NCBI Reference Sequence Research Articles

Genomic analysis of Enterobacter cloacae complex from Southern Thailand reveals insights into multidrug resistance genotypes and genetic diversity

In this study, we conducted a comprehensive investigation into the Enterobacter cloacae complex (ECC), a group of notorious pathogens responsible for various hospital-acquired infections. We aimed to gain critical insights into antimicrobial resistance profiles and genomic diversity among 17 ECC isolates, which were previously collected as part of a short-term surveillance effort for 6 months in 2019. We identified two novel sequence types (ST-1936 in E. bugandensis PSU30 and ST-1937 in E. roggenkampii PSU45) among the 14 distinct STs identified in our ECC isolates. Furthermore, our expanded investigation revealed 296 novel STs within the NCBI Reference Sequence database. We identified six isolates carrying the mcr-9 gene, highlighting a significant concern in antimicrobial resistance (AMR). These genes confer a reduced susceptibility to colistin, a critical last-resort drug for the treatment of multidrug-resistant (MDR) infection. In addition to the AMR complexity, we found that three isolates carried the blaNDM gene on IncN2 plasmids, further emphasizing the urgency of monitoring and managing ECC-related infections. Our study provided evidence of intra-hospital transmission involving E. asburiae isolates PSU37, PSU39, and PSU40, all collected from the nasopharynx of three individuals in the intensive care unit (ICU) of the same hospital. These findings highlight the need for stringent infection control measures to prevent similar outbreaks and emphasize the importance of effective surveillance and management strategies to address ECC-related challenges.

Highly efficient biosynthesis of 6′-sialyllactose in a metabolically engineered plasmid-free Escherichia coli using a novel α2,6-sialyltransferase from Nicoletella semolina

6′-Sialyllactose (6′-SL), one of the most prevalent sialylated human milk oligosaccharides (HMOs), has recently garnered significant attention due to its promising health effects for infants. In this study, the 6′-SL biosynthetic pathway in EZAK (E. coli BL21(DE3) ΔlacZΔnanAΔnanK) was initially constructed by introducing a plasmid expressing the precursor CMP-Neu5Ac synthesis pathway genes neuBCA. A novel α2,6-sialyltransferase Ev6ST (NCBI Reference Sequence: WP_132500470) was selected by introducing plasmids expressing various α2,6-sialyltransferase-encoding genes and subsequent comparisons of the yields of 6′-SL. Subsequently, by integrating neuBCA and ev6st individually or in combination on the chromosome of EZAK, the high-yielding plasmid-free strain EZAKBEP with an extracellular yield of 5.68 g/L. In the 5 L bioreactor, fed-batch fermentation resulted in an extracellular yield of 15.35 g/L of 6′-SL. This work successfully screened a high-efficiency α2,6-sialyltransferase Ev6ST and constructed a high-yielding strain EZAKBEP under plasmid-free conditions, which is the highest yield of shake flask fermentation to date, and provides some reference significance for subsequent research.

Inconsistencies in the published rabbit ribosomal rRNAs: a proposal for uniformity in sequence and site numbering.

Examination of all publicly available Oryctolagus cuniculus (rabbit) ribosome cryo-EM structures reveals numerous confusing inconsistencies. First, there are a plethora of single nucleotide differences among the various rabbit 28S and 18S rRNA structures. Second, two nucleotides are absent from the NCBI Reference Sequence for the 18S rRNA gene. Moving forward, we propose using the Broad Institute's rabbit whole genome shotgun sequence and numbering to reduce modeling ambiguity and improve consistency between ribosome models.

Unveiling the Hidden Enemies: Morphological and Molecular Characterization of Pod Rot Pathogens in Groundnut (Arachis hypogea L.) in India

Pod rot of groundnut is a complex disease caused by multiple pathogens causes significant economic losses. Present study was undertaken to know the pathogens associated with the disease. In Chittoor, Andhra Pradesh, roving survey against pod rot of groundnut revealed association of three fungal species: Sclerotium rolfsii, Rhizoctonia bataticola and Fusarium spp. Molecular analysis with modified CTAB method and performing PCR using ITS1 and ITS4 primers yielded DNA bands of 650-700 bp, 550-600 bp, and 500-600 bp for Sclerotium, Rhizoctonia and Fusarium species, respectively. Sequencing of the rDNA ITS region confirmed the identities of the pathogens as Sclerotium rolfsii, Rhizoctonia bataticola, Fusarium solani, Fusarium oxysporum, and Fusarium keratoplasticum, based on similarity with NCBI reference sequences. This study confirms that Sclerotium rolfsii, Rhizoctonia bataticola and Fusarium spp. are the primary causal agents of pod rot disease in Andhra Pradesh.

Sequence Alignment between TRIM33 Gene and Human Noncoding RNAs: A Potential Explanation for Paraneoplastic Dermatomyositis.

This computational analysis investigated sequence complementarities between the TRIM33 gene and human noncoding (nc)RNAs and characterized their interactions in the context of paraneoplastic dermatomyositis. TRIM33 FASTA sequence (NCBI Reference Sequence: NC_000001.11) was used for BLASTN analysis against Human GRCh38 in the Ensembl.org database. Retrieved ncRNAs showing hits to TRIM33 were searched in the GeneCards.org database and further analyzed through RNAInter, QmRLFS-finder, Spliceator, and NcPath enrichment analysis. A total of 100 hits were found, involving the lncRNAs NNT-AS1, MKLN1-AS, LINC01206, and PAXBP1-AS1, whose dysregulation has been reported in either cancer or dermatomyositis. Additionally, the lncRNAs NNT-AS1 and PAXBP1-AS1 may interact with microRNA-142-3p, reducing its expression and increasing that of TRIM33. Sequence complementarity affected only TRIM33 intron 1, possibly resulting in alternatively spliced isoforms of TIF1γ with increased immunogenicity. The results also revealed nucleotide alignment between TRIM33 and the gene regulatory elements of 28 ncRNA genes involved in immune pathways. This pivotal study demonstrates sequence complementarity between TRIM33 and human ncRNAs dysregulated in cancer and dermatomyositis. This scenario may lead to the overproduction of more immunogenic TIF1γ variants in tumors and the stimulation of autoimmunity. Further experimental analyses using targeted methods such as Western blot or Chip-Seq are required to confirm these data.

Probing the Association of EPCAM Gene Single Nucleotide Polymorphisms with Genetic Disorders

Background: Mutations in the EPCAM gene can lead to the absence of this protein from epithelial cell surfaces, resulting in various disorders such as Lynch syndrome, congenital tufting enteropathy, cholestatic liver disease, and a range of cancers. Therefore, analyzing mutations in this gene is significant from diagnostic and prognostic perspectives. This study examines the effect of various mutations in the EPCAM gene on different attributes of the encoded protein. Objective: The objective of this study was to analyze the impact of single nucleotide polymorphisms (SNPs) in the EPCAM gene on the protein's structural and functional properties, aiming to identify potential predictive biomarkers for EPCAM-associated diseases. Methods: The study focused on the EPCAM gene transcript EPCAM-201, with ENSEMBL transcript ID ENST00000263735.9 and NCBI Reference Sequence NM_002354.3. The gene sequence was retrieved from the NCBI database, and SNPs were selected from the ENSEMBL database. A total of 21 variants were selected to design twenty-one cases. These cases were analyzed using various bioinformatics tools, including EXPASY for nucleotide sequence translation, HOPE server for 3D structure analysis, CELLO2GO for sub-cellular localization, and PROTPARAM for physicochemical parameter prediction. Data collection adhered to the principles outlined in the Declaration of Helsinki. Statistical analyses were conducted using SPSS version 25, with descriptive statistics and appropriate tests to determine the significance of the observed variations. Results: Analysis revealed that sixteen SNPs, namely rs994384264, rs1280024892, rs1294456118, rs149875996, rs767811939, rs750826481, rs754293486, rs748292053, rs1460762372, rs1294456118, rs149875996, rs754293486, rs748292053, rs771063031, rs776854951, and rs267606785, significantly altered the 3D structure of mutated proteins. Among these SNPs, rs994384264 and rs149875996 also affected the sub-cellular localization of proteins. Additionally, four mutations—rs1280024892, rs1460762372, rs987919056, and rs771029207—caused alterations in extinction coefficient, isoelectric point (pI), aliphatic index, and instability index. Conclusion: These single nucleotide variants might serve as predictive biomarkers for EPCAM-associated diseases, aiding in diagnosis and prognosis. Variants predicted in this study require further experimental validation to confirm their clinical utility.

Phylogenetic Analysis and Lipoxygenase (LOX) Gene Family Variation in The Pistachio

Lipoxygenases (LOX) gene family is a type of nonheme iron-containing dioxygenases, which has a very important aspect in plant development and fruit quality. LOX gene, which is responsible for lipid oxidation, the main role for the biosynthesis pathway of unsaturated fatty acids. Although some studies have investigated the LOX gene family in several species including arabidopsis, soybean, peanut and apple, there is no information from Pistachio; and the phylogeny of this gene family in the Pistacia genus is still not determined. In this study, Arabidopsis thaliana LOX1 gene (NCBI Reference Sequence: NM_104376.3) was selected and used as a query sequence for performing a BLASTN search. Among all sequence query which was found by NCBI platform, 9 sequences were selected for further analysis. Phylogenetic tree of full-length LOX gene sequences from the Pistacia genus was constructed using the Maximum Likelihood method with MEGA software. By using phylogenic analysis, we identified variations in gene structure and revealed the phylogenetic evolutionary relationship of this gene family. Additionally, this may serve as a reference value for assessing the genetic relationships among various LOX genes in Pistacia genus species. This variations provides us the possibility the design the primer to achieve us to find the exact LOX gene in Pistacia genus and future research on the evolutionary history and transgenic research on LOX genes.

Diverse Xylaria in the Ecuadorian Amazon and their mode of wood degradation

BackgroundXylaria is a diverse and ecologically important genus in the Ascomycota. This paper describes the xylariaceous fungi present in an Ecuadorian Amazon Rainforest and investigates the decay potential of selected Xylaria species. Fungi were collected at Yasuní National Park, Ecuador during two collection trips to a single hectare plot divided into a 10-m by 10-m grid, providing 121 collection points. All Xylaria fruiting bodies found within a 1.2-m radius of each grid point were collected. Dried fruiting bodies were used for culturing and the internal transcribed spacer region was sequenced to identify Xylaria samples to species level. Agar microcosms were used to assess the decay potential of three selected species, two unknown species referred to as Xylaria 1 and Xylaria 2 and Xylaria curta, on four different types of wood from trees growing in Ecuador including balsa (Ochroma pyramidale), melina (Gmelina arborea), saman (Samanea saman), and moral (Chlorophora tinctoria). ANOVA and post-hoc comparisons were used to test for differences in biomass lost between wood blocks inoculated with Xylaria and uninoculated control blocks. Scanning electron micrographs of transverse sections of each wood and assay fungus were used to assess the type of degradation present.Results210 Xylaria collections were sequenced, with 106 collections belonging to 60 taxa that were unknown species, all with less than 97% match to NCBI reference sequences. Xylaria with sequence matches of 97% or greater included X. aff. comosa (28 isolates), X. cuneata (9 isolates) X. curta and X. oligotoma (7 isolates), and X. apiculta (6 isolates)., All Xylaria species tested were able to cause type 1 or type 2 soft rot degradation in the four wood types and significant biomass loss was observed compared to the uninoculated controls. Balsa and melina woods had the greatest amount of biomass loss, with as much as 60% and 25% lost, respectively, compared to the controls.ConclusionsXylaria species were found in extraordinary abundance in the Ecuadorian rainforest studied. Our study demonstrated that the Xylaria species tested can cause a soft rot type of wood decay and with the significant amount of biomass loss that occurred within a short incubation time, it indicates these fungi likely play a significant role in nutrient cycling in the Amazonian rainforest.

Mutational analysis of catalytic site domain of CCHFV L RNA segment

Crimean-Congo haemorrhagic fever virus (CCHFV) has tripartite RNA genome and is endemic in various countries of Asia, Africa and Europe. The present study is focused on mutation profiling of CCHFV L segment and phylogenetic clustering of protein dataset into six CCHFV genotypes. Phylogenetic tree rooted with NCBI reference sequence (YP_325663.1) indicated less divergence from genotype III and the sequences belonging to same genotypes have shown less divergence among each other. Mutation frequency at 729 mutated positions was calculated and 563, 49, 33, 46 and 38 amino acid positions were found to be mutated at mutation frequency intervals of 0-0.2, 0.21-0.4, 0.41-0.6, 0.61-0.8 and 0.81-1.0 respectively. Thirty-eight highly frequent mutations (0.81-1.0 interval) were found in all genotypes and mapping in L segment (encoded for RdRp) revealed four mutations (V2074I, I2134T/A, V2148A and Q2695H/R) in catalytic site domain and no mutation in OTU domain. Molecular dynamic simulation and in silico analysis showed that catalytic site domain displayed large deviation and fluctuation upon introduction of these point mutations. Overall study provides strong evidence that OTU domain is highly conserved and less prone to mutation whereas point mutations recorded in catalytic domain have affected the stability of protein and were found to be persistent in the large population.

Biochemical and breakpoint cluster region-c-ABL oncogene 1 polymorphism study among Iraqi patients with chronic myeloid leukemia

BACKGROUND: Chronic myeloid leukemia (CML) has been well recognized as an exemplary instance of a malignant disease characterized by a distinctive molecular occurrence, namely the presence of the breakpoint cluster region (BCR)-c-ABL oncogene 1 (ABL1) oncogene. The Philadelphia chromosome gives rise to an anomalous fusion gene characterized by atypical kinase activity, resulting in the accumulation of reactive oxygen species and genetic instability that holds significance in the advancement of diseases. OBJECTIVE: The objective of this study was to investigate the detection rate of BCR-ABL1 polymorphism and BCR protein level in a group of Iraqi patients with CML. MATERIALS AND METHODS: This study has been carried out on 150 specimens, 120 patients subjected to CML included 20 patients diagnosed as newly diagnosis CML and 100 patients treated with CML. In addition to 30 apparently healthy persons as a control group (normal persons) from the National Center of Hematology/Mustansiryiah University/Baghdad, 65 out of 100 patients on imatinib while 35 nonimatinib (nilotinib and bosutinib). Fresh whole blood and serum were obtained from all patients and controls. We used total DNA genomic extraction extracted from ethylenediaminetetraacetic acid blood for genetic detection of Bcr/Abl Genes Polymorphism by sequencing technique in patients with CML and apparently control groups and used serum for biochemical tests include urea, lactate dehydrogenase (LDH), aspartate transaminase (AST), alanine transaminase (ALT), and creatinine using biochemicals methods (colorimetric and kinetic), respectively, as well as detection BCR protein level using sandwich enzyme-linked immunosorbent assays technique. RESULTS: According to age and sex, the patients’ groups were matching with the control group. Regarding the biochemical parameters (urea creatinine, ALT, AST, and LDH) serum level, there are no significant differences among new diagnosis CML, patients respond to treatments and failure group except in serum level of creatinine between new diagnosis CML group and failure group, there are significant differences (P = 0.01). The present results showed that DNA polymorphism distribution was according to C\C; G\C; A\T; and A\A were 32%, 26%, 18%, and 24%, respectively, in patients with CML and 28%; 20%;12%; and 40%, respectively, in the control group. There are significant statistical differences (P < 0.05) between different groups according to the genotyping of BCR\ABL, the results obtained from the sequenced 429 bp fragments, and the detailed positions of the observed variations are described in the NCBI reference sequences (rs766724113). The samples were submitted in NCBI, and the accession number of nucleotide sequences of BCR\ABL as new recording: LC 775148, LC 775149, and LC 775150, while regarding with BCR protein, there are significant differences in level between new diagnosis CML and CML on treatment and control groups, P < 0.001 for each comparison while there are no significant differences between treated group and control group (P = 0.729). CONCLUSION: The present results indicate that BCR-ABL1 polymorphism and BCR protein level in a group of Iraqi patients with CML may play a role in the tumor biology of the examined subset of CML and may contributed to their development.

A novel lncRNA MDHDH suppresses glioblastoma multiforme by acting as a scaffold for MDH2 and PSMA1 to regulate NAD+ metabolism and autophagy

BackgroundTo identify potential targets related to nicotinamide adenine dinucleotide (NAD+) metabolism in gliomas, we used RNA immunoprecipitation to identify a novel long noncoding RNA renamed malate dehydrogenase degradation helper (MDHDH) (NONCODE annotation ID: NONHSAT138800.2, NCBI Reference Sequence: NR_028345), which bound to MDH2 (malate dehydrogenase 2), that is downregulated in glioblastoma multiforme (GBM) and associated with metabolic regulation. However, its underlying mechanisms in the progression of GBM have not been well studied.MethodsTo investigate the clinical significance of MDHDH, we analyzed its expression levels in publicly available datasets and collected clinical samples from Shandong Provincial Hospital, affiliated with Shandong University. Functional assays, including FISH/CISH, CCK8, EdU, wound healing, and transwell assays, were used to determine the cellular/subcellular localization, tissue expression profile and anti-oncogenic role of MDHDH. Furthermore, RNA pulldown, mass spectrometry RNA immunoprecipitation, coimmunoprecipitation, JC-1 probe, and cell energy-production assays were used to determine the mechanisms of MDHDH in the development of GBM. Animal experiments were conducted to determine the antitumorigenic role of MDHDH in GBM in vivo.ResultsIn public datasets, MDHDH expression was significantly downregulated in GBM and LGG compared with GTEx normal brain tissues. The results of the tissue microarray showed that the MDHDH expression level negatively correlated with the tumor grade. Altered MDHDH expression led to significant changes in the proliferation, migration and invasion of GBM cells both in vitro and in vivo.Mechanistically, we found that MDHDH directly bound to MDH2 and PSMA1 (20S proteasomal core subunit alpha-type 1) as a molecular scaffold and accelerated the degradation of MDH2 by promoting the binding of ubiquitinated MDH2 to the proteasome. The degradation of MDH2 subsequently led to changes in the mitochondrial membrane potential and NAD+/NADH ratio, which impeded glycolysis in glioma cells.ConclusionsIn conclusion, this study broadened our understanding of the functions of lncRNAs in GBM. We demonstrated that the tumor suppressor MDHDH might act as a clinical biomarker and that the overexpression of MDHDH might be a novel synergistic strategy for enhancing metabolism-based, epigenetic-based, and autophagy regulation-based therapies with clinical benefits for glioblastoma multiforme patients.

Proteomic Analysis of Urine from Patients with Plasmodium vivax Malaria Unravels a Unique Plasmodium vivax Protein That Is Absent from Plasmodium falciparum.

Five species of Plasmodium cause malaria in humans and two of them, P. vivax and P. falciparum, pose the greatest threat. Rapid antigen detection tests (RADT) have been used for many years to diagnose and distinguish malaria caused by these two parasites. P. falciparum malaria can single-handedly be diagnosed using an RADT, which detects the unique P. falciparum specific histidine-rich protein 2 (HRP2). Unfortunately, there is no RADT that can single-handedly diagnose P. vivax malaria because no specific marker of this parasite has yet been described. Here, we report the discovery of a unique P. vivax protein (Vir14, NCBI Reference Sequence: XP_001612449.1) that has no sequence similarity with proteins of P. falciparum and no significant similarities with proteins of other species of Plasmodium. We propose that this protein could be an outstanding candidate molecule for the development of a promising RADT that can single-handedly and specifically diagnose P. vivax malaria.

Chemical Links Between Redox Conditions and Estimated Community Proteomes from 16S rRNA and Reference Protein Sequences.

Environmental influences on community structure are often assessed through multivariate analyses in order to relate microbial abundances to separately measured physicochemical variables. However, genes and proteins are themselves chemical entities; in combination with genome databases, differences in microbial abundances directly encode for chemical variability. We predicted that the carbon oxidation state of estimated community proteomes, obtained by combining taxonomic abundances from published 16S rRNA gene sequencing datasets with reference microbial proteomes from the NCBI Reference Sequence (RefSeq) database, would reflect environmental oxidation-reduction conditions. Analysis of multiple datasets confirms the geobiochemical predictions for environmental redox gradients in hydrothermal systems, stratified lakes and marine environments, and shale gas wells. The geobiochemical signal is largest for the steep redox gradients associated with hydrothermal systems and between injected water and produced fluids from shale gas wells, demonstrating that microbial community composition can be a chemical proxy for environmental redox gradients. Although estimates of oxidation state from 16S amplicon and metagenomic sequences are correlated, the 16S-based estimates show stronger associations with redox gradients in some environments.

An Improved Genome Sequence Resource of Bipolaris maydis, Causal Agent of Southern Corn Leaf Blight.

An Improved Genome Sequence Resource of Bipolaris maydis, Causal Agent of Southern Corn Leaf Blight.

Whole-Genome Sequence Resource of Fusarium oxysporum Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from Tetrastigma hemsleyanum

Whole-Genome Sequence Resource of <i>Fusarium oxysporum</i> Strain TH15, a Plant Growth Promoting Endophytic Fungus Isolated from <i>Tetrastigma hemsleyanum</i>

Fusion of the HMGA2 and BNC2 Genes in Uterine Leiomyoma With t(9;12)(p22;q14).

The translocation t(9;12) (p22;q14~15) has been reported in lipomas, pleomorphic adenomas, a myolipoma, two chondroid hamartomas, and two uterine leiomyomas. In lipomas and pleomorphic adenomas, the translocation fuses HMGA2 (12q14) with the NFIB gene from 9p22; in myolipoma, it fuses HMGA2 with C9orf92 from 9p22; and in chondroid hamartomas, fluorescence in situ hybridization (FISH) investigations showed the chromosomal aberration to cause intragenic rearrangement of HMGA2. The translocation's molecular consequence in a uterine leiomyoma is described here. A typical leiomyoma was investigated using banding cytogenetics, FISH, RNA sequencing, reverse transcription polymerase chain reaction and Sanger sequencing. A single translocation, t(9;12)(p22;q14) leading to an HMGA2::BNC2 chimera, was found in tumor cells. A sequence of the untranslated part of exon 5 of HMGA2 (nucleotide 1035 in the NCBI reference sequence NM_003483.4) had fused with a sequence from the untranslated part of exon 7 of BNC2 from 9p22 (nucleotide 9284 in reference sequence NM_017637.6). At the molecular level, the t(9;12)(p22;q14~15) found in several benign tumors appears to be heterogeneous fusing HMGA2 with either BNC2, C9orf92 or NFIB which all three map close to one another within a 3 Mbp region in 9p22. Because the fusion point in HMGA2 in the present tumor lays downstream from the first Let-7 miRNA consensus binding site, we conclude that deletion of the first Let-7 miRNA binding site is not important for the transcriptional upregulation of HMGA2 caused by the genomic rearrangement.

A Review on the Methods for Identification of Mutations in the Tumour Suppressor Gene Retinoblastoma RB1

Retinoblastoma is the most common intraocular cancer of childhood. RB1 is the gene responsible for causing retinoblastoma, spans more than 180 kilobases (kb) located on chromosome 13q14, which consist of 27 exons. Retinoblastoma in children may either be hereditary or non-hereditary. Mutations in RB1 gene are mostly point mutations of non-sense or missense type but could also be of frameshift type. These mutations can be identified from both blood and tumour samples by Sanger sequencing and other molecular identification techniques such as Multiplex Ligation-dependent Probe Amplification (MLPA). ‘Fragile’ codons are codons which gets point mutated to form stop codons so that the resulting protein will be incomplete or immature. In RB1, fragile codons get mutated predominantly and lead to the truncation of RB1 protein. The frequent mutations that predominantly occur in the arginine (CGA) codon, wherein changes in the single nucleotide results in the stop (UGA) codon, than any other fragile codon. The present paper reviews the role of RB1 mutations in retinoblastoma and the methods to identify it. We also make an attempt to identify the fragile codons in the RB genome based on the NCBI reference sequence NM_000321.2

First Report of Pectobacterium polaris Causing Soft Rot and Black Leg of Potato in Russia.

Blackleg and soft rot of potato (Solanum tuberosum) were monitored in the Central European part of Russia within a period of 2012- 2019. Symptoms included decay of tubers, blackening of stem vascular bundles, and partial yellowing of leaves. The disease causes serious potato yield losses in the field and storage. Pectobacterium parmentieri, P. brasiliense, P. versatile (syn. Ca. Pectobacterium maceratum), P. carotovorum, P. atrosepticum, Dickeya dianthicola, and D. solani are considered as main causal agents of soft rot and blackleg disease in Russia (Voronina et al. 2019, Ngoc Ha et al., 2019, Shirshikov et al. 2018, Kornev et al. 2012). Potato plant samples collected in commercial fields in routine plant health assay were used for bacteria isolation on crystal violet pectate agar (CVP) (Helias et al. 2012) as described previously (Voronina et al. 2019). Bacterial colonies producing pitting on CVP were re-isolated and purified on nutrient broth yeast extract medium. DNA of bacterial isolates was extracted, and polymerase chain reaction (PCR) amplifications were performed using gapA primers (Cigna et al. 2017) followed by sequencing. DNA sequence alignment showed that the isolates F099, F100, F106, F109, and F118 were identical (deposited as part of NCBI Ref.Seq. for F109 NZ_RRYS01000004.1, locus KHDHEBDM_RS06360) and grouped together with the type strain Pectobacterium polaris NIBIO1006T (CP017481), a new species described as a potato pathogen (Dees et al. 2017). These strains were negative in diagnostic PCR assays using specific primers Y45/Y46 for the detection of P. atrosepticum, Br1f and L1r for P. brasiliense (Duarte et al. 2004), and ADE1/ADE2 for Dickeya sp. (Nassar et al. 1996). To further validate the identification, strain F109 of P. polaris was selected for genome sequencing. The genome of P. polaris strain F109, (NCBI Reference Sequence NZ_RRYS00000000.1) reveals >99% sequence similarity with type strain P. polaris IPO_1606 (GenBank accession GCA_902143345.1). The strain F109 was deposited to All-Russian Collection of Microorganisms under number VKM V-3420. Thus, the characterization of five isolates provided evidence that a previously unreported pathogen was present in the surveyed fields. The isolates were uniform in genetic and physiological properties; they were gram negative, facultative anaerobes with pectinolytic activity, negative for oxidase, urease, indole production, gelatin liquefaction. All isolates were catalase positive, produced acid from lactose, rhamnose, saccharose, xylose, and trehalose, and were tolerant to 5% NaCl, unable to utilize malonate and citrate. All the isolates grew at 37°C. All isolates caused soft rot symptoms on 10 inoculated potato tubers. They produced typical black leg rot symptoms in young potato plants inoculated with 107 CFU/ml of the pathogen by stem injection and incubated at 25°C for 48 h. The bacteria were re-isolated successfully from symptomatic potato and pathogen confirmed by gapA sequencing to complete Koch's postulates. To our knowledge, this is the first report of blackleg and soft rot caused by P. polaris on potato in the Russian Federation. According to the data of commercial diagnostic laboratory "PhytoEngineering" (Moscow region), P. polaris occurred in 5% potato seed stocks harvested in 2017-2019 in the Moscow region. This finding may indicate that new Pectobacterium strains have adapted to a diverse environment, which is consistent with widespread distribution of commercial seed potatoes. The author(s) declare no conflict of interest. Funding: This work was supported by Russian Science Foundation grant #16-16-00073.

Screening, cloning, enzymatic properties of a novel thermostable cellulase enzyme, and its potential application on water hyacinth utilization.

Cellulose is the cheapest, natural, renewable organic substance that is used as a carbon source in various fields. Water hyacinth, an aquatic plant rich in cellulose, is often used as a raw material in fuel production. However, natural cellulase can be hardly used in industrial production on account of its low thermal stability and activity. In this study, a metagenomic library was constructed. Then, a new cellulase gene, cel1029, was screened by Congo red staining and expressed in the prokaryotic system. Enzymatic properties of Cel1029 were explored, including optimum temperature and pH, thermal and pH stability, and tolerance against organic solvents, metal ions, and salt solutions. Finally, its ability of degrading water hyacinth was identified and evaluated. Cel1029 displayed high homology with endoglucanase in the glycoside hydrolase family 5 (GH5) and had high stability across a broad temperature range. More than 86% of its enzymatic activities were retained between 4 and 60 °C after 24 h of incubation. Single-factor analysis and orthogonal design were further conducted to determine the optimal conditions for the highest reducing sugar yield of water hyacinth. Interestingly, Cel1029 efficiently transformed water hyacinth with a reducing sugar yield of 430.39 mg/g in 22 h. These findings may open the door for significant industrial applications of a novel GH5 cellulase (NCBI Reference Sequence: MK051001, Cel1029) and help identify more efficient methods to degrade cellulose-rich plants.

First complete genome sequence of carnation latent virus, the type member of the genus Carlavirus.

The genus Carlavirus (family Betaflexiviridae, order Tymovirales) currently includes 53 species recognized by the ICTV. The NCBI GenBank database has 43 full-length carlavirus genome sequences (7,890 to 9,073 nt). Surprisingly, the type species Carnation latent virus is not associated with a complete genome sequence of a carnation latent virus (CLV) isolate; GenBank only has accessions with 1313 or fewer nucleotides. The goal of this study was to determine the full-length genome sequence of CLV. Naturally infected greenhouse-grown 'Kiwi Lace' carnation plants that tested positive for CLV by ELISA and RT-PCR were used as source plants for high-throughput sequencing, completed by 5' and 3' RACE and validated by Sanger sequencing of CLV-specific RT-PCR-generated amplicons. The complete CLV-KL sequence (MN450069) was determined to be 8,513 nt in length. In pairwise analysis, the genome shares 40-46% identity with recognized carlaviruses and the six in silico-translated proteins have 15-62% amino acid sequence identity to their respective carlavirus orthologs. The CLV-KL coat protein shares 98.4% identity with the NCBI reference sequence CLV-UK. In phylogenetic analysis, CLV clusters with butterbur mosaic virus, coleus vein necrosis virus, and garlic common latent virus. This is the first report of the full genome sequence of an isolate of carnation latent virus, the type member of the genus Carlavirus.