Full-Length Genome of an Ogataea polymorpha Strain CBS4732 ura3Δ Reveals Large Duplicated Segments in Subtelomeric Regions

BACKGROUND: Currently, methylotrophic yeasts (e.g., Pichia pastoris, Ogataea polymorpha, and Candida boindii) are subjects of intense genomics studies in basic research and industrial applications. In the genus Ogataea, most research is focused on three basic O. polymorpha strains-CBS4732, NCYC495, and DL-1. However, the relationship between CBS4732, NCYC495, and DL-1 remains unclear, as the genomic differences between them have not be exactly determined without their high-quality complete genomes. As a nutritionally deficient mutant derived from CBS4732, the O. polymorpha strain CBS4732 ura3Δ (named HU-11) is being used for high-yield production of several important proteins or peptides. HU-11 has the same reference genome as CBS4732 (noted as HU-11/CBS4732), because the only genomic difference between them is a 5-bp insertion. RESULTS: In the present study, we have assembled the full-length genome of O. polymorpha HU-11/CBS4732 using high-depth PacBio and Illumina data. Long terminal repeat retrotransposons (LTR-rts), rDNA, 5′ and 3′ telomeric, subtelomeric, low complexity and other repeat regions were exactly determined to improve the genome quality. In brief, the main findings include complete rDNAs, complete LTR-rts, three large duplicated segments in subtelomeric regions and three structural variations between the HU-11/CBS4732 and NCYC495 genomes. These findings are very important for the assembly of full-length genomes of yeast and the correction of assembly errors in the published genomes of Ogataea spp. HU-11/CBS4732 is so phylogenetically close to NCYC495 that the syntenic regions cover nearly 100% of their genomes. Moreover, HU-11/CBS4732 and NCYC495 share a nucleotide identity of 99.5% through their whole genomes. CBS4732 and NCYC495 can be regarded as the same strain in basic research and industrial applications. CONCLUSION: The present study preliminarily revealed the relationship between CBS4732, NCYC495, and DL-1. Our findings provide new opportunities for in-depth understanding of genome evolution in methylotrophic yeasts and lay the foundations for the industrial applications of O. polymorpha CBS4732, NCYC495, DL-1, and their derivative strains. The full-length genome of O. polymorpha HU-11/CBS4732 should be included into the NCBI RefSeq database for future studies of Ogataea spp

Quantification of Enterohemorrhagic Escherichia coli O157:H7 proteome using TMT-Based Analysis

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible for diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). EHEC infection is distributed worldwide and numerous outbreaks of diseases caused by enterohemorrhagic have been reported. To promote a comprehensive insight into the molecular basis of EHEC O157:H7 physiology and pathogenesis, the combined proteome of EHEC O157:H7 strains, Clade 8 and Clade 6 isolated from cattle in Argentina, and the standard EDL933 (clade 3) strain has been analyzed. TMT (Tandem Mass Tags)-based quantitative proteomic and emPAI analyses were performed to estimate the protein abundance in EHEC proteome. 2,234 non-redundant proteins of EHEC O157:H7 were identified. A comparison of this result with in silico data of EHEC O157:H7 genome showed that approximately 40% of the predicted proteome of this pathogen were covered. According to the emPAI analysis, 85 proteins were among the most abundant (e.g. GAPDH, FliC H-antigen, Enolase, and GroEL). Tellurite resistance proteins were also highly abundant. COG analysis showed that although most of the identified proteins are related to cellular metabolism, the majority of the most abundant proteins are associated with translation processes. A KEGG enrichment analysis revealed that Glycolysis / Gluconeogenesis was the most significant pathway. On the other hand, the less abundant detected proteins are those related to DNA processes, cell respiration and prophage. Among the proteins that composed the Type III Secretion System, the most abundant protein was EspA. Altogether, the results show a subset of important proteins that contribute to physiology and pathogenicity of EHEC O157:H7.IMPORTANCE The study of the abundance of proteins present within a complex mixture of proteins in a cell, under different conditions, can provide important information about the activities of individual protein components and protein networks that are cornerstones for the comprehension of physiological adaptations in response to biological demands promoted by environmental changes. We generated a comprehensive and accurate quantitative list of EHEC O157:H7 proteome, which provides a description of the most abundant proteins produced by this pathogen that were related to physiology and pathogenesis of EHEC. This study provides information and extends the understanding on functional genomics and the biology of this pathogen.Fil: Marques Da Silva, Wanderson. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Bei, Jinlong. No especifíca;Fil: Amigo, Natalia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Valacco, Maria Pia. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Amadio, Ariel. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Santa Fe. Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Zhang, Qi. No especifíca;Fil: Wu, Xiuju. No especifíca;Fil: Yu, Ting. No especifíca;Fil: Larzabal, Mariano. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Chen, Zhuang. No especifíca;Fil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Quantification of enterohemorrhagic Escherichia coli O157:H7 protein abundance by high-throughput proteome

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a human pathogen responsible fordiarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS). To promote a comprehensiveinsight into the molecular basis of EHEC O157:H7 physiology and pathogenesis,the combined proteome of EHEC O157:H7 strains, Clade 8 and Clade 6 isolated from cattlein Argentina, and the standard EDL933 (clade 3) strain has been analyzed. From shotgunproteomic analysis a total of 2,644 non-redundant proteins of EHEC O157:H7 were identified,which correspond approximately 47% of the predicted proteome of this pathogen. Normalizedspectrum abundance factor analysis was performed to estimate the proteinabundance. According this analysis, 50 proteins were detected as the most abundant ofEHEC O157:H7 proteome. COG analysis showed that the majority of the most abundantproteins are associated with translation processes. A KEGG enrichment analysis revealedthat Glycolysis / Gluconeogenesis was the most significant pathway. On the other hand, theless abundant detected proteins are those related to DNA processes, cell respiration andprophage. Among the proteins that composed the Type III Secretion System, the mostabundant protein was EspA. Altogether, the results show a subset of important proteins thatcontribute to physiology and pathogenicity of EHEC O157:H7.Fil: Marques Da Silva, Wanderson. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Bei, Jinlong. Guangdong Academy Of Agricultural Sciences; ChinaFil: Amigo, Natalia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; ArgentinaFil: Valacco, Maria Pia. Universidad de Buenos Aires; ArgentinaFil: Amadio, Ariel Fernando. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Zhang, Qi. Guangdong Academy Of Agricultural Sciences; ChinaFil: Wu, Xiuju. Guangdong Academy Of Agricultural Sciences; ChinaFil: Yu, Ting. Guangdong Academy Of Agricultural Sciences; ChinaFil: Larzabal, Mariano. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; ArgentinaFil: Chen, Zhuang. Guangdong Academy Of Agricultural Sciences; ChinaFil: Cataldi, Ángel Adrián. Instituto Nacional de Tecnología Agropecuaria. Centro Regional Buenos Aires; Argentin

A Phenotypic characterization of two isolates of a multidrug resistant outbreak strain of mycobacterium tuberculosis with opposite epidemiological fitness

Tuberculosis (TB) is an infectious disease, caused by Mycobacterium tuberculosis, primarily affecting the lungs. The M. tuberculosis strain of the Haarlem family named M was responsible for a large multidrug-resistant TB (MDR-TB) outbreak in Buenos Aires. This outbreak started in the early 1990s and in the mid 2000s still accounted for 29% of all MDR-TB cases in Argentina. By contrast, a clonal variant of strain M, named 410, has caused a single tuberculosis case since the onset of the outbreak. The molecular bases of the high epidemiological fitness of the M strain remain unclear. To assess its unique molecular properties, herein, we performed a comparative protein and lipid analysis of a representative clone of the M strain (Mp) and the nonprosperous M variant 410. We also evaluated their growth in low pH. The variant 410 had higher levels of latency proteins under standard conditions and delayed growth at low pH, suggesting that it is more sensitive to stress stimuli than Mp. Moreover, Mp showed higher levels of mycolic acids covalently attached to the cell wall and lower accumulation of free mycolic acids in the outer layer than the 410 strain. The low expression of latency proteins together with the reduced content of surface mycolic acids may facilitate Mp to evade the host immune responses.Fil: Bei, Jinlong. Guangdong Academy of Agricultural Sciences; ChinaFil: Bigi, María Mercedes. Universidad de Buenos Aires. Facultad de Agronomía; ArgentinaFil: Lima, Analía. Instituto Pasteur de Montevideo; UruguayFil: Zhang, Qi. Guangdong Academy of Agricultural Sciences; ChinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Lopez, Beatriz. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; ArgentinaFil: Yu, Ting. Guangdong Academy of Agricultural Sciences; ChinaFil: Wang, Zhilin. Guangdong Academy of Agricultural Sciences; ChinaFil: Dai, Zhangyan. Guangdong Academy of Agricultural Sciences; ChinaFil: Chen, Zhuang. Guangdong Academy of Agricultural Sciences; ChinaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Sasiain, María del Carmen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Ritacco, Gloria Viviana. Dirección Nacional de Instituto de Investigación. Administración Nacional de Laboratorio e Instituto de Salud "Dr. C. G. Malbrán"; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: de la Barrera, Silvia Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Soria, Marcelo Abel. Universidad de Buenos Aires. Facultad de Agronomía. Departamento de Biología Aplicada y Alimentos. Cátedra de Microbiología Agrícola; ArgentinaFil: Durán, Rosario. Instituto Pasteur de Montevideo; UruguayFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

The complete chloroplast genome of Callicarpa macrophylla Vahl.

Callicarpa macrophylla Vahl. belongs to the family Lamiaceae. Its root is a widely used Yao Medicine (YM) to treat internal and external bleeding at the Yao minority areas in southern China. Here, we provide the complete chloroplast genome of C. macrophylla which was collected from Laibin city in Guangxi, China. The total length of the chloroplast genome is 154,141 bp, including a large single-copy (LSC) region, a small single-copy (SSC) region, and a pair of inverted repeats (IRs) regions which are separated by the LSC and SSC, with lengths of 84,904 bp, 17,839 bp, and 25,699 bp, respectively. One hundred and thirty-one genes were identified, including 89 protein-coding genes, 34 tRNA genes, and eight rRNA genes. The overall GC content is 38%. Phylogenetic analysis revealed that C. macrophylla is closely related to C. integerrima var. chinensis

Characterization of AcMNPV with a deletion of ac69 gene

<p>ORF69 (Ac69) of <em>Autographa californica</em> multiple nucleopolyhedrovirus (Ac<em>M</em>NPV) is conserved in some baculovirus genomes. Although it has been shown that Ac69 has cap 0-dependent methyltransferase activity and is not required for budded virus production in <em>Spodoptera frugiperda</em> Sf-9 cells, its role in occlusion-derived virus synthesis and virus oral infectivity is not known. This paper describes generation of an <em>ac69</em> knockout Ac<em>M</em>NPV bacmid mutant and analyses of the influence of <em>ac69</em> deletion on the viral infectivity in Sf-9 cells and <em>Trichoplusia ni</em> larvae so as to investigate the role of <em>ac69 in the viral life cycle. Results indicated that ac69</em> deletion has little effect on the production rates and morphogenesis of budded virus and occlusion-derived virus in Sf-9 cells. In addition, animal experiment revealed that the deletion mutant did not affect Ac<em>M</em>NPV infectivity for <em>Trichoplusia ni</em> larvae in LD₅₀ and LT₅₀ bioassay when administered orally. These results suggest that <em>ac69</em> may be dispensable for viral infectivity both in vitro and in vivo.</p

High efficient development of green kinetic hydrate inhibitors via combined molecular dynamic simulation and experimental test approach

The development of environmental friendly low dose hydrate inhibitors like kinetic hydrate inhibitors (KHIs) is of great significance for the flow assurance in oil &amp; gas production and transportation. In this work, a combined molecular dynamic simulation and experimental verification approach was adopted to increase the efficiency of KHIs development. The inhibition effect of a series of copolymers (N-vinylpyrrolidone and N-acrylate) on hydrate growth was studied by using both molecular dynamics simulation and experimental approaches. The simulation results demonstrated that introduction of hydrophobic ester and butyl group in PVP is beneficial for the inhibition. The length of the alkyl chain of ester group played an important role in improving inhibition performance. PVP-A, the one being introduced butyl ester group into PVP gets the best inhibition effect. In addition, inhibitors can restrict methane bubbles to re-dissolve into the liquid phase, thereby inhibiting the growth of methane hydrate. Increasing the interaction between KHIs and methane can also improve the inhibitory effect of KHIs. The experimental results confirm the reliability of the molecular dynamics simulation

Development of Poly(vinylpyrrolidone)-<i>co</i>-poly(cyclohexyl vinyl ether) as Kinetic Hydrate Inhibitors through Molecular Simulation and Experiment

Due to their low dosage and cost-effectiveness, kinetic hydrate inhibitors (KHIs) have recently garnered increased attention as an effective method for mitigating the harm caused by hydrate plugging. Hence, the development of efficient KHIs holds significant importance in addressing the safety issues of oil and gas flow. In this work, molecular simulation was used for the precise design of KHIs and helped in the development of KHIs. The simulation results reveal that cyclohexyl ether groups strengthen poly(vinylpyrrolidone)’s ability to inhibit methane hydrate formation, and an optimal inhibitory performance of the polymer was achieved when the ratio of pyrrolidone groups to cyclohexyl ether groups on the polymer chain was 1:1. Subsequently, based on simulation results, KHIs were precisely designed and synthesized. The experimental results demonstrate that the polymer chain with a 1:1 ratio of pyrrolidone groups to cyclohexyl ether groups exhibits the strongest inhibitory effect, comparable to the commercial inhibitor Inhibex 501, thereby validating the molecular simulation results. Our results suggest that employing molecular simulation for the precise development of hydrate inhibitors holds great potential, and in the future, it can significantly enhance the efficiency of developing KHIs