DNA-directed RNA Polymerase Subunit Research Articles

Core Genome of Deinococcota Phylum from 72 Strains Across 40 Species Consist of Only One Gene, Beta Subunit of DNA-Directed RNA Polymerase

Core Genome of Deinococcota Phylum from 72 Strains Across 40 Species Consist of Only One Gene, Beta Subunit of DNA-Directed RNA Polymerase

Genetic and phenotypical diversity of Pseudomonas syringae population in the Russian Federation.

Proteobacteria comprising species of Pseudomonas syringae group cause diseases of many plants around the world. The phytopathogen has a complex taxonomic structure, which is constantly being revised due to the emergence of new molecular and biochemical diagnostic methods. Here for the first time, we describe the genetic and phenotypic diversity of 57 strains of Pseudomonas syringae isolated from affected soybeans, cereals, sunflowers, and other plants in the Russian Federation from 1950 to 2019. Genetic diversity was assessed by Multi Locus Sequence Analysis (MLSA) using fragments of the genes of glyceraldehyde-3-phosphate dehydrogenase (gapdh), the DNA-directed RNA polymerase subunit D (rpoD), gyrase (topoisomerase) B subunit (gyrB), and citrate synthase I (gltA). The synthesis of syringomycin and coronatine by bacteria was assessed by the reaction of susceptible yeast culture, seedlings of barley, tomato, and sunflower, and by presence of toxin genes confirmed by PCR test. The pathogenicity of the strains was confirmed on seedlings of dicotyledonous and monocotyledonous plants of peas, soybean, sunflowers, barley and wheat, as the most affected crops. The sensitivity of bacteria to 10 antibiotics of the main mechanisms of activity and two bactericidal commercial products was tested by standard disc method. The obtained results showed a high genetic homogeneity of the Russian population of P. syringae, which infects various agricultural crops, and an increase in the proportion of antibiotic-resistant strains over the years.

First Report of Root Rot on Paris polyphylla var. yunnanensis caused by Fusarium solani in Yunnan, China.

Paris polyphylla var. yunnanensis is a perennial herb with diverse chemical components having wide-ranging pharmacological effects. The demand for P. polyphylla var. yunnanensis as a raw material increases greatly and currently exceeds 1,000 tons per year (Zhou et al. 2021). In September 2021, root rot was observed on P. polyphylla var. yunnanensis in Mangshi, Yunnan province, China. Average disease incidences in the fields reached 15%, with diseased plants exhibiting yellowing and wilting leaves, as well as browning and rotting roots. Cross sections (5 × 5 mm2) cut from the margin of symptomatic and asymptomatic root tissues were surface-sterilized for 30 s with 75% ethanol, followed by 180 s with 1% sodium hypochlorite. After rinsing thrice with sterile distilled water, the fragments were transferred to potato dextrose agar (PDA) plates and incubated at 28°C in the dark. Ten isolates were obtained, and single spore isolation was performed. These isolates showed similar morphological characters, with colonies ranging in color from white to pale cream and sparse mycelia. Conidia were produced on the top or side of phialides. Microconidia were oval or reniform, 0- or 1-septate, with a diameter of 5.1-10.7 µm × 1.6-3.9 µm (average 7.6 μm × 2.8 µm) (n=30). The macroconidia were straight to slightly curved or sickle-shaped, 3- to 5-septate, with a diameter of 15.1-27.9 μm × 2.8-4.0 μm (average 21.0 μm × 3.6 μm). Chlamydospores were smooth, nearly round, and 3.3-6.6 (average 4.9) μm in diameter. Genomic DNA were extracted from mycelia of the two isolates. The nuclear ribosomal internal transcribed spacer (ITS), translation elongation factor 1 alpha (EF1α), and the second largest subunit of nuclear DNA-directed RNA polymerase II (RPB2) were amplified with the primer pairs of ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone and Kohn 1999), and fRPB2-5F/fRPB2-7cR (Liu et al. 1999), respectively. These two isolates exhibited the same nucleotide sequences (ITS, OP646781; EF1α, OP661172; RPB2, OP661173), with BLASTn analyses showing 100%, 99.66%, and 99.65% identity, respectively, with Fusarium solani (syn. Neocosmospora solani) (Crespo et al. 2019) strain NRRL 43474 (ITS, EF453097; EF1α, EF452945; RPB2, EF469984). A phylogenetic tree was constructed using MEGAX based on the nucleotide sequences of ITS, EF1α, and RPB2, using the maximum likelihood method. The isolate was classified into the F. solani clade. According to the morphology and sequence analyses, the isolate was identified as F. solani (Chehri et al. 2015), and named PpFs1. To test the pathogenicity of the isolate PpFs1, the roots of four years old P. polyphylla var. yunnanensis plants were dipped in 107 spore/mL suspension filtered from potato dextrose broth (PDB) for 30 min, while control roots were dipped in sterile water. After inoculation, all plants were transplanted in pots filled with sterile soil and kept at 25°C with a 12/12-h light/darkness photoperiod. Six plants were used for each treatment, and repeated thrice. Two months after inoculation, the infected plants showed wilted leaves and rotted roots, while controls remained asymptomatic. PpFs1, identified by morphology and ITS, was re-isolated from infected plants, and was found to comply with Koch's postulates. To the best of our knowledge, F. oxysporum and F. concentricum causes Paris polyphylla var. Chinensis stem rot in China. But this is the first report of root rot on P. polyphylla var. yunnanensis being caused by F. solani in Yunnan, China.

First Report of Basal Rot of Onion Caused by Fusarium oxysporum f. sp. cepae in Saudi Arabia.

Onion (Allium cepa L.) is a globally important crop worldwide including Saudi Arabia. In November 2020, 2-month-old onion plants (cv. Redwing) in commercial fields within the Sajir area of Riyadh region (∼ 1.4 ha), showed symptoms of yellowing, wilting, stunting, bulb discoloration, rot in the basal parts of bulb and decrease in roots. In the advanced stages, the affected plants collapsed and died. The incidence of symptomatic plants ranged from 30 to 65% in the surveyed fields. To isolate the pathogen, symptomatic onion plants (n =20) were sampled. Diseased tissues from roots and bulbs were cut into small pieces (4 × 4 mm), sanitized with 1% sodium hypochlorite solution for 2 min, submerged in 70% alcohol for 20 s, then rinsed with sterile water, before plating on potato dextrose agar (PDA) medium. The plates were incubated at 25°C for 6 days. Subcultures of the mycelia grown out of the diseased tissues produced purplish pink fungal colonies on PDA. On carnation leaf agar, cultures were characteristic of Fusarium oxysporum as described by Leslie and Summerell (2006), with the presence of unicellular microconidia (3.8 to 7.8 × 1.7 to 2.5 μm, n= 50) without septa in false heads or short monophialides and slightly curved macroconidia (16.3 to 28 × 4.2 to 6.1 μm, n= 50) with two to four septa. Older mycelia developed many chlamydospores that were single or in short chains. To further confirm the pathogen identification, DNA was extracted from single-spore cultures of three representative isolates using the DNeasy Plant Mini kit (QIAGEN, Hilden, Germany). Three different fungal nuclear regions of internal transcribed spacer (ITS), elongation factor 1-α, (TEF1-α) and the second largest subunit of DNA-directed RNA polymerase II (RPB2) DNA were amplified by PCR and sequenced with the following primers: ITS4 and ITS5 (White et al. 1990); EF-1 and EF-2 (O'Donnell et al. 2008); and fRPB2-5F and fRPB2-7cR (Liu et al. 1999), respectively. Phylogenetic analysis based on the alignment of the ITS, TEF1-α, and RPB2 sequences using MEGA7 placed these isolates in the F. oxysporum clade. The ITS, TEF1-α, and RPB2 sequences of an isolate FOC-OR9 were submitted to GenBank (OL721757, OL764494, and OL764495 respectively). To confirm the forma specialis cepae, a fragment of the F. oxysporum f. sp. cepae gene Secreted In Xylem 3 (SIX3) was amplified by PCR (Kalman et al. 2020). The SIX3 amplicon (∼ 277-bp) was sent for sequencing, and the sequence was submitted to GenBank (OL828265). BLASTn analysis of the sequences showed 100% identity with F. oxysporum f. sp. cepae (KP746408). To fulfill Koch's postulates, pathogenicity tests were performed with healthy onion bulbs cv. "Redwing" of 100-150 g each. Prior to inoculation of onion bulbs, the dry bulb scales, one of the fleshy inner scales, as well as the roots were removed. Bulbs were then surface sterilized (as described above) and injected with 20 µl of a conidial suspension (106 spores/ ml) into the basal plate of each bulb and approximately 1 cm deep into the tissue. Six bulbs were inoculated for each isolate, placed in a mesh bag, and incubated at 28 °C in the dark. Six bulbs injected with sterile water and six non-inoculated bulbs served as controls. At the 4th week post inoculation, necrotic rot symptoms and brown discoloration were observed on the basal plates of these inoculated bulbs (similar symptoms to those observed in the field), while control treatments showed no symptoms. The pathogen was re-isolated from the basal plates onto PDA and identified morphologically and molecularly as F. oxysporum f. sp. cepae, thus fulfilling Koch's postulates. The test was repeated twice. This pathogen was previously reported causing onion basal rot in United Kingdom (Taylor, et al., 2013). To our knowledge, this is the first report of basal rot in onion caused by F. oxysporum f. sp. cepae in Saudi Arabia. It is recommended that preventive management should be considered as this disease may cause significant economic losses for onion growers in Saudi Arabia. Also, Fusarium mycotoxin contamination of onion bulb could pose a public health risk.

Isolation and evaluation of Bacillus subtilis RSS-1 as a potential biocontrol agent against Sclerotinia sclerotiorum on oilseed rape

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is one of the most important diseases of oilseed rape in the world. Because of the absence of resistant varieties and the disadvantages of chemical control, application of antifungal microbes has become an eco-friendly and effective measure to control this disease. In this study, Bacillus subtilis strain RSS-1, isolated from soil samples, was identified based on morphological, physiological and biochemical tests, and DNA gyrase subunit A (gyrA), gyrB, DNA-directed RNA polymerase subunit beta (rpoB) and rpoC gene sequence analysis. It significantly inhibited mycelial growth and sclerotial production of S. sclerotiorum in vitro. In greenhouse experiments, all three tested concentrations (106, 107, 108 cfu mL−1) of cell fermentation broth and culture filtrate significantly reduced the severity of sclerotinia stem rot on oilseed rape (P < 0.05). RSS-1 was more effective at reducing disease severity when applied 24 h before inoculation with S. sclerotiorum than at 24 h post inoculation, suggesting that RSS-1 should be applied as a prophylactic rather than a curative biological agent. Colonization tests indicated that the population density of RSS-1 on rapeseed leaves significantly decreased (P < 0.05) over 6 days. However, RSS-1 could stably colonize in rhizospheric soil of rapeseed over 30 days. Challenge inoculation tests showed that RSS-1 significantly inhibited the activities of polygalacturonase and cellulase and accumulation of oxalic acid during the S. sclerotiorum infection. These results suggest that RSS-1 was a potential biological agent for controlling sclerotinia stem rot caused by S. sclerotiorum on oilseed rape.

Mycobacterium senriense sp. nov., a slowly growing, non-scotochromogenic species, isolated from sputum of an elderly man.

A slowly growing mycobacteria, identified as strain TY59T, was isolated from sputum of an elderly man with pneumonia. Sequencing of the 16S rRNA gene indicated that this strain was similar to members of the Mycobacterium avium complex and closely related species. Strain TY59T has highest 16S rRNA gene sequence similarities to the type strains of Mycobacterium colombiense (99.80 % sequence similarity), Mycobacterium vulneris (99.74 %), Mycobacterium timonense (99.54 %), Mycobacterium avium subsp. avium (99.54 %) and Mycobacterium avium subsp. silvaticum (99.54 %). Analysis of the internal transcribed spacer (ITS) and DNA-directed RNA polymerase subunit beta (rpoB) sequences gave similar results to the 16S rRNA gene analysis. The closest species to strain TY59T were M. colombiense and M. vulneris with 97.90-98.25 % identity in ITS and 96.4-96.6 % in rpoB. The strain's 65 kDa heat shock protein (hsp65) gene was different from those of M. vulneris, M. colombiense and M. avium subsp. silvaticum with 72.4-74.2 % identity. Average nucleotide identity results showed a 93.4 % match to M. vulneris as the maximum value. Phenotypically, the non-chromogenicity, rough colonies, growth at 42 °C, negative results for nitrate reduction, β-glucosidase and Tween 80 hydrolysis, and positive results for catalase activity set this strain apart from closely related species. We propose that Mycobacterium senriense sp. nov. is a novel species of slowly growing mycobacteria. The type strain is TY59T (RIMD 1371001T=CIP 111917T).

Mycobacterium tuberculosis Affects Protein and Lipid Content of Circulating Exosomes in Infected Patients Depending on Tuberculosis Disease State.

Tuberculosis (TB), which is caused by the bacterium Mycobacterium tuberculosis (Mtb), is still one of the deadliest infectious diseases. Understanding how the host and pathogen interact in active TB will have a significant impact on global TB control efforts. Exosomes are increasingly recognized as a means of cell-to-cell contact and exchange of soluble mediators. In the case of TB, exosomes are released from the bacillus and infected cells. In the present study, a comprehensive lipidomics and proteomics analysis of size exclusion chromatography-isolated plasma-derived exosomes from patients with TB lymphadenitis (TBL) and treated as well as untreated pulmonary TB (PTB) was performed to elucidate the possibility to utilize exosomes in diagnostics and knowledge building. According to our findings, exosome-derived lipids and proteins originate from both the host and Mtb in the plasma of active TB patients. Exosomes from all patients are mostly composed of sphingomyelins (SM), phosphatidylcholines, phosphatidylinositols, free fatty acids, triacylglycerols (TAG), and cholesterylesters. Relative proportions of, e.g., SMs and TAGs, vary depending on the disease or treatment state and could be linked to Mtb pathogenesis and dormancy. We identified three proteins of Mtb origin: DNA-directed RNA polymerase subunit beta (RpoC), Diacyglycerol O-acyltransferase (Rv2285), and Formate hydrogenase (HycE), the latter of which was discovered to be differently expressed in TBL patients. Furthermore, we discovered that Mtb infection alters the host protein composition of circulating exosomes, significantly affecting a total of 37 proteins. All TB patients had low levels of apolipoproteins, as well as the antibacterial proteins cathelicidin, Scavenger Receptor Cysteine Rich Family Member (SSC5D), and Ficolin 3 (FCN3). When compared to healthy controls, the protein profiles of PTB and TBL were substantially linked, with 14 proteins being co-regulated. However, adhesion proteins (integrins, Intercellular adhesion molecule 2 (ICAM2), CD151, Proteoglycan 4 (PRG4)) were shown to be more prevalent in PTB patients, while immunoglobulins, Complement component 1r (C1R), and Glutamate receptor-interacting protein 1 (GRIP1) were found to be more abundant in TBL patients, respectively. This study could confirm findings from previous reports and uncover novel molecular profiles not previously in focus of TB research. However, we applied a minimally invasive sampling and analysis of circulating exosomes in TB patients. Based on the findings given here, future studies into host–pathogen interactions could pave the way for the development of new vaccines and therapies.

Proteome wide screening for identification of putative drug target gene in Nautella italica and structure-based ligand screening for therapeutic candidates

In silico based subtractive genomic approaches were employed to identify the key drug targets for an opportunistic pathogen Nautella italica, a member of the marine Roseobacter clade that causes bleaching disease in the temperate-marine red macro algae, Delisea pulchra. The aim of this study is to propose new active ligands against bleaching disease seen in algae. Using comparative and subtractive genomic approach, a set of 21 proteins were identified as the therapeutic drug target proteins for algal bleaching. This core set of drug targets has been analyzed for network topology using string network analysis and major hub gene identified by CytoHubba was rpoB (DNA directed RNA Polymerase subunit beta). The three-dimensional structure of rpoB was built by comparative modelling and used to perform a virtual screening of Zinc database by DOCK Blaster server. The 50 top scored compounds were screened for toxicity analysis by OSIRIS Data Warrior and ECOSAR tool. Further refinement by autodock program revealed two compounds ZINC49821385 and ZINC97218938 with the best binding energy of -7.07 and -6.79 respectively. These results indicated that 5-(4- isopropylphenyl)furan-2-carboxamide (ZINC ID 49821385) could be one of the potential ligand to treat bleaching disease in algae.

First Report of Fusarium Root and Stem Rot Caused by Fusarium oxysporum f. sp. radicis-cucumerinum on Greenhouse Cucumbers in Saudi Arabia.

Cucumber (Cucumis sativus L.) is an important vegetable crop in Saudi Arabia. During May 2018, 45 - 60% of 5-month-old cucumber plants showed symptoms of a previously unknown wilt in commercial greenhouses around Al Kharj area of Riyadh region. Symptoms consisted of crown and root rot, wilting and stem disintegration, along with yellowish brown to brown external discoloration extended throughout the affected tissues. As the disease progressed, a pinkish-orange mycelial growth was often observed at the basis of affected stems while vessels were discolored. Subsequently, the affected plants were collapsed and died. Crown, stem, and root fragments (4 × 4 mm) were cut from symptomatic tissues, surface sterilized in 2.5% NaOCl, cultured on potato dextrose agar (PDA) with 25 mg/liter of streptomycin sulfate, and incubated at 26°C in darkness for 6 days. Single-spored cultures produced white mycelium with pink, white, or purple pigmentation in the center. The mycelium produced sporodochia. Macroconidia were mainly slightly curved with three to five septa. Microconidia were single-celled oval and produced on short lateral phialides. Chlamydospores were single or in short chains. Morphologically, the isolated fungus was characterized as Fusarium oxysporum (Leslie and Summerell 2006). To further confirm the fungus identification, DNA was extracted from a single-spored culture. Three different fungal nuclear regions of internal transcribed spacer (ITS), elongation factor 1-α, (TEF1-α) and the second largest subunit of DNA-directed RNA polymerase II (rpb2) with the following primers: ITS4 and ITS5 (White et al. 2017), EF-1 and EF-2 (O'Donnell et al. 2008), and fRPB2-5F and fRPB2-7cR (Liu et al. 1999), respectively. The ITS, TEF1-α, and rpb2 sequences of the isolate FCKSU17 were submitted to GenBank (MT232918, MW471131, and MW449833 respectively). Phylogenetic analysis based on the alignment of the ITS, TEF1-α, and rpb2 sequences using MEGA7 placed this strain in the F. oxysporum clade. To confirm the forma specialis radicis-cucumerinum, amplification with the specific primers ForcF1/ForcR2 was conducted (Lievens et al. 2007). The amplified fragment (∼ 250-bp) was sent for sequencing, and the sequence was submitted to GenBank (MW471132). BLASTn analysis of the sequences showed 100% identity with F. oxysporum radicis-cucumerinum (KP746408). To fulfill Koch's postulates, pathogenicity test was conducted on 7-day-old plants of cucumber cultivar Beit Alpha grown into pots filled with soil mix (2:1 sandy loam-peat moss, vol/vol). The plants were inoculated through drenching with 100 ml of conidial suspension in sterile distilled water (106 spores/ml) per pot. Control plants were treated with sterile distilled water. Each treatment included 10 replicates (pots), with two plants per pot. The pathogenicity test was repeated once. Cucumber plants inoculated with the fungus showed early wilting symptoms within the first 2 weeks post inoculation. At the 6th week post inoculation, 90 to 100% of the inoculated plants developed typical symptoms. No symptoms were observed on the control plants. The pathogen was successfully re-isolated from the inoculated wilted plants and identified morphologically. To our knowledge, this is the first report of F. oxysporum f.sp. radicis-cucumerinum on cucumber in Saudi Arabia. It is recommended that preventive management should be considered as this disease may cause significant economic losses on cucumbers in Saudi Arabia.

Developing species-specific quantitative real-time polymerase chain reaction primers for detecting Lautropia mirabilis

This study aimed to develop Lautropia mirabilis -specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis . The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/ RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

Long-term exposure of Daphnia magna to polystyrene microplastic (PS-MP) leads to alterations of the proteome, morphology and life-history

In the past years, the research focus on the effects of MP on aquatic organisms extended from marine systems towards freshwater systems. An important freshwater model organism in the MP field is the cladoceran Daphnia, which plays a central role in lacustrine ecosystems and has been established as a test organism in ecotoxicology. To investigate the effects of MP on Daphnia magna, we performed a chronic exposure experiment with polystyrene MP under strictly standardized conditions. Chronic exposure of D. magna to PS microparticles led to a significant reduction in body length and number of offspring. To shed light on underlying molecular mechanisms induced by microplastic ingestion in D. magna, we assessed the effects of PS-MP at the proteomic level, as proteins, e.g., enzymes, are especially relevant for an organism's physiology. Using a state-of-the-art mass spectrometry based approach, we were able to identify 28,696 different peptides, which could be assigned to 3784 different proteins. Using a customized bioinformatic workflow, we identified 41 proteins significantly altered in abundance (q-value <0.05) in the PS exposed D. magna. Among the proteins increased in the PS treated group were several sulfotransferases, involved in basic biochemical pathways, as well as GABA transaminase catalyzing the degradation of the neurotransmitter GABA. In the abundance decreased group, we found essential proteins such as the DNA-directed RNA polymerase subunit and other proteins connected to biotic and inorganic stress and reproduction. Strikingly, we further identified several digestive enzymes that are significantly downregulated in the PS treated animals, which could have interfered with the affected animal's nutrient supply. This may explain the altered morphological and life history traits of the PS exposed daphnids. Our results indicate that long-term exposure to PS microplastics, which are frequently detected in environmental samples, may affect the fitness of daphnids.

Genetic characterization, mechanisms and dissemination risk of antibiotic resistance of multidrug-resistant Rothia nasimurium

Rothia nasimurium is part of the commensal flora of humans and other animals and has recently received increasing attention for its multidrug-resistance (MDR) and pathogenicity. Currently, no systematic reports characterize the genetics, mechanisms, and dissemination risks of antibiotic resistance in MDR R. nasimurium. Here, we present the first report outlining a MDR strain of R. nasimurium, E1706032a, isolated from ducks exhibiting clinical sickness. Phylogenetic analysis indicates that E1706032a mostly likely originated in the commensal bacteria of Amazona aestiva in Florida. E1706032a is resistant to beta-lactams, aminoglycosides, macrolides, sulfonamides, fluoroquinolones, rifamycins, tetracyclines, lincosamides and chloramphenicol. Genetic sequences related to drug resistance were detected, including resistance genes (aac(6')-Ib, ant(3″)-Ia, sul1, dfrA7, erm(X)), efflux pumps (tetZ, qacEΔ1, cmx, phosphate ABC transporter ATP-binding protein), and resistance-related proteins (hydrolase of the metallo-beta-lactamase (MBLs), mycinamicin resistance protein (myrA), DNA-directed RNA polymerase subunit beta (rpoB) variants, etc). E1706032a carries an IS481-like element, IS5564 and IS6-like elements, and IS6100 along with several novel transposases of the IS3 family. E1706032a also carries the class 1 integron gene IntI1, which is downstream adjacent to the gene cassettes aac(6')-Ib, tetZ, dfrA27, ant(3″)-Ia, qacEΔ1, sul1, cmx and upstream adjacent to gene tnpA of IS6100. Genetic analysis suggests that E1706032a carries wide antibiotic resistance and dissemination potential through movable elements and thus has the potential to cause difficult-to-treat infections in animals and humans. The dissemination of E1706032a from parrots in Florida to ducks in eastern China indicates a cross-regional public health infection risk that should be evaluated for risk of global spreading.

Proteomics and transcriptomic analysis of Micrococcus luteus strain AS2 under arsenite stress and its potential role in arsenic removal.

The proteomics and transcriptomic analysis of Micrococcus luteus strain AS2 was carried out through 2D gel electrophoresis and RT-PCR. Seven protein spots were selected randomly from the gel and identified through mass spectrometry. Four proteins including putative metal-dependent hydrolase TatD, thioredoxin reductase, DNA-directed RNA polymerase subunit alpha and chaperone protein DnaK were upregulated while superoxide dismutase [Mn], 3-oxoacyl-[acyl-carrier-protein] reductase FabG, and putative alkyl/aryl-sulfatase YjcS were down-regulated under arsenite stress. No significant difference was observed in aioB gene expression analysis in the presence and absence of arsenite. The optimum arsenite processing ability was determined at 37°C (90%) and at pH 7 (92%). The maximum metal processing ability was determined at 250mM arsenite/L (90%) while the minimum was estimated at 1250mM arsenite/L (42%). The maximum arsenite removal ability of strain AS2 determined after 8 days was 68 and 82% from wastewater and distilled water, and the organism can be a good bioresource for green chemistry to eradicate environmental arsenite.

Description of Allocanariomyces and Parachaetomium, two new genera, and Achaetomium aegilopis sp. nov. in the Chaetomiaceae

We describe Allocanariomyces tritici gen et sp. nov. and Achaetomium aegilopis sp. nov. as seed endophytes of Triticum boeoticum and Aegilops triuncialis, respectively, in the west and northwestern Iran using morphological traits and sequences of the internal transcribed spacer regions 1 and 2 including the intervening 5.8S nuclear ribosomal DNA (ITS), partial nuclear 28S ribosomal DNA (LSU), β-tubulin (TUB2), and the second-largest subunit of DNA-directed RNA polymerase II (RPB2) gene. Chaetomium carinthiacum, C. iranianum, and C. truncatulum are also combined here under the new genus, Parachaetomium. Allocanariomyces is differentiated from Canariomyces, its closest relative, by having solitary and glabrous ascomata, cells of the perithecial wall forming a textura epidermoidea, stalked asci, densely granular ascospores with a distinct subapical germ pore, and producing only solitary conidia. Parachaetomium is characterized by distinctly ostiolate ascomata and equi- or inequilaterally fusiform, typically less than 13-μm-long ascospores with an oblique or subapical germ pore. Achaetomium aegilopis is mainly distinguished from A. strumarium, its closest relative, by possessing brown, ascomata, hyaline ascomatal hairs covered with hyaline crystals, hyaline chlamydospores, and lacking an anamorph.

First report of Fusarium proliferatum causing necrotic leaf lesions and bulb rot on storage onion (Allium cepa) in southwestern Idaho.

In September 2014, a high rate of bulb rot (5-15% depending on producer) was reported across all cultivars developing early in the storage season in the onion producing region of southwestern Idaho. Spanish yellow onion bulbs cv. Vaquero displaying tan to light brown necrotic rot were obtained. The bulb rot originated in the neck and spread to successive scales (Figure 1). In August 2015, onion cv. Redwing and Vaquero were observed to have wet necrotic lesions developing on leaves in the field (Figure 2). Margins of necrotic tissue, 1-2 cm3, were excised, surface sterilized, plated on water agar medium and incubated at 24°C. Hyphal growth was sub-cultured from eight strains (A- D in 2014; E-H in 2015) to fresh potato dextrose agar to obtain pure cultures. Cultures were characteristic of Fusarium species as described by Nelson et al. (1983) with the presence of microconidia formed on polyphialides with macroconidia present. Primers ITS4-A1 and ITS5 primers (White et al. 1990); EF-1 and EF-2 (O'Donnell et al. 1998); and fRPB2-5F and fRPB2-7cR (Liu et al. 1999) were used to amplify regions of the ITS, elongation factor 1-α and the second largest subunit of DNA-directed RNA polymerase II. Amplicons were sequenced and analyzed using BLAST (https://www.ncbi.nlm.nih.gov/) and in combination using Pairwise DNA Alignment and Polyphasic Identification (http://www.westerdijkinstitute.nl/Fusarium/DefaultInfo.aspx?Page=Home) as described by O'Donnell et al. 2015. Analysis indicated that these strains are Fusarium proliferatum, which is part of the F. fujikuroi species complex (O'Donnell et al. 1998). Similarity (99.5%) was observed in pairwise analyses and the polyphasic identification clustering to representative F. proliferatum strain NRRL 22944 and others. Sequences were submitted to Genbank and registered accession numbers are found in Table 1. To complete Koch's postulates, cv. Vaquero onion bulbs were surface sterilized and injected with 3 × 105 microconidia into the shoulder of each bulb. Five bulbs were inoculated for each isolate, placed in a mesh bag, and incubated at 30°C in the dark. Five bulbs injected with sterile water and five non-inoculated bulbs served as controls. After 14 days, each bulb was sliced vertically down the center and inspected for rot. All eight strains induced tan to light brown necrotic rot symptoms in each inoculated bulb. No symptoms were observed for the water inoculated and the non-inoculated onion bulbs. A fungus was isolated from the necrotic tissue and confirmed to be F. proliferatum as described above. Ten µl aliquots containing 1 × 105 microconidia of F. proliferatum strains (C, E-H) were applied to leaves in triplicate of 12-week-old onion plants (cv. Vaquero) wounded with a 21-gauge needle. Water controls were included. Within three days lesions, with light chlorosis, began to form and quickly spread on the leaves. A fungus was isolated and confirmed to be F. proliferatum as described above. This is the first extensive description and identification of F. proliferatum causing bulb rot in storage in Idaho (Mohan et al. 1997). In addition, this is the first report of the fungus causing leaf infection in the field. These findings confirm F. proliferatum as the causal agent of the high incidence of bulb rot observed in 2014 and 2015. This bulb rot continues to occur in southwestern Idaho and since the pathogen can cause leaf infections growers are encouraged to be vigilant for both leaf lesions during the growing season and bulb rot in storage.

In-Silico Proteomic Exploratory Quest: Crafting T-Cell Epitope Vaccine Against Whipple's Disease.

Whipple’s disease is one of the rare maladies in terms of spread but very fatal one as it is linked with many disorders (like Gastroenteritis, Endocarditis etc.). Also, current regimens include less effective drugs which require long duration follows up. This exploratory study was conducted to commence the investigation for crafting multi target epitope vaccine against its bacterial pathogen Tropheryma whipplei. The modern bioinformatics tools like VaxiJen, NETMHCII PAN 3.2, ALLERGEN-FP, PATCH-DOCK, TOXIC-PRED, MHCPRED and IEDB were deployed, which makes the study more intensive in analyzing proteome of T. whipplei as these methods are based on robust result generating statistical algorithms ANN, HMM, and ML. This Immuno-Informatics approach leads us in the prediction of two epitopes: VLMVSAFPL and IRYLAALHL interacting with 4 and 6 HLA DRB1 alleles of MHC Class II respectively. VLMVSAFPL epitope is a part of DNA-directed RNA polymerase subunit beta, and IRYLAALHL epitope is a part of membranous protein insertase YidC of this bacterium. Molecular-Docking and Molecular-Simulation analysis yields the perfect interaction based on Atomic contact energy, binding scores along with RMSD values (0 to 1.5 Ǻ) in selection zone. The IEDB (Immune epitope database) population coverage analysis exhibits satisfactory relevance with respect to world population.

A New Record of Epicoccum draconis Isolated from the Soil in Korea

A fungal isolate US-18-11 was isolated from the soil in Uiseong, Korea. The mycelium growth measured after 7 days of incubation at 22℃ on malt extract agar (MEA) and oatmeal agar (OA) media was 42-43 mm and 41-44 mm in diameter, respectively. The fungal colony formed white to dull green aerial mycelia that were floccose with regular margins and olivaceous black with leaden gray patches on the reverse side. The conidia were hyaline to brown in color, ellipsoidal to ovoid, guttulate, abundant, globose, solitary, or confluent measuring 3.2-7.2×1.1-2.3 μm. A BLAST search of the large subunit (LSU), internal transcribed spacer (ITS) region, second largest subunit of DNA-directed RNA polymerase II (RPB2) and β-tubulin (TUB2) gene sequences revealed that the isolate US-18-11 has similarities of 99, 100, 97, and 99% with those of Epicoccum draconis CBS 186.83, respectively. A neighbor-joining phylogenetic tree constructed based on the concatenated dataset of above-mentioned sequences showed that isolate US-18-11 clustered with Epicoccum draconis CBS 186.83 in the same clade. Based on the results of morphological, cultural, and phylogenetic analysis, the isolate US-18-11 was identical to the previously described E. draconis CBS 186.83. To our knowledge, this is the first report of E. draconis in Korea.

Transcriptome analysis of the thermosensitive genic male-sterile line provides new insights into fertility alteration in rice (Oryza sativa)

Thermosensitive genic male-sterile (TGMS) lines have been widely used for rice breeding to produce hybrid rice. Here, RNA-Seq was performed to investigate the expression profiles of young panicle from rice TGMS line at the microspore mother cell and meiosis stages under sterile and fertile conditions. A total of 1070 differentially expressed genes (DEGs) were identified, which were enriched in protein folding, protein binding, regulation of transcription, transcription factor activity and metabolic related processes. Further analyses showed hub genes UbL40s were predicted to interact with proteolysis-related genes and DNA-directed RNA polymerase subunit, and HSPs interacted with kinases to play important roles in regulating fertility alteration. It suggested that besides UbL40s, DNA-directed RNA polymerase subunit, kinases and HSPs might be involved in fertility alteration in rice TGMS lines. These findings provided new insights into fertility alteration in rice TGMS line, which will benefit for further application of TGMS in rice breeding.

Homology modeling and in silico toxicity assessment of potential inhibitors of cytidylate kinase from Mycobacterium tuberculosis

Cytidylate kinase (CMK) plays a central role in the phosphorylation of ATP to nucleoside diphosphates in the purine and pyrimidine pathway in Mycobacterium tuberculosis (MTB). The enzyme is specific to bacteria, fungi, and plants and it shares the same pathway with a DNA-directed RNA polymerase subunit beta, the known target of rifampicin (RIF). Therefore, it serves as an attractive alternative target to inhibit the purine and pyrimidine pathway in MTB. In this study, the novel inhibitors of CMK were identified using in silico approach. Homology modeling was used to build the three-dimensional (3D) structure of CMK using program implemented in Modeller 9.16 and based on the template (3R20) obtained from Mycobacterium smegmatis. Structural analysis of CMK showed that it has P-loop or core in the ATP binding domain, nucleoside monophosphate binding (NMP) site, and a LID domain. Inhibition of any of the residues that formed the nucleoside binding site or ATP binding site blocks the catalytic activity of the CMK. Eleven thousand four hundred and fifty-four compounds that had an affinity to bind to the CMK with minimum binding energies were obtained through virtual screening from Zinc and PubChem databases using PyRx 8.0. These compounds were further filtered for Lipinski rule of five, molecular docking analysis, and ADMET properties (absorption distribution, metabolism, excretion, and toxicity). Four compounds (ZINC95485891 = − 8.80 kcal/mol, ZINC95485880 = − 8.44 kcal/mol, ZINC95485900 = − 8.24 kcal/mol, and ZINC95486231 = − 8.20 kcal/mol) with good binding energies and desirable pharmacokinetic properties were selected. These compounds were further subjected to molecular dynamic (MD) simulation and molecular mechanics generalized Born and surface area (MM-GBSA) analyses. The results of the analyses showed that all the four ligands formed a stable complex and had good free binding energy after 50 ns MD simulation. Therefore, these compounds were considered as possible inhibitors of MTB after experimental validation.

Interactions of protein translocation machinery components of Streptococcus mutans

BackgroundPivotal virulence properties of the cariogenic bacterium Streptococcus mutans depend on secreted and integral membrane proteins. Bacterial protein trafficking involves the signal recognition particle (SRP) pathway components Ffh, scRNA and FtsY, the SecYEG translocon, the SecA motor protein, and membrane‐localized YidC chaperone/insertases. Unlike Escherichia coli, S. mutans survives loss of the SRP pathway. In addition, S. mutans has two yidC paralogs. The phenotype of a DyidC2 mutant largely parallels that of Dffh, while that of DyidC1 is far less severe. Also in S. mutans, YlxM acts as an accessory factor of the SRP that modulates GTP hydrolysis activities of Ffh and FtsY. The goal of this study is to identify interactions among S. mutans transport machinery components to better understand the respective functions of YidC1 and YidC2 alone and in concert with the SRP, ribosome, and/or Sec translocon that can affect the destiny of translocated proteins.MethodsA chemical cross‐linking approach was employed, whereby whole cell lysates (wcl) were treated with formaldehyde followed by Western blotting using anti‐Ffh, FtsY, YidC1 and YidC2 antibodies and mass spectrometry (MS) analysis of corresponding gel‐shifted bands. Wcl from WT and DyidC2 strains were also reacted with a‐YidC2 antibodies coupled to magnetic Dynabeads™ and co‐captured proteins identified by MS. In addition, biolayer interferometry (BLI) was used to evaluate specific interactions of the C‐terminal tails of YidC1 and YidC2, engineered as glutathione‐S‐transferase fusion proteins (GST‐C1 and GST‐C2), with the SRP components Ffh and YlxM. The same GST‐constructs were also subjected to 2D Difference Gel Electrophoresis (DIGE), in which each was reacted with wcl, captured with glutathione affinity resin, and labelled individually with fluorescent dyes. Spots unique to the GST‐C1 and/or GST‐C2 samples were analysed by MS.Results and ConclusionBand‐shift patterns detected with anti‐Ffh, FtsY and YidC2 antisera were similar to one another and distinct from that of anti‐YidC1. Cross‐linked proteins included elongation factor Tu, DNA‐directed RNA polymerase subunit b, and HSP 70. Immunocaptured proteins from anti‐YidC2 Dynabeads™ included two small, transmembrane proteins, LemA and SMU_1719c, which may represent translocation substrates. Both GST‐C1 and GST‐C2 interacted with Ffh by BLI with binding influenced by YlxM. Over one hundred spots were identified by DIGE. Among differentially identified proteins in the GST‐C1 sample were DnaK, RNA polymerase subunit d, and cell division protein FtsH. Ribosomal proteins S7 and L13 were prevalent in spots of the GST‐C2 sample, whereas ribosomal protein L2, DNA polymerase III g/t subunit, and the Cas5 homolog were all more abundant in spots from GST‐C1 and GST‐C2 samples compared to GST. This information suggests a coupled cellular machinery that integrates transcription and translation with protein transport and folding.Support or Funding InformationNIH R01DE008007This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.