterilized grape (Vitis vinifera L.). 16S rRNA gene sequence analyses showed that the isolate was grouped within the genus Brevundimonas, displaying the highest similarity with Brevundimonas lenta DS-18T (97.9%) and Brevundimonas kwangchunensis KSL-102T (97.8%) and less than 97.5% similarity with other members of Brevundimonas. The strain GR-TSA-9T was a gram negative, rod shaped, facultatively anaerobic, catalase and oxidase positive, and motile bacterium. Its growth occurred at 10–37°C (optimally 25–30°C), at pH 7.0–8.0, and in NaCl 0–1% (optimally 0%). It contained ubiquinone-10 as a respiratory quinone, and the major cellular fatty acids (>10% of the total) were C16:0 (14.2%) and summed feature 8 (C18:1ω7c and/or C18:1ω6c, 65.6%). The polar lipids present in the strain were phosphoglycolipids, phosphatidylglycerol, 1,2-di-O-acyl-3-O-[d-glucopyranosyl-(1→4)-α-d-glucopyranuronosyl]glycerol, and unidentified lipids (L1, L2, and L4). The strain had one 2,976,716bp circular chromosome with a G+C content of 66.4%. The digital DNA–DNA hybridization value between strain GR-TSA-9T and B. lenta DS-18T was 20.9%, while the average nucleotide identity value was 76.7%. In addition, the dDDH and ANI values to other members in this genus, whose genome sequences are available, are less than 21.1 and 77.6%. Genome annotation predicted the presence of some gene clusters related to tyrosine degradation and pyomelanin formation. Strain GR-TSA-9T produced a brown melanin-like pigment in the presence of L-tyrosine-containing media. The highest pigment production (0.19g/L) was observed in tryptic soy broth with 1.0mg/ml L-tyrosine at 25°C for 6days of culture. Biophysical characterization by ultraviolet (UV)–visible spectroscopy, Fourier-transform infrared spectroscopy, and electrospray ionization mass spectrometry confirmed that the pigment was pyomelanin. Additionally, melanized GR-TSA-9T cells could protect the cells against UVC exposure. The phylogenetic, genomic, phenotypic, and chemotaxonomic features indicated that strain GR-TSA-9T represents a novel melanin-producing species of Brevundimonas. The type strain was GR-TSA-9T (KCTC 82386T=CGMCC 1.18820T).

A novel bacterial isolate designated as strain AGMB01083T was isolated from Korean cow faeces deposited in the National Institute of Animal Science (Wanju, Republic of Korea). The bacterium is obligate anaerobic, Gram-strain-positive, and motile. Cells are straight or curved rod-shaped, flagella and spores are observed. Growth occurs between 20–40 °C (temperature optimum of 35 °C), at pH 7–9 (pH optimum of 7), and in the presence of 0.5–1.0 % (w/v) NaCl. Based on the 16S rRNA gene sequence analysis, the strain belongs to the genus Anaerosporobacter and is most closely related to A. mobilis HY-37-4T (=KCTC5027T, similarity, 95.7 %). The DNA G+C content is 36.2 mol%, determined by the whole-genome sequence. The average nucleotide identity value between strain AGMB01083T and strain A. mobilis HY-37-4T is 75.5 %, below the interspecies identity threshold value. The major cellular fatty acids (>10 %) of strain AGMB01083T are C16 : 0, C16 : 0 dimethyl acetal (DMA), and C16 : 0 3-OH. Based on the phylogenetic, phenotypic, biochemical, chemotaxonomic, and genomic characterization, strain AGMB01083T is proposed to be a novel species, named Anaerosporobacter faecicola, in the genus Anaerosporobacter . The type strain is AGMB01083T (=KCTC 15857T=NBRC 114517T).

An obligately anaerobic, non-motile, Gram-negative and rod-shaped strain (AGMB03916T) was isolated from faeces of a 2-week-old piglet raised at the National Institute of Animal Science in Wanju, Republic of Korea. Growth of strain AGMB03916T occurred at 30–45 °C (optimum, 37 °C), at pH 6–9 (optimum, pH 8) and in the presence of 0.5–1.0 % (w/v) NaCl. Based on the results of 16S rRNA gene sequence analyses, strain AGMB03916T was closely related to two validly published species of the genus Phocaeicola , Phocaeicola plebeius and Phocaeicola coprocola . The 16S rRNA gene sequence similarity of strain AGMB03916T compared to P. plebeius M12T (=KCTC 5793T) and P. coprocola M16T (=KCTC 5443T) were 96.3 and 95.0 %, respectively. The genomic DNA G+C content of strain AGMB03916T was 46.4 mol%. The average nucleotide identity values between strain AGMB03916T and the reference strains were 74.9–78.5 %. Cells were able to utilize d-glucose, lactose, sucrose, maltose, salicin, aesculin hydrolysis, cellobiose and raffinose. The major end product of metabolism was acetate. The major cellular fatty acids (>10 %) of the isolate were iso-C15 : 0, anteiso-C15 : 0, C16 : 0, C16 : 0 3-OH and summed feature 11 (iso-C17 : 0 3-OH and/or C18 : 2 DMA). On the basis of the genotypic, biochemical, chemotaxonomic, phenotypic and phylogenetic data, strain AGMB03916T represents a novel species of the genus Phocaeicola , for which the name Phocaeicola faecicola sp. nov. is proposed. The type strain is AGMB03916T (=KCTC 25014T=GDMCC 1.2574T).

An obligately anaerobic, Gram-stain-positive, non-motile, non-spore-forming and rod-shaped strain AGMB00832T was isolated from swine faeces. Phylogenetic analysis based on the 16S rRNA gene, together with the housekeeping genes, gyrB and rpoD, revealed that strain AGMB00832T belonged to the genus Faecalicatena and was most closely related to Faecalicatena orotica KCTC 15331T. In biochemical analysis, strain AGMB00832T was shown to be negative for catalase, oxidase and urease. Furthermore, the isolate was positive for β-glucosidase, β-glucuronidase, glutamic acid decarboxylase, proline arylamidase, acid phosphatase and naphthol-AS-BI-phosphohydrolase. The major cellular fatty acids (> 10%) of the isolate were C14:0, C16:0 and C18:1ω11t DMA. Based on the whole genome sequence analysis, the DNA G + C content of strain AGMB00832T was 44.2 mol%, and the genome size and numbers of rRNA and tRNA genes were 5,175,159 bp, 11 and 53, respectively. The average nucleotide identity and digital DNA–DNA hybridization values between strain AGMB00832T and related strains were ≤ 77.4 and 22.5%, respectively. Furthermore, the genome analysis revealed the presence of genes for alkaline shock protein 23 and cation/proton antiporters, which may facilitate growth of strain AGMB00832T in alkaline culture condition. On the basis of polyphasic taxonomic approach, strain AGMB00832T represents a novel species within the genus Faecalicatena, for which the name Faecalicatena faecalis sp. nov. is proposed. The type strain is AGMB00832T (= KCTC 15946T = NBRC 114613T).

An obligately anaerobic, Gram-positive, non-motile, coccus-shaped bacterial strain designated AGMB00490T was isolated from swine faeces. 16S rRNA gene sequence-based phylogenetic analysis indicated that the isolate belongs to the genus Peptoniphilus and that the most closely related species is Peptoniphilus gorbachii WAL 10418T (=KCTC 5947T, 97.22 % 16S rRNA gene sequence similarity). Whole genome sequence analysis determined that the DNA G+C content of strain AGMB00490T was 31.2 mol% and moreover that the genome size and numbers of tRNA and rRNA genes were 2 129 517 bp, 34 and 10, respectively. Strain AGMB00490T was negative for oxidase and urease; positive for catalase, indole production, arginine arylamidase, leucine arylamidase, tyrosine arylamidase and histidine arylamidase; and weakly positive for phenylalanine arylamidase and glycine arylamidase. The major cellular fatty acids (>10 %) of the isolate were determined to be C16 : 0 and C18 : 1 ω9c. Strain AGMB00490T produced acetic acid as a major end product of metabolism. Accordingly, phylogenetic, physiologic and chemotaxonomic analyses revealed that strain AGMB00490T represents a novel species for which the name Peptoniphilus faecalis sp. nov. is proposed. The type strain is AGMB00490T (=KCTC 15944T=NBRC 114159T).

A gram-negative colonizer of the oral cavity, Fusobacterium nucleatum not only interacts with many pathogens in the oral microbiome but also has the ability to spread to extraoral sites including placenta and amniotic fluid, promoting preterm birth. To date, however, the molecular mechanism of interspecies interactions-termed coaggregation-by F. nucleatum and how coaggregation affects bacterial virulence remain poorly defined. Here, we employed genome-wide transposon mutagenesis to uncover fusobacterial coaggregation factors, revealing the intertwined function of a two-component signal transduction system (TCS), named CarRS, and a lysine metabolic pathway in regulating the critical coaggregation factor RadD. Transcriptome analysis shows that CarR modulates a large regulon including radD and lysine metabolic genes, such as kamA and kamD, the expression of which are highly up-regulated in the DeltacarR mutant. Significantly, the native culture medium of DeltakamA or DeltakamD mutants builds up abundant amounts of free lysine, which blocks fusobacterial coaggregation with streptococci. Our demonstration that lysine-conjugated beads trap RadD from the membrane lysates suggests that lysine utilizes RadD as its receptor to act as a metabolic inhibitor of coaggregation. Lastly, using a mouse model of preterm birth, we show that fusobacterial virulence is significantly attenuated with the DeltakamA and DeltacarR mutants, in contrast to the enhanced virulence phenotype observed upon diminishing RadD (DeltaradD or DeltacarS mutant). Evidently, F. nucleatum employs the TCS CarRS and environmental lysine to modulate RadD-mediated interspecies interaction, virulence, and nutrient acquisition to thrive in the adverse environment of oral biofilms and extraoral sites.

Controlled RNA degradation is a crucial process in bacterial cell biology for maintaining proper transcriptome homeostasis and adaptation to changing environments. mRNA turnover in many Gram-positive bacteria involves a specialized ribonuclease called RNase J (RnJ). To date, however, nothing is known about this process in the diphtheria-causative pathogen Corynebacterium diphtheriae, nor is known the identity of this ribonuclease in this organism. Here, we report that C. diphtheriae DIP1463 encodes a predicted RnJ homolog, comprised of a conserved N-terminal β-lactamase domain, followed by β-CASP and C-terminal domains. A recombinant protein encompassing the β-lactamase domain alone displays 5′-exoribonuclease activity, which is abolished by alanine-substitution of the conserved catalytic residues His186 and His188. Intriguingly, deletion of DIP1463/rnj in C. diphtheriae reduces bacterial growth and generates cell shape abnormality with markedly augmented cell width. Comparative RNA-seq analysis revealed that RnJ controls a large regulon encoding many factors predicted to be involved in biosynthesis, regulation, transport, and iron acquisition. One upregulated gene in the ∆rnj mutant is ftsH, coding for a membrane protease (FtsH) involved in cell division, whose overexpression in the wild-type strain also caused cell-width augmentation. Critically, the ∆rnj mutant is severely attenuated in virulence in a Caenorhabditis elegans model of infection, while the FtsH-overexpressing and toxin-less strains exhibit full virulence as the wild-type strain. Evidently, RNase J is a key ribonuclease in C. diphtheriae that post-transcriptionally influences the expression of numerous factors vital to corynebacterial cell physiology and virulence. Our findings have significant implications for basic biological processes and mechanisms of corynebacterial pathogenesis.

An obligate anaerobic, Gram-stain-positive, non-spore forming, non-motile, catalase and oxidase-negative, coccoid-shaped bacterium designated AGMB00486T was isolated from swine faeces. The optimal growth of the isolate occurred at pH 8.0 and 37 ℃. Furthermore, the growth was observed in the presence of up to 4% (w/v) NaCl but not at salinity levels higher than 5%. The phylogenetic analysis based on the 16S rRNA gene sequences revealed that strain AGMB00486T was a member of the genus Anaerococcus and that the isolate was most closely related to Anaerococcus vaginalis KCTC 15028T (96.7% 16S rRNA gene sequence similarity) followed by Anaerococcus hydrogenalis KCTC 15014T (96.7%) and Anaerococcus senegalensis KCTC 15435T (96.3%). Whole-genome sequence analysis determined that the DNA G+C content of strain AGMB00486T was 30.1 mol%, and the genome size, numbers of tRNA and rRNA genes were 2,268,866 bp, 47 and 8, respectively. The average nucleotide identity values between strain AGMB00486T and the three related type strains were 77.0, 77.4 and 77.2%, respectively. The major cellular fatty acids (> 10%) of strain AGMB00486T were C14:0, C16:0 and C16:0 DMA. Accordingly, these distinct phenotypic and phylogenetic properties revealed that strain AGMB00486T represents a novel species, for which the name Anaerococcus faecalis sp. nov. is proposed. The type strain is AGMB00486T (= KCTC 15945T = CCTCC AB 202009T).

We demonstrated successful overexpression of porcine reproductive and respiratory syndrome virus (PRRSV)-derived GP4D and GP5D antigenic proteins in Arabidopsis. Pigs immunized with transgenic plants expressing GP4D and GP5D proteins generated both humoral and cellular immune responses to PRRSV. Porcine reproductive and respiratory syndrome virus (PRRSV) causes PRRS, the most economically significant disease affecting the swine industry worldwide. However, current commercial PRRSV vaccines (killed virus or modified live vaccines) show poor efficacy and safety due to concerns such as reversion of virus to wild type and lack of cross protection. To overcome these problems, plants are considered a promising alternative to conventional platforms and as a vehicle for large-scale production of recombinant proteins. Here, we demonstrate successful production of recombinant protein vaccine by expressing codon-optimized and transmembrane-deleted recombinant glycoproteins (GP4D and GP5D) from PRRSV in planta. We generated transgenic Arabidopsis plants expressing GP4D and GP5D proteins as candidate antigens. To examine immunogenicity, pigs were fed transgenic Arabidopsis leaves expressing the GP4D and GP5D antigens (three times at 2-week intervals) and then challenged with PRRSV at 6-week post-initial treatment. Immunized pigs showed significantly lower lung lesion scores and reduced viremia and viral loads in the lung than pigs fed Arabidopsis leaves expressing mYFP (control). Immunized pigs also had higher titers of PRRSV-specific antibodies and significantly higher levels of pro-inflammatory cytokines (TNF-α and IL-12). Furthermore, the numbers of IFN-γ+-producing cells were higher, and those of regulatory T cells were lower, in GP4D and GP5D immunized pigs than in control pigs. Thus, plant-derived GP4D and GP5D proteins provide an alternative platform for producing an effective subunit vaccine against PRRSV.

Background/Aims: South Korean soldiers are exposed to similar environmental factors. In this study, we sought to evaluate the gut microbiome of healthy young male soldiers (HYMS) and to identify the primary factors influencing the microbiome composition. Methods: We prospectively collected stool from 100 HYMS and performed next-generation sequencing of the 16S rRNA genes of fecal bacteria. Clinical data, including data relating to the diet, smoking, drinking, and exercise, were collected. Results: The relative abundances of the bacterial phyla Firmicutes, Actinobacteria, Bacteroidetes, and Proteobacteria were 72.3%, 14.5%, 8.9%, and 4.0%, respectively. Fifteen species, most of which belonged to Firmicutes (87%), were detected in all examined subjects. Using cluster analysis, we found that the subjects could be divided into the two enterotypes based on the gut microbiome bacterial composition. Compared with enterotype 2 subjects, subjects classified as enterotype 1 tended to be characterized by higher frequencies of potentially harmful lifestyle habits (current smoker: 55.6% vs 36.6%, p=0.222; heavy drinker: 16.7% vs 3.7%, p=0.120; insufficient physical activity: 27.8% vs 14.6%, p=0.318). We identified a significant difference in the microbiome compositions of current and noncurrent smokers (p=0.008); the former differed from the latter mainly in a relatively lower abundance of Bifidobacterium species and a higher abundance of Negativicutes. Conclusions: A high abundance of Actinobacteria and low abundance of Bacteroidetes were the main features distinguishing the gut microbiomes of HYMS, and current smokers could be differentiated from noncurrent smokers by their lower abundance of Bifidobacterium and higher abundance of Negativicutes. (Gut Liver 2021;15:243-252)

An obligately anaerobic, Gram-stain-negative, non-motile, non-spore-forming, and coccobacilli-shaped bacterial strain, designated KGMB03119T, was isolated from human faeces from a Korean. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the isolate was a member of the genus Sutterella and most closely related to Sutterlla wadsworthensis KCTC 15691T (96.8% 16S rRNA gene sequence similarity). The DNA G + C content of strain KGMB03119T was 58.3 mol% as determined from its whole genome sequence. Strain KGMB03119T was asaccharolytic, catalase-positive, oxidase- and urease-negative. Furthermore, the isolate was positive for alkaline phosphatase, leucine arylamidase, acid phosphatase, arginine arylamidase, alanine arylamidase, and glycine arylamidase. The major cellular fatty acids (> 10%) of the isolate were C18:1ω9c and C16:0. Methylmenaquinone-5 (MMK-5, 100%) was the predominant isoprenoid quinone in the isolate. Based on the phylogenetic, physiological, and chemotaxonomic characteristics, strain KGMB03119T represents a novel species, for which the name Sutterella faecalis sp. nov. is proposed. The type strain is KGMB03119T (= KCTC 15823T = NBRC 114254T).

A strictly anaerobic bacterium, designated as strain KGMB-03357T, was isolated from the faeces of a healthy Korean selected by Bundang Seoul National University based on health status. Cells of strain KGMB03357T are Gram-stain-positive, non-motile, non-spore-forming, and observed as straight or curved rods. The isolate grew at 10-45°C (optimum temperature of 40°C) and a pH range of 5.1-10.5 (optimum pH of 6.8). Analysis of phylogenetic trees based on the 16S rRNA gene sequences revealed that strain KGMB03357T forms a lineage within the genus Anaerotignum, and is most closely related to Anaerotignum lactatifermentans G17T (= KCTC 15066T, 96.1%), Anaerotignum propionicum DSM 1682T (= KCTC 5582T, 94.9%), Anaerotignum neopropionicum DSM 03847T (= KCTC 15564T, 94.9%), and Anaerotignum aminivorans SH021T (= KCTC 15705T, 94.8%). The ANI values between strain KGMB 03357T and members of the genus Anaerotignum were 73.3-71.0%, which are below the ANI criterion for interspecies identity. The DNA G + C content based on the whole-genome sequence is 47.3 mol%. The major cellular fatty acids of strain KGMB03357T are C16:0, C18:0, C18∶1 cis 9, and anteiso-C15∶0. Strain KGMB03357T contains meso-diaminopimelic acid as the diagnostic amino acid in the cell wall peptidoglycan. Based on the phenotypic, phylogenetic, and genomic properties, strain KGMB 03357T represents a novel species of the genus Anaerotignum, for which the name Anaerotignum faecicola sp. nov. is proposed. The type strain is KGMB03357T (= KCTC 15736T = DSM 107953T).

A novel actinobacterial strain, designated KGMB04489T, was isolated from the faeces of a healthy Korean. Cells of the strain were strictly anaerobic, Gram-stain-positive and short-rod-shaped. On the basis of 16S rRNA gene sequence similarity, strain KGMB04489T belonged to the genus Olsenella and was most closely related to Olsenella scatoligenes SK9K4T (94.3 %), Olsenella uli ATCC 49627T (93.5 %), Olsenella umbonata lac31T (93.4 %) and Olsenella profusa D315A-29T (93.3 %). The major end product was lactic acid. The DNA G+C content was 65.5 mol%. The major cellular fatty acids of strain KGMB04489T were C18 : 1 cis9, C18 : 1 cis9 DMA and C16 : 0. Strain KGMB04489T contained meso-diaminopimelic acid as the diamino acid in the peptidoglycan. The polar lipids consisted of an unidentified phospholipid, six unidentified glycolipids and an unidentified lipid. Based on phylogenetic, physiological and chemotaxonomic characteristics, strain KGMB04489T is considered to represent a novel species within the genus Olsenella , for which the name Olsenella faecalis sp. nov. is proposed. The type strain is KGMB04489T (=KCTC 15699T=CCUG 72345T).

A Gram-stain-positive, obligately anaerobic, non-motile, nonspore-forming, and rod-shaped bacterial strain, designated KGMB01110T, was isolated from a faecal sample of a healthy male in South Korea. Phylogenetic analysis based on 16S rRNA gene showed that strain KGMB01110T belonged to Clostridium cluster XIVa and was most closely related to Mediterraneibacter glycyrrhizinilyticus KCTC 5760T (95.9% 16S rRNA gene sequence similarity). The DNA G + C content of strain KGMB01110T based on its whole genome sequence was 44.1 mol%. The major cellular fatty acids (> 10%) of the isolate were C14:0 and C16:0. The strain KGMB01110T was positive for arginine dihydrolase, β-galactosidase-6-phosphatase, and alkaline phosphatase. The strain KGMB01110T also produced acid from D-glucose and D-rhamnose, and hydrolyzed gelatin and aesculin. Furthermore, HPLC analysis and UV-tests of culture supernatant revealed that the strain KGMB01110T produced butyrate as the major end product of glucose fermentation. Based on the phylogenetic and phenotypic characteristics, strain KGMB01110T represent a novel species of the genus Mediterraneibacter in the family Lachnospiraceae. The type strain is KGMB01110T (= KCTC 15684T = CCUG 72830T).

Fusobacterium nucleatum is a key member of the human oral biofilm. It is also implicated in preterm birth and colorectal cancer. To facilitate basic studies of fusobacterial virulence, we describe here a versatile transposon mutagenesis procedure and a pilot screen for mutants defective in biofilm formation. Out of 10 independent biofilm-defective mutants isolated, the affected genes included the homologs of the Escherichia coli cell division proteins FtsX and EnvC, the electron transport protein RnfA, and four proteins with unknown functions. Next, a facile new gene deletion method demonstrated that nonpolar, in-frame deletion of ftsX or envC produces viable bacteria that are highly filamentous due to defective cell division. Transmission electron and cryo-electron microscopy revealed that the ΔftsX and ΔenvC mutant cells remain joined with apparent constriction, and scanning electron microscopy (EM) uncovered a smooth cell surface without the microfolds present in wild-type cells. FtsX and EnvC proteins interact with each other as well as a common set of interacting partners, many with unknown function. Last, biofilm development is altered when cell division is blocked by MinC overproduction; however, unlike the phenotypes of ΔftsX and ΔenvC mutants, a weakly adherent biofilm is formed, and the wild-type rugged cell surface is maintained. Therefore, FtsX and EnvC may perform novel functions in Fusobacterium cell biology. This is the first report of an unbiased approach to uncover genetic determinants of fusobacterial biofilm development. It points to an intriguing link among cytokinesis, cell surface dynamics, and biofilm formation, whose molecular underpinnings remain to be elucidated., IMPORTANCE Little is known about the virulence mechanisms and associated factors in F. nucleatum, due mainly to the lack of convenient genetic tools for this organism. We employed two efficient genetic strategies to identify F. nucleatum biofilm-defective mutants, revealing FtsX and EnvC among seven biofilm-associated factors. Electron microscopy established cell division defects of the ΔftsX and ΔenvC mutants, accompanied with a smooth cell surface, unlike the microfold, rugged appearance of wild-type bacteria. Proteomic studies demonstrated that FtsX and EnvC interact with each other as well as a set of common and unique interacting proteins, many with unknown functions. Importantly, blocking cell division by MinC overproduction led to formation of a weakly adherent biofilm, without alteration of the wild-type cell surface. Thus, this work links cell division and surface dynamics to biofilm development and lays a foundation for future genetic and biochemical investigations of basic cellular processes in this clinically significant pathogen.

Trypanosomes are protistan parasites that diverged early in evolution from most eukaryotes. Their streamlined genomes are packed with arrays of tandemly linked genes that are transcribed polycistronically by RNA polymerase (pol) II. Individual mRNAs are processed from pre-mRNA by spliced leader (SL) trans splicing and polyadenylation. While there is no strong evidence that general transcription factors are needed for transcription initiation at these gene arrays, a RNA pol II transcription pre-initiation complex (PIC) is formed on promoters of SLRNA genes, which encode the small nuclear SL RNA, the SL donor in trans splicing. The factors that form the PIC are extremely divergent orthologues of the small nuclear RNA-activating complex, TBP, TFIIA, TFIIB, TFIIH, TFIIE and Mediator. Here, we functionally characterized a heterodimeric complex of unannotated, nuclear proteins that interacts with RNA pol II and is essential for PIC formation, SL RNA synthesis in vivo, SLRNA transcription in vitro, and parasite viability. These functional attributes suggest that the factor represents TFIIF although the amino acid sequences are too divergent to firmly make this conclusion. This work strongly indicates that early-diverged trypanosomes have orthologues of each and every general transcription factor, requiring them for the synthesis of SL RNA.

Dynein light chain LC8 is highly conserved among eukaryotes and has both dynein-dependent and dynein-independent functions. Interestingly, LC8 was identified as a subunit of the class I transcription factor A (CITFA), which is essential for transcription by RNA polymerase I (Pol I) in the parasite Trypanosoma brucei. Given that LC8 has never been identified with a basal transcription factor and that T. brucei relies on RNA Pol I for expressing the variant surface glycoprotein (VSG), the key protein in antigenic variation, we investigated the CITFA-specific role of LC8. Depletion of LC8 from mammalian-infective bloodstream trypanosomes affected cell cycle progression, reduced the abundances of rRNA and VSG mRNA, and resulted in rapid cell death. Sedimentation analysis, coimmunoprecipitation of recombinant proteins, and bioinformatic analysis revealed an LC8 binding site near the N terminus of the subunit CITFA2. Mutation of this site prevented the formation of a CITFA2-LC8 heterotetramer and, in vivo, was lethal, affecting assembly of a functional CITFA complex. Gel shift assays and UV cross-linking experiments identified CITFA2 as a promoter-binding CITFA subunit. Accordingly, silencing of LC8 or CITFA2 resulted in a loss of CITFA from RNA Pol I promoters. Hence, we discovered an LC8 interaction that, unprecedentedly, has a basal function in transcription.

Alkyl hydroperoxide reductase E (AhpE) is a member of the peroxidase family of enzymes that catalyse the reduction of peroxides, however its structural and functional roles are still unclear in details. In this study, we used the Thermococcus kodakarensis AhpE-like protein as a model to investigate structure–function relationships including the molecular properties of DNA binding activity. Multiple sequence alignment, structural comparison and biochemical analyses revealed that TkAhpE includes conserved peroxidase residues in the active site, and exhibits peroxidase activity with structure-dependent holdase chaperone function. Following electrophoretic mobility shift assays and electron microscopy analysis demonstrated distinctive binding features of TkAhpE to the DNA showing that their dimeric conformer can bind to the double-stranded DNA, but not to the single-stranded DNA, indicating its striking molecular features to double-stranded DNA-specific interactions. Based on our results, we provided that TkAhpE is a multifunctional peroxidase displaying structure-dependent molecular chaperone and DNA binding activities.

Summary Conserved from yeast to humans, TFIIH is essential for RNA polymerase II transcription and nucleotide excision repair (NER). TFIIH consists of a core that includes the DNA helicase Xeroderma pigmentosum B (XPB) and a kinase subcomplex. Trypanosoma brucei TFIIH harbours all core complex components and is indispensable for RNA polymerase II transcription of spliced leader RNA genes (SLRNAs). Kinetoplastid organisms, however, possess two highly divergent XPB paralogues with only the larger being identified as a TFIIH subunit in T. brucei. Here we show that a knockout of the gene for the smaller paralogue, termed XPB-R (R for repair) resulted in viable cultured trypanosomes that grew slower than normal. XPB-R depletion did not affect transcription in vivo or in vitro and XPB-R was not found to occupy the SLRNA promoter which assembles a RNA polymerase II transcription pre-initiation complex including TFIIH. However, XPB-R−/− cells were much less tolerant than wild-type cells to UV light- and cisplatin-induced DNA damage, which require NER. Since XPB-R−/− cells were not impaired in DNA base excision repair, XPB-R appears to function specifically in NER. Interestingly, several other protists possess highly divergent XPB paralogues suggesting that XPBs specialized in transcription or NER exist beyond the Kinetoplastida.

Conserved from yeast to mammals, phosphorylation of the heptad repeat sequence Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7) in the carboxy-terminal domain (CTD) of the largest RNA polymerase II (RNA Pol II) subunit, RPB1, mediates the enzyme's promoter escape and binding of RNA-processing factors, such as the m(7)G capping enzymes. The first critical step, Ser(5) phosphorylation, is carried out by cyclin-dependent kinase 7 (CDK7), a subunit of the basal transcription factor TFIIH. Many early-diverged protists, such as the lethal human parasite Trypanosoma brucei, however, lack the heptad repeats and, apparently, a CDK7 ortholog. Accordingly, characterization of trypanosome TFIIH did not identify a kinase component. The T. brucei CTD, however, is phosphorylated and essential for transcription. Here we show that silencing the expression of T. brucei cdc2-related kinase 9 (CRK9) leads to a loss of RPB1 phosphorylation. Surprisingly, this event did not impair RNA Pol II transcription or cotranscriptional m(7)G capping. Instead, we observed that CRK9 silencing led to a block of spliced leader (SL) trans splicing, an essential step in trypanosome mRNA maturation, that was caused by hypomethylation of the SL RNA's unique cap4.

Trypanosoma brucei is the only organism known to have evolved a multifunctional RNA polymerase I (pol I) system that is used to express the parasite's ribosomal RNAs, as well as its major cell surface antigens, namely, the variant surface glycoprotein (VSG) and procyclin, which are vital for establishing successful infections in the mammalian host and the tsetse vector, respectively. Thus far, biochemical analyses of the T. brucei RNA pol I transcription machinery have elucidated the subunit structure of the enzyme and identified the class I transcription factor A (CITFA). CITFA binds to RNA pol I promoters, and its CITFA-2 subunit was shown to be absolutely essential for RNA pol I transcription in the parasite. Tandem affinity purification (TAP) of CITFA revealed the subunits CITFA-1 to -6, which are conserved only among kinetoplastid organisms, plus the dynein light chain DYNLL1. Here, by tagging CITFA-6 instead of CITFA-2, a complex was purified that contained all known CITFA subunits, as well as a novel proline-rich protein. Functional studies carried out in vivo and in vitro , as well as a colocalization study, unequivocally demonstrated that this protein is a bona fide CITFA subunit, essential for parasite viability and indispensable for RNA pol I transcription of ribosomal gene units and the active VSG expression site in the mammalian-infective life cycle stage of the parasite. Interestingly, CITFA-7 function appears to be species specific, because expression of an RNA interference (RNAi)-resistant CITFA-7 transgene from Trypanosoma cruzi could not rescue the lethal phenotype of silencing endogenous CITFA-7 .

Nitric oxide (NO) is known for its role in the activation of plant defense responses. To examine the involvement and mode of action of NO in plant defense responses, we introduced calmodulin-dependent mammalian neuronal nitric oxide synthase (nNOS), which controls the CaMV35S promoter, into wild-type and NahG tobacco plants. Constitutive expression of nNOS led to NO production and triggered spontaneous induction of leaf lesions. Transgenic plants accumulated high amounts of H(2)O(2), with catalase activity lower than that in the wild type. nNOS transgenic plants contained high levels of salicylic acid (SA), and they induced an array of SA-, jasmonic acid (JA)-, and/or ethylene (ET)-related genes. Consequently, NahG co-expression blocked the induction of systemic acquired resistance (SAR)-associated genes in transgenic plants, implying SA is involved in NO-mediated induction of SAR genes. The transgenic plants exhibited enhanced resistance to a spectrum of pathogens, including bacteria, fungi, and viruses. Our results suggest a highly ranked regulatory role for NO in SA-, JA-, and/or ET-dependent pathways that lead to disease resistance.

Dynein light chain LC8 is highly conserved among eukaryotes and has both dynein-dependent and dynein-independent functions. Interestingly, LC8 was identified as a subunit of the class I transcription factor A (CITFA), which is essential for transcription by RNA polymerase I (Pol I) in the parasite Trypanosoma brucei. Given that LC8 has never been identified with a basal transcription factor and that T. brucei relies on RNA Pol I for expressing the variant surface glycoprotein (VSG), the key protein in antigenic variation, we investigated the CITFA-specific role of LC8. Depletion of LC8 from mammalian-infective bloodstream trypanosomes affected cell cycle progression, reduced the abundances of rRNA and VSG mRNA, and resulted in rapid cell death. Sedimentation analysis, coimmunoprecipitation of recombinant proteins, and bioinformatic analysis revealed an LC8 binding site near the N terminus of the subunit CITFA2. Mutation of this site prevented the formation of a CITFA2-LC8 heterotetramer and, in vivo, was lethal, affecting assembly of a functional CITFA complex. Gel shift assays and UV cross-linking experiments identified CITFA2 as a promoter-binding CITFA subunit. Accordingly, silencing of LC8 or CITFA2 resulted in a loss of CITFA from RNA Pol I promoters. Hence, we discovered an LC8 interaction that, unprecedentedly, has a basal function in transcription.

Genome annotation suggested that early-diverged kinetoplastids possess a reduced set of basal transcription factors. More recent work, however, on the lethal parasite Trypanosoma brucei identified extremely divergent orthologs of TBP, TFIIA, TFIIB, and TFIIH which, together with the small nuclear RNA-activating protein complex, form a transcription preinitiation complex (PIC) at the spliced leader (SL) RNA gene (SLRNA) promoter. The SL RNA is a small nuclear RNA and a trans splicing substrate for the maturation of all pre-mRNAs which is metabolized continuously to sustain gene expression. Here, we identified and biochemically characterized a novel TFIIH-associated protein complex in T. brucei (Med-T) consisting of nine subunits whose amino acid sequences are conserved only among kinetoplastid organisms. Functional analyses in vivo and in vitro demonstrated that the complex is essential for cell viability, SLRNA transcription, and PIC integrity. Molecular structure analysis of purified Med-T and Med-T/TFIIH complexes by electron microscopy revealed that Med-T corresponds to the mediator head module of higher eukaryotes. These data therefore show that mediator is a basal factor for small nuclear SL RNA gene transcription in trypanosomes and that the basal transcription function of mediator head is a characteristic feature of eukaryotes which developed early in their evolution.

In trypanosomatid parasites, spliced leader (SL) trans splicing is an essential nuclear mRNA maturation step which caps mRNAs posttranscriptionally and, in conjunction with polyadenylation, resolves individual mRNAs from polycistronic precursors. While all trypanosomatid mRNAs are trans spliced, intron removal by cis splicing is extremely rare and predicted to occur in only four pre-mRNAs. trans - and cis -splicing reactions are carried out by the spliceosome, which consists of U-rich small nuclear ribonucleoprotein particles (U snRNPs) and of non-snRNP factors. Mammalian and yeast spliceosome complexes are well characterized and found to be associated with up to 170 proteins. Despite the central importance of trans splicing in trypanosomatid gene expression, only the core RNP proteins and a few snRNP-specific proteins are known. To characterize the trypanosome spliceosomal protein repertoire, we conducted a proteomic analysis by tagging and tandem affinity-purifying the canonical core RNP protein SmD1 in Trypanosoma brucei and by identifying copurified proteins by mass spectrometry. The set of 47 identified proteins harbored nearly all spliceosomal snRNP factors characterized in trypanosomes thus far and 21 proteins lacking a specific annotation. A bioinformatic analysis combined with protein pull-down assays and immunofluorescence microscopy identified 10 divergent orthologues of known splicing factors, including the missing U1-specific protein U1A. In addition, a novel U5-specific, and, as we show, an essential splicing factor was identified that shares a short, highly conserved N-terminal domain with the yeast protein Cwc21p and was thus tentatively named U5-Cwc21. Together, these data strongly indicate that most of the identified proteins are components of the spliceosome.

Calmodulin (CaM) is a ubiquitous Ca2+-binding protein known to regulate diverse cellular functions by modulating the activity of various target proteins. We isolated a cDNA encoding AtWRKY7, a novel CaM-binding transcription factor, from an Arabidopsis expression library with horseradish peroxidase-conjugated CaM. CaM binds specifically to the Ca2+-dependent CaM-binding domain (CaMBD) of AtWRKY7, as shown by site-directed mutagenesis, a gel mobility shift assay, a split-ubiquitin assay, and a competition assay using a Ca2+/CaM-dependent enzyme. Furthermore, we show that the CaMBD of AtWRKY7 is a conserved structural motif (C-motif) found in group IId of the WRKY protein family.

Reversible phosphorylation is a key mechanism for the control of intercellular events in eukaryotic cells. In animal cells, Ca2+/CaM-dependent protein phosphorylation and dephosphorylation are implicated in the regulation of a number of cellular processes. However, little is known on the functions of Ca2+/CaM-dependent protein kinases and phosphatases in Ca2+ signaling in plants. From an Arabidopsis expression library, we isolated cDNA encoding a dual specificity protein phosphatase 1, which is capable of hydrolyzing both phosphoserine/threonine and phosphotyrosine residues of the substrates. Using a gel overlay assay, we identified two Ca2+-dependent CaM binding domains (CaMBDI in the N terminus and CaMBDII in the C terminus). Specific binding of CaM to two CaMBD was confirmed by site-directed mutagenesis, a gel mobility shift assay, and a competition assay using a Ca2+/CaM-dependent enzyme. At increasing concentrations of CaM, the biochemical activity of dual specificity protein phosphatase 1 on the p-nitrophenyl phosphate (pNPP) substrate was increased, whereas activity on the phosphotyrosine of myelin basic protein (MBP) was inhibited. Our results collectively indicate that calmodulin differentially regulates the activity of protein phosphatase, dependent on the substrate. Based on these findings, we propose that the Ca2+ signaling pathway is mediated by CaM cross-talks with a protein phosphorylation signal pathway in plants via protein dephosphorylation.

Proliferation arrest and distinct developmental stages alter and decrease general translation yet maintain ongoing translation. The factors that support translation in these conditions remain to be characterized. We investigated an altered translation factor in three cell states considered to have reduced general translation: immature Xenopus laevis oocytes, mouse ES cells, and the transition state of proliferating mammalian cells to quiescence (G0) upon growth-factor deprivation. Our data reveal a transient increase of eukaryotic translation initiation factor 5B (eIF5B), the eukaryotic ortholog of bacterial initiation factor IF2, in these conditions. eIF5B promotes 60S ribosome subunit joining and pre-40S subunit proofreading. eIF5B has also been shown to promote the translation of viral and stress-related mRNAs and can contribute indirectly to supporting or stabilizing initiator methionyl tRNA (tRNA-Met(i)) association with the ribosome. We find that eIF5B is a limiting factor for translation in these three conditions. The increased eIF5B levels lead to increased eIF5B complexes with tRNA-Met(i) upon serum starvation of THP1 mammalian cells. In addition, increased phosphorylation of eukaryotic initiation factor 2α, the translation factor that recruits initiator tRNA-Meti for general translation, is observed in these conditions. Importantly, we find that eIF5B is an antagonist of G0 and G0-like states, as eIF5B depletion reduces maturation of G0-like, immature oocytes and hastens early G0 arrest in serum-starved THP1 cells. Consistently, eIF5B overexpression promotes maturation of G0-like immature oocytes and causes cell death, an alternative to G0, in serum-starved THP1 cells. These data reveal a critical role for a translation factor that regulates specific cell-cycle transition and developmental stages.

Ovalbumin (OVA), one of the members of the serpin-superfamily, is the major protein in chicken eggs. Many studies have demonstrated the polymerization ability of OVA but the detailed molecular mechanisms demonstrating its conformational changes accompanying fibril formation are still unclear. This study revealed nitric oxide (NO) induced conformational changes and oligomerization of egg white OVA, resulting in polymerized fibrils. Electron microscopic analysis showed that NO treatment to OVA under mild acidic condition resulted in morphological changes, producing structures similar to the long protein fibrils found in egg white. Spectroscopic analysis and mass spectrometry found that NO-treated OVA contains increased number of β-sheet, indicating transition from α-helixes to β-sheets, and S-nitrosylation of OVA cysteine residue 367. Structural modeling showed that S-nitrosocysteine, Cys367NO, is located in the amyloidogenic core region of the C-terminal region, nearby the N-terminal core region where the α-to-β transition is induced. Such results provide a potential mechanism for non-disease-associated fibril formation of OVA.

Trypanosoma brucei has a multifunctional RNA polymerase (pol) I that transcribes ribosomal gene units (RRNA) and units encoding its major cell surface proteins variant surface glycoprotein (VSG) and procyclin. Previous analysis of tandem affinity-purified, transcriptionally active RNA pol I identified ten subunits including an apparently trypanosomatid-specific protein termed RPA31. Another ortholog was identified in silico. No orthologs of the yeast subunit doublet RPA43/RPA14 have been identified yet. Instead, a recent report presented evidence that RPB7, the RNA pol II paralog of RPA43, is an RNA pol I subunit and essential for RRNA and VSG transcription in bloodstream form trypanosomes (Penate et al., 2009, EMBO Rep. 10:252–257). Revisiting this attractive hypothesis, we were unable to detect a stable interaction between RPB7 and RNA pol I in either reciprocal co-immunoprecipitation or tandem affinity purification. Furthermore, immunodepletion of RPB7 from extract virtually abolished RNA pol II transcription in vitro but had no effect on RRNA or VSG ES promoter transcription in the same reactions. Accordingly, chromatin immunoprecipitation analysis revealed cross-linking of RPB7 to known RNA pol II transcription units but not to the VSG ES promoter or to the 18S rRNA coding region. Interestingly, RPB7 did crosslink to the RRNA promoter but so did the RNA pol II-specific subunit RPB9 suggesting that RNA pol II is recruited to this promoter. Overall, our data led to the conclusion that RNA pol I transcription in T. brucei does not require the RNA pol II subunit RPB7.

Spliceosomal small nuclear ribonucleoproteins (snRNPs) in trypanosomes contain either the canonical heptameric Sm ring (U1, U5, spliced leader snRNPs), or variant Sm cores with snRNA-specific Sm subunits (U2, U4 snRNPs). Searching for specificity factors, we identified SMN and Gemin2 proteins that are highly divergent from known orthologs. SMN is splicing-essential in trypanosomes and nuclear-localized, suggesting that Sm core assembly in trypanosomes is nuclear. We demonstrate in vitro that SMN is sufficient to confer specificity of canonical Sm core assembly and to discriminate against binding to nonspecific RNA and to U2 and U4 snRNAs. SMN interacts transiently with the SmD3B subcomplex, contacting specifically SmB. SMN remains associated throughout the assembly of the Sm heteroheptamer and dissociates only when a functional Sm site is incorporated. These data establish a novel role of SMN, mediating snRNP specificity in Sm core assembly, and yield new biochemical insight into the mechanism of SMN activity.

Trypanosoma brucei is a member of the early-diverged, protistan family Trypanosomatidae and a lethal parasite causing African Sleeping Sickness in humans. Recent studies revealed that T. brucei harbors extremely divergent orthologues of the general transcription factors TBP, TFIIA, TFIIB and TFIIH and showed that these factors are essential for initiating RNA polymerase II-mediated synthesis of spliced leader (SL) RNA, a trans splicing substrate and key molecule in trypanosome mRNA maturation. In yeast and metazoans, TFIIH is composed of a core of seven conserved subunits and the ternary cyclin-activating kinase (CAK) complex. Conversely, only four TFIIH subunits have been identified in T. brucei. Here, we characterize the first protistan TFIIH which was purified in its transcriptionally active form from T. brucei extracts. The complex consisted of all seven core subunits but lacked the CAK sub-complex; instead it contained two trypanosomatid-specific subunits, which were indispensable for parasite viability and SL RNA gene transcription. These findings were corroborated by comparing the molecular structures of trypanosome and human TFIIH. While the ring-shaped core domain was surprisingly congruent between the two structures, trypanosome TFIIH lacked the knob-like CAK moiety and exhibited extra densities on either side of the ring, presumably due to the specific subunits.

Aurora B kinase is a key component of the chromosomal passenger complex (CPC), which regulates chromosome segregation and cytokinesis. An ortholog of Aurora B was characterized in Trypanosoma brucei (TbAUK1), but other conserved components of the complex have not been found. Here we identified four novel TbAUK1 associated proteins by tandem affinity purification and mass spectrometry. Among these four proteins, TbKIN-A TbKIN-B are novel kinesin homologs, whereas TbCPC1 and TbCPC2 are hypothetical proteins without any sequence similarity to those known CPC components from yeasts and metazoans. RNAI-mediated silencing of each of the four genes led to loss of spindle assemply, chromosome segregation and cytokinesis. TbKIN-A localizes to the mitotic spindle and TbKIN B to the spindle midzone during mitosis, whereas TbCPC1, TbCPC2 and TbAUK1 display the dynamic localization pattern of a CPC. After mitosis, the CPC disappears from the central spindle and re-localizes at a dorsal mid-point of the mother cell, where the anterior tip of the daughter cell is tethered, to start cell division toward the posterior end, indicating a most unusual CPC-initiated cytokinesis in a eukaryote. Copyright: © 2008 Li et al.

Trypanosomatid parasites share a gene expression mode which differs greatly from that of their human and insect hosts. In these unicellular eukaryotes, protein-coding genes are transcribed polycistronically and individual mRNAs are processed from precursors by spliced leader (SL) trans splicing and polyadenylation. In trans splicing, the SL RNA is consumed through a transfer of its 5′-terminal part to the 5′ end of mRNAs. Since all mRNAs are trans spliced, the parasites depend on strong and continuous SL RNA synthesis mediated by RNA polymerase II. As essential factors for SL RNA gene transcription in Trypanosoma brucei , the general transcription factor (GTF) IIB and a complex, consisting of the TATA-binding protein-related protein 4, the small nuclear RNA-activating protein complex, and TFIIA, were recently identified. Although T. brucei TFIIA and TFIIB are extremely divergent to their counterparts in other eukaryotes, their characterization suggested that trypanosomatids do form a class II transcription preinitiation complex at the SL RNA gene promoter and harbor orthologues of other known GTFs. TFIIH is a GTF which functions in transcription initiation, DNA repair, and cell cycle control. Here, we investigated whether a T. brucei TFIIH is important for SL RNA gene transcription and found that silencing the expression of the highly conserved TFIIH subunit XPD in T. brucei affected SL RNA gene synthesis in vivo, and depletion of this protein from extract abolished SL RNA gene transcription in vitro. Since we also identified orthologues of the TFIIH subunits XPB, p52/TFB2, and p44/SSL1 copurifying with TbXPD, we concluded that the parasite harbors a TFIIH which is indispensable for SL RNA gene transcription.

Calmodulin (CaM) regulates diverse cellular functions by modulating the activities of a variety of enzymes and proteins. However, direct modulation of transcription factors by CaM has been poorly understood. In this study, we isolated a putative transcription factor by screening a rice cDNA expression library by using CaM:horse-radish peroxidase as a probe. This factor, which we have designated OsCBT (Oryza sativa CaM-binding transcription factor), has structural features similar to Arabidopsis AtSRs/AtCAMTAs and encodes a 103-kDa protein because it contains a CG-1 homology DNA-binding domain, three ankyrin repeats, a putative transcriptional activation domain, and five putative CaM-binding motifs. By using a gel overlay assay, gel mobility shift assays, and site-directed mutagenesis, we showed that OsCBT has two different types of functional CaM-binding domains, an IQ motif, and a Ca2+-dependent motif. To determine the DNA binding specificity of OsCBT, we employed a random binding site selection method. This analysis showed that OsCBT preferentially binds to the sequence 5′-TWCG(C/T)GTKKKKTKCG-3′ (W and K represent A or C and T or G, respectively). OsCBT was able to bind this sequence and activate β-glucuronidase reporter gene expression driven by a minimal promoter containing tandem repeats of these sequences in Arabidopsis leaf protoplasts. Green fluorescent protein fusions of two putative nuclear localization signals of OsCBT, a bipartite and a SV40 type, were predominantly localized in the nucleus. Most interestingly, the transcriptional activation mediated by OsCBT was inhibited by co-transfection with a CaM gene. Taken together, our results suggest that OsCBT is a transcription activator modulated by CaM.

The Ca2+-binding protein calmodulin mediates cellular Ca2+ signals in response to a wide array of stimuli in higher eukaryotes. Plants express numerous CaM isoforms. Transcription of one soybean (Glycine max) CaM isoform, SCaM-4, is dramatically induced within 30 min of pathogen or NaCl stresses. To characterize the cis-acting element(s) of this gene, we isolated an approximately 2-kb promoter sequence of the gene. Deletion analysis of the promoter revealed that a 130-bp region located between nucleotide positions −858 and −728 is required for the stressors to induce expression of SCaM-4. A hexameric DNA sequence within this region, GAAAAA (GT-1 cis-element), was identified as a core cis-acting element for the induction of the SCaM-4 gene. The GT-1 cis-element interacts with an Arabidopsis GT-1-like transcription factor, AtGT-3b, in vitro and in a yeast selection system. Transcription of AtGT-3b is also rapidly induced within 30 min after pathogen and NaCl treatment. These results suggest that an interaction between a GT-1 cis-element and a GT-1-like transcription factor plays a role in pathogen- and salt-induced SCaM-4 gene expression in both soybean and Arabidopsis.