Menu

Auto Tag: by SMRT

July 7, 2019

Bioinformatics analysis and characterization of highly efficient polyvinyl alcohol (PVA)-degrading enzymes from the novel PVA degrader Stenotrophomonas rhizophila QL-P4.

Polyvinyl alcohol (PVA) is used widely in industry, and associated environmental pollution is a serious problem. Herein, we report a novel, efficient PVA degrader, Stenotrophomonas rhizophila QL-P4, isolated from fallen leaves from virgin forest in the Qinling Mountains. The complete genome was obtained using single-molecule real-time (SMRT) technology and corrected using Illumina sequencing. Bioinformatics analysis revealed eight PVA/OVA (vinyl alcohol oligomer)-degrading genes. Of these, seven genes were predicted to be involved in the classical intracellular PVA/OVA degradation pathway, and one (BAY15_3292) was identified as a novel PVA oxidase. Five PVA/OVA-degrading enzymes were purified and characterised. Among which, BAY15_1712, a PVA dehydrogenase (PVADH), displayed high catalytic efficiency towards PVA and OVA substrate. All reported PVADHs only have PVA-degrading ability. Most importantly, we discovered a novel PVA oxidase (BAY15_3292) that exhibited highest PVA-degrading efficiency than the reported PVADHs. Further investigation indicated that BAY15_3292 plays a crucial role in PVA degradation in S. rhizophila QL-P4. Knocking out BAY15_3292 resulted in a significant decline in PVA-degrading activity in S. rhizophila QL-P4. Interestingly, we found that BAY15_3292 possesses exocrine activity, which distinguishes it from classical PVADHs. Transparent circle experiments further proved that BAY15_3292 greatly affects extracellular PVA degradation in S. rhizophila QL-P4. The exocrine characteristics of BAY15_3292 facilitate its potential application to PVA bioremediation. In addition, we report three new efficient secondary alcohol dehydrogenases (SADHs) with OVA-degrading ability in S. rhizophila QL-P4, compared with only one OVA-degrading SADH as reported previously.Importance With the widespread application of PVA in industry, PVA-related environmental pollution is an increasingly serious issue. Because PVA is difficult to degrade, it accumulates in aquatic environments and causes chronic toxicity to aquatic organisms. Biodegradation of PVA, as an economical and environment-friendly method, has attracted much interest. To date, effective and applicable PVA-degrading bacteria/enzymes have not been reported. Herein, we report a new efficient PVA degrader (S. rhizophila QL-P4) that has five PVA/OVA-degrading enzymes with high catalytic efficiency, among which BAY15_1712 is the only reported PVADH with both PVA- and OVA-degrading abilities. Importantly, we discovered a novel PVA oxidase (BAY15_3292) that is not only more efficient than other reported PVA-degrading PVADHs, but also has exocrine activity. Overall, our findings provide new insight into PVA-degrading pathways in microorganisms, and suggest S. rhizophila QL-P4 and its enzymes have potential for application to PVA bioremediation to reduce or eliminate PVA-related environmental pollution. Copyright © 2017 American Society for Microbiology.

Read more
July 7, 2019

Convergence of plasmid architectures drives emergence of multi-drug resistance in a clonally diverse Escherichia coli population from a veterinary clinical care setting.

The purpose of this study was to determine the plasmid architecture and context of resistance genes in multi-drug resistant (MDR) Escherichia coli strains isolated from urinary tract infections in dogs. Illumina and single-molecule real-time (SMRT) sequencing were applied to assemble the complete genomes of E. coli strains associated with clinical urinary tract infections, which were either phenotypically MDR or drug susceptible. This revealed that multiple distinct families of plasmids were associated with building an MDR phenotype. Plasmid-mediated AmpC (CMY-2) beta-lactamase resistance was associated with a clonal group of IncI1 plasmids that has remained stable in isolates collected up to a decade apart. Other plasmids, in particular those with an IncF replicon type, contained other resistance gene markers, so that the emergence of these MDR strains was driven by the accumulation of multiple plasmids, up to 5 replicons in specific cases. This study indicates that vulnerable patients, often with complex clinical histories provide a setting leading to the emergence of MDR E. coli strains in clonally distinct commensal backgrounds. While it is known that horizontally-transferred resistance supplements uropathogenic strains of E. coli such as ST131, our study demonstrates that the selection of an MDR phenotype in commensal E. coli strains can result in opportunistic infections in vulnerable patient populations. These strains provide a reservoir for the onward transfer of resistance alleles into more typically pathogenic strains and provide opportunities for the coalition of resistance and virulence determinants on plasmids as evidenced by the IncF replicons characterised in this study. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Read more
July 7, 2019

N6-adenine DNA methylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena.

DNA N6-methyladenine (6mA) is newly rediscovered as a potential epigenetic mark across a more diverse range of eukaryotes than previously realized. As a unicellular model organism, Tetrahymena thermophila is among the first eukaryotes reported to contain 6mA modification. However, lack of comprehensive information about 6mA distribution hinders further investigations into its function and regulatory mechanism. In this study, we provide the first genome-wide, base pair-resolution map of 6mA in Tetrahymena by applying single-molecule real-time (SMRT) sequencing. We provide evidence that 6mA occurs mostly in the AT motif of the linker DNA regions. More strikingly, these linker DNA regions with 6mA are usually flanked by well-positioned nucleosomes and/or H2A.Z-containing nucleosomes. We also find that 6mA is exclusively associated with RNA polymerase II (Pol II)-transcribed genes, but is not an unambiguous mark for active transcription. These results support that 6mA is an integral part of the chromatin landscape shaped by adenosine triphosphate (ATP)-dependent chromatin remodeling and transcription.© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

Read more
July 7, 2019

Methylomic and phenotypic analysis of the ModH5 phasevarion of Helicobacter pylori.

The Helicobacter pylori phase variable gene modH, typified by gene HP1522 in strain 26695, encodes a N6-adenosine type III DNA methyltransferase. Our previous studies identified multiple strain-specific modH variants (modH1 – modH19) and showed that phase variation of modH5 in H. pylori P12 influenced expression of motility-associated genes and outer membrane protein gene hopG. However, the ModH5 DNA recognition motif and the mechanism by which ModH5 controls gene expression were unknown. Here, using comparative single molecule real-time sequencing, we identify the DNA site methylated by ModH5 as 5′-Gm6ACC-3′. This motif is vastly underrepresented in H. pylori genomes, but overrepresented in a number of virulence genes, including motility-associated genes, and outer membrane protein genes. Motility and the number of flagella of H. pylori P12 wild-type were significantly higher than that of isogenic modH5 OFF or ?modH5 mutants, indicating that phase variable switching of modH5 expression plays a role in regulating H. pylori motility phenotypes. Using the flagellin A (flaA) gene as a model, we show that ModH5 modulates flaA promoter activity in a GACC methylation-dependent manner. These findings provide novel insights into the role of ModH5 in gene regulation and how it mediates epigenetic regulation of H. pylori motility.

Read more
July 7, 2019

Complete genome sequencing and genomic characterization of two Escherichia coli strains co-producing MCR-1 and NDM-1 from bloodstream infection.

We previously described the discovery of two Escherichia coli isolates (EC1002 and EC2474) co-harbouring mcr-1 and bla NDM-1 genes, which were recovered from bloodstream infection in China. More importantly, these antibiotic resistance genes were located on different plasmids and signaling the potential spread of pandrug-resistant bacteria. Here, the complete genome sequences of both isolates were determined using Pacbio RS II and Illumina HiSeq2000 systems. The genome of EC1002 consists of a 5,177,501 base pair chromosome and four circular plasmids, while the genome of EC2474 consists of a 5,013,813 base pair chromosome and three plasmids. The plasmid replicon type of pEC1002_NDM and pEC2474_NDM were identified as IncA/C2 and IncF, respectively. The genetic environment of bla NDM-1 in this study was similar to bla NDM-carrying plasmids detected in China, although the overall nucleotide identity and query coverage were variable. The plasmid replicon type of pEC1002_MCR and pEC2474_MCR were identified as IncI2 and IncHI2, respectively. Two different genetic strategies for mcr-1 gene spread were observed in this study and bla NDM-1 genes were also found transferred by two different mobile genetic elements in two plasmids. The findings of this study further support that the diversified transfer mechanisms of bla NDM-1 and mcr-1 present in Enterobacteriaceae.

Read more
July 7, 2019

Chromosome level assembly and secondary metabolite potential of the parasitic fungus Cordyceps militaris.

Cordyceps militaris is an insect pathogenic fungus that is prized for its use in traditional medicine. This and other entomopathogenic fungi are understudied sources for the discovery of new bioactive molecules. In this study, PacBio SMRT long read sequencing technology was used to sequence the genome of C. militaris with a focus on the genetic potential for secondary metabolite production in the genome assembly of this fungus.This is first chromosome level assembly of a species in the Cordyceps genera. In this seven chromosome assembly of 33.6 Mba there were 9371 genes identified. Cordyceps militaris was determined to have the MAT 1-1-1 and MAT 1-1-2 mating type genes. Secondary metabolite analysis revealed the potential for at least 36 distinct metabolites from a variety of classes. Three of these gene clusters had homology with clusters producing desmethylbassianin, equisetin and emericellamide that had been studied in other fungi.Our assembly and analysis has revealed that C. militaris has a wealth of gene clusters for secondary metabolite production distributed among seven chromosomes. The identification of these gene clusters will facilitate the future study and identification of the secondary metabolites produced by this entomopathogenic fungus.

Read more
July 7, 2019

Comparative genomic analysis of two clonally related multidrug resistant Mycobacterium tuberculosis by Single Molecule Real Time Sequencing.

Background: Multidrug-resistant tuberculosis (MDR-TB) is posing a major threat to global TB control. In this study, we focused on two consecutive MDR-TB isolated from the same patient before and after the initiation of anti-TB treatment. To better understand the genomic characteristics of MDR-TB, Single Molecule Real-Time (SMRT) Sequencing and comparative genomic analyses was performed to identify mutations that contributed to the stepwise development of drug resistance and growth fitness in MDR-TB underin vivochallenge of anti-TB drugs.Result:Both pre-treatment and post-treatment strain demonstrated concordant phenotypic and genotypic susceptibility profiles toward rifampicin, pyrazinamide, streptomycin, fluoroquinolones, aminoglycosides, cycloserine, ethionamide, and para-aminosalicylic acid. However, although both strains carried identical missense mutations atrpoBS531L,inhAC-15T, andembBM306V, MYCOTB Sensititre assay showed that the post-treatment strain had 16-, 8-, and 4-fold elevation in the minimum inhibitory concentrations (MICs) toward rifabutin, isoniazid, and ethambutol respectively. The results have indicated the presence of additional resistant-related mutations governing the stepwise development of MDR-TB. Further comparative genomic analyses have identified three additional polymorphisms between the clinical isolates. These include a single nucleotide deletion at nucleotide position 360 ofrv0888in pre-treatment strain, and a missense mutation atrv3303c(lpdA)V44I and a 6-bp inframe deletion at codon 67-68 inrv2071c(cobM)in the post-treatment strain. Multiple sequence alignment showed that these mutations were occurring at highly conserved regions among pathogenic mycobacteria. Using structural-based and sequence-based algorithms, we further predicted that the mutations potentially have deleterious effect on protein function.Conclusion:This is the first study that compared the full genomes of two clonally-related MDR-TB clinical isolates during the course of anti-TB treatment. Our work has demonstrated the robustness of SMRT Sequencing in identifying mutations among MDR-TB clinical isolates. Comparative genome analysis also suggested novel mutations atrv0888, lpdA, andcobMthat might explain the difference in antibiotic resistance and growth pattern between the two MDR-TB strains.

Read more
July 7, 2019

Study of mesophilic Aeromonas salmonicida A527 strain sheds light on the species’ lifestyles and taxonomic dilemma.

The Gram-negative bacterium Aeromonas salmonicida contains five subspecies: salmonicida, smithia, achromogenes, masoucida and pectinolytica. Pectinolytica is a mesophilic subspecies with the ability to thrive at a wide range of temperatures, including 37°C, while the four other subspecies are psychrophilic, restricted to lower temperatures. The psychrophilic subspecies are known to infect a wide range of fishes. However, there is no evidence of pathogenicity for the mesophilic subspecies pectinolytica. Study of the differences between the mesophilic and psychrophilic subspecies is hampered by the lack of completely sequenced and closed genomes from the mesophilic subspecies. A previous study reported that insertion sequences, which can induce genomic rearrangements at temperatures around 25°C, could be one of the determinants explaining the differences in lifestyle (mesophilic or psychrophilic) between the subspecies. In this study, the genome of mesophilic strain A527 of A. salmonicida was sequenced, closed and analyzed to investigate the mesophilic-psychrophilic discrepancy. This reference genome supports the hypothesis that insertion sequences are major determinants of the lifestyle differences between the A. salmonicida subspecies. Moreover, the phylogenetic analysis performed to position strain A527 within the taxonomy raises an issue regarding the intraspecies structure of A. salmonicida.© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Read more
July 7, 2019

Genome mining of astaxanthin biosynthetic genes from Sphingomonas sp. ATCC 55669 for heterologous overproduction in Escherichia coli.

As a highly valued keto-carotenoid, astaxanthin is widely used in nutritional supplements and pharmaceuticals. Therefore, the demand for biosynthetic astaxanthin and improved efficiency of astaxanthin biosynthesis has driven the investigation of metabolic engineering of native astaxanthin producers and heterologous hosts. However, microbial resources for astaxanthin are limited. In this study, we found that the a-Proteobacterium Sphingomonas sp. ATCC 55669 could produce astaxanthin naturally. We used whole-genome sequencing to identify the astaxanthin biosynthetic pathway using a combined PacBio-Illumina approach. The putative astaxanthin biosynthetic pathway in Sphingomonas sp. ATCC 55669 was predicted. For further confirmation, a high-efficiency targeted engineering carotenoid synthesis platform was constructed in E. coli for identifying the functional roles of candidate genes. All genes involved in astaxanthin biosynthesis showed discrete distributions on the chromosome. Moreover, the overexpression of exogenous E. coli idi in Sphingomonas sp. ATCC 55669 increased astaxanthin production by 5.4-fold. This study described a new astaxanthin producer and provided more biosynthesis components for bioengineering of astaxanthin in the future. © 2015 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Read more
July 7, 2019

hybridSPAdes: an algorithm for hybrid assembly of short and long reads.

Recent advances in single molecule real-time (SMRT) and nanopore sequencing technologies have enabled high-quality assemblies from long and inaccurate reads. However, these approaches require high coverage by long reads and remain expensive. On the other hand, the inexpensive short reads technologies produce accurate but fragmented assemblies. Thus, a hybrid approach that assembles long reads (with low coverage) and short reads has a potential to generate high-quality assemblies at reduced cost.We describe hybridSPAdes algorithm for assembling short and long reads and benchmark it on a variety of bacterial assembly projects. Our results demonstrate that hybridSPAdes generates accurate assemblies (even in projects with relatively low coverage by long reads) thus reducing the overall cost of genome sequencing. We further present the first complete assembly of a genome from single cells using SMRT reads.hybridSPAdes is implemented in C++?as a part of SPAdes genome assembler and is publicly available at http://bioinf.spbau.ru/en/spades CONTACT: d.antipov@spbu.ruSupplementary information: supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Read more
July 7, 2019

Complete genome and methylome sequences of two Salmonella enterica spp.

Salmonella enterica is responsible for major foodborne outbreaks worldwide. It can cause gastroenteritis characterized by diarrhea, vomiting, and fever. Salmonella infections raise public health concerns along with consequential economic impacts. In this report, we announce the first complete genome sequences of Salmonella enterica subsp. enterica serovar Choleraeuis (S. Choleraeuis) ATCC 10708 and Salmonella enterica subsp. enterica serovar Pullorum (S. Pullorum) ATCC 9120, isolated from patients with diarrhea. Copyright © 2016 Yao et al.

Read more
July 7, 2019

Adaptive engineering of a hyperthermophilic archaeon on CO and discovering the underlying mechanism by multi-omics analysis.

The hyperthermophilic archaeon Thermococcus onnurineus NA1 can grow and produce H2 on carbon monoxide (CO) and its H2 production rates have been improved through metabolic engineering. In this study, we applied adaptive evolution to enhance H2 productivity. After over 150 serial transfers onto CO medium, cell density, CO consumption rate and H2 production rate increased. The underlying mechanism for those physiological changes could be explained by using multi-omics approaches including genomic, transcriptomic and epigenomic analyses. A putative transcriptional regulator was newly identified to regulate the expression levels of genes related to CO oxidation. Transcriptome analysis revealed significant changes in the transcript levels of genes belonging to the categories of transcription, translation and energy metabolism. Our study presents the first genome-scale methylation pattern of hyperthermophilic archaea. Adaptive evolution led to highly enhanced H2 productivity at high CO flow rates using synthesis gas produced from coal gasification.

Read more
July 7, 2019

Whole genome sequence of Pantoea ananatis R100, an antagonistic bacterium isolated from rice seed.

Pantoea ananatis is a group of bacteria, which was first reported as plant pathogen. Recently, several papers also described its biocontrol ability. In 2003, P. ananatis R100, which showed strong antagonism against several plant pathogens, was isolated from rice seeds. In this study, whole genome sequence of this strain was determined by SMRT Cell technology. The total genome size of R100 is 4,857,861bp with 4659 coding genes (CDS), 82 tRNAs and 22 rRNAs. The genome sequence of R100 may shed a light on the research of antagonism P. ananatis. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

Read more

Subscribe for blog updates:

Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.