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Auto Tag: Escherichia coli

July 7, 2019

Complete genome analysis of Thermus parvatiensis and comparative genomics of Thermus spp. provide insights into genetic variability and evolution of natural competence as strategic survival attributes.

Thermophilic environments represent an interesting niche. Among thermophiles, the genus Thermus is among the most studied genera. In this study, we have sequenced the genome of Thermus parvatiensis strain RL, a thermophile isolated from Himalayan hot water springs (temperature >96°C) using PacBio RSII SMRT technique. The small genome (2.01 Mbp) comprises a chromosome (1.87 Mbp) and a plasmid (143 Kbp), designated in this study as pTP143. Annotation revealed a high number of repair genes, a squeezed genome but containing highly plastic plasmid with transposases, integrases, mobile elements and hypothetical proteins (44%). We performed a comparative genomic study of the group Thermus with an aim of analysing the phylogenetic relatedness as well as niche specific attributes prevalent among the group. We compared the reference genome RL with 16 Thermus genomes to assess their phylogenetic relationships based on 16S rRNA gene sequences, average nucleotide identity (ANI), conserved marker genes (31 and 400), pan genome and tetranucleotide frequency. The core genome of the analyzed genomes contained 1,177 core genes and many singleton genes were detected in individual genomes, reflecting a conserved core but adaptive pan repertoire. We demonstrated the presence of metagenomic islands (chromosome:5, plasmid:5) by recruiting raw metagenomic data (from the same niche) against the genomic replicons of T. parvatiensis. We also dissected the CRISPR loci wide all genomes and found widespread presence of this system across Thermus genomes. Additionally, we performed a comparative analysis of competence loci wide Thermus genomes and found evidence for recent horizontal acquisition of the locus and continued dispersal among members reflecting that natural competence is a beneficial survival trait among Thermus members and its acquisition depicts unending evolution in order to accomplish optimal fitness.

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July 7, 2019

SMRT Sequencing revealed mitogenome characteristics and mitogenome-wide DNA modification pattern in Ophiocordyceps sinensis.

Single molecule, real-time (SMRT) sequencing was used to characterize mitochondrial (mt) genome of Ophiocordyceps sinensis and to analyze the mt genome-wide pattern of epigenetic DNA modification. The complete mt genome of O. sinensis, with a size of 157,539 bp, is the fourth largest Ascomycota mt genome sequenced to date. It contained 14 conserved protein-coding genes (PCGs), 1 intronic protein rps3, 27 tRNAs and 2 rRNA subunits, which are common characteristics of the known mt genomes in Hypocreales. A phylogenetic tree inferred from 14 PCGs in Pezizomycotina fungi supports O. sinensis as most closely related to Hirsutella rhossiliensis in Ophiocordycipitaceae. A total of 36 sequence sites in rps3 were under positive selection, with dN/dS >1 in the 20 compared fungi. Among them, 16 sites were statistically significant. In addition, the mt genome-wide base modification pattern of O. sinensis was determined in this study, especially DNA methylation. The methylations were located in coding and uncoding regions of mt PCGs in O. sinensis, and might be closely related to the expression of PCGs or the binding affinity of transcription factor A to mtDNA. Consequently, these methylations may affect the enzymatic activity of oxidative phosphorylation and then the mt respiratory rate; or they may influence mt biogenesis. Therefore, methylations in the mitogenome of O. sinensis might be a genetic feature to adapt to the cold and low PO2 environment at high altitude, where O. sinensis is endemic. This is the first report on epigenetic modifications in a fungal mt genome.

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July 7, 2019

Complete genome sequencing and targeted mutagenesis reveal virulence contributions of Tal2 and Tal4b of Xanthomonas translucens pv. undulosa ICMP11055 in bacterial leaf streak of wheat

Bacterial leaf streak caused by Xanthomonas translucens pv. undulosa (Xtu) is an important disease of wheat (Triticum aestivum) and barley (Hordeum vulgare) worldwide. Transcription activator-like effectors (TALEs) play determinative roles in many of the plant diseases caused by the different species and pathovars of Xanthomonas, but their role in this disease has not been characterized. ICMP11055 is a highly virulent Xtu strain from Iran. The aim of this study was to better understand genetic diversity of Xtu and to assess the role of TALEs in bacterial leaf streak of wheat by comparing the genome of this strain to the recently completely sequenced genome of a U.S. Xtu strain, and to several other draft X. translucens genomes, and by carrying out mutational analyses of the TALE (tal) genes the Iranian strain might harbor. The ICMP11055 genome, including its repeat-rich tal genes, was completely sequenced using single molecule, real-time technology (Pacific Biosciences). It consists of a single circular chromosome of 4,561,583 bp, containing 3,953 genes. Whole genome alignment with the genome of the United States Xtu strain XT4699 showed two major re-arrangements, nine genomic regions unique to ICMP11055, and one region unique to XT4699. ICMP110055 harbors 26 non-TALE type III effector genes and seven tal genes, compared to 25 and eight for XT4699. The tal genes occur singly or in pairs across five scattered loci. Four are identical to tal genes in XT4699. In addition to common repeat-variable diresidues (RVDs), the tal genes of ICMP11055, like those of XT4699, encode several RVDs rarely observed in Xanthomonas, including KG, NF, Y*, YD, and YK. Insertion and deletion mutagenesis of ICMP11055 tal genes followed by genetic complementation analysis in wheat cv. Chinese Spring revealed that Tal2 and Tal4b of ICMP11055 each contribute individually to the extent of disease caused by this strain. A largely conserved ortholog of tal2 is present in XT4699, but for tal4b, only a gene with partial, fragmented RVD sequence similarity can be found. Our results lay the foundation for identification of important host genes activated by Xtu TALEs as targets for the development of disease resistant varieties.

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July 7, 2019

Comparative sequence analysis of multidrug-resistant IncA/C plasmids from Salmonella enterica

Determinants of multidrug resistance (MDR) are often encoded on mobile elements, such as plasmids, transposons, and integrons, which have the potential to transfer among foodborne pathogens, as well as to other virulent pathogens, increasing the threats these traits pose to human and veterinary health. Our understanding of MDR among Salmonella has been limited by the lack of closed plasmid genomes for comparisons across resistance phenotypes, due to difficulties in effectively separating the DNA of these high-molecular weight, low-copy-number plasmids from chromosomal DNA. To resolve this problem, we demonstrate an efficient protocol for isolating, sequencing and closing IncA/C plasmids from Salmonella sp. using single molecule real-time sequencing on a Pacific Biosciences (Pacbio) RS II Sequencer. We obtained six Salmonella enterica isolates from poultry, representing six different serovars, each exhibiting the MDR-Ampc resistance profile. Salmonella plasmids were obtained using a modified mini preparation and transformed with Escherichia coli DH10Br. A Qiagen Large-Construct kit™ was used to recover highly concentrated and purified plasmid DNA that was sequenced using PacBio technology. These six closed IncA/C plasmids ranged in size from 104 to 191 kb and shared a stable, conserved backbone containing 98 core genes, with only six differences among those core genes. The plasmids encoded a number of antimicrobial resistance genes, including those for quaternary ammonium compounds and mercury. We then compared our six IncA/C plasmid sequences: first with 14 IncA/C plasmids derived from S. enterica available at the National Center for Biotechnology Information (NCBI), and then with an additional 38 IncA/C plasmids derived from different taxa. These comparisons allowed us to build an evolutionary picture of how antimicrobial resistance may be mediated by this common plasmid backbone. Our project provides detailed genetic information about resistance genes in plasmids, advances in plasmid sequencing, and phylogenetic analyses, and important insights about how MDR evolution occurs across diverse serotypes from different animal sources, particularly in agricultural settings where antimicrobial drug use practices vary.

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July 7, 2019

Complete genome sequence of Pseudomonas antarctica PAMC 27494, a bacteriocin-producing psychrophile isolated from Antarctica.

Antimicrobial-producing, cold-adapted microorganisms have great potential for biotechnological applications in food, pharmaceutical, and cosmetic industries. Pseudomonas antarctica PAMC 27494, a psychrophile exhibiting antimicrobial activity, was isolated from an Antarctic freshwater sample. Here we report the complete genome of P. antarctica PAMC 27494. The strain contains a gene cluster encoding microcin B which inhibits DNA regulations by targeting the DNA gyrase. PAMC 27494 may produce R-type pyocins and also contains a complete set of proteins for the biosynthesis of adenosylcobalamin and possibly induces plant growth by supplying pyrroloquinoline quionone molecules. Copyright © 2017 Elsevier B.V. All rights reserved.

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July 7, 2019

Hydroxy-pentanones production by Bacillus sp. H15-1 and its complete genome sequence

Acyloins are useful organic compounds with reactive adjacent hydroxyl group and carbonyl group. Current research is usually constrained to acetoin (i.e. 3-hydroxy-2-butanone) and the biological production of other acyloins was scarcely reported. In this study, two hydroxy-pentanone metabolites (3-hydroxy-2-pentanone and 2-hydroxy-3-pentanone) of Bacillus sp. H15-1 were identified by gas chromatography–mass spectrometry and authentic standards. Then the complete genome of this strain was sequenced and de novo assembled to a single circular chromosome of 4,162,101 bp with a guanine-cytosine content of 46.3%, but no special genes were found for the biosynthesis of the hydroxy-pentanones. Since hydroxy-pentanones are the homologues of acetoin, the two genes alsD and alsS (encoding a-acetolactate decarboxylase and a-acetolactate synthase, respectively) responsible for acetoin formation in this strain were respectively expressed in Escherichia coli. The purified enzymes were found to be capable of transforming pyruvate and 2-oxobutanoate to the two hydroxy-pentanones. This study extends the knowledge on the biosynthesis of acyloins and provides helpful information for further utilizing Bacillus sp. H15-1 as a source of valuable acyloins.

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July 7, 2019

Comparative genomic and regulatory analyses of natamycin production of Streptomyces lydicus A02.

Streptomyces lydicus A02 is used by industry because it has a higher natamycin-producing capacity than the reference strain S. natalensis ATCC 27448. We sequenced the complete genome of A02 using next-generation sequencing platforms, and to achieve better sequence coverage and genome assembly, we utilized single-molecule real-time (SMRT) sequencing. The assembled genome comprises a 9,307,519-bp linear chromosome with a GC content of 70.67%, and contained 8,888 predicted genes. Comparative genomics and natamycin biosynthetic gene cluster (BGC) analysis showed that BGC are highly conserved among evolutionarily diverse strains, and they also shared closer genome evolution compared with other Streptomyces species. Forty gene clusters were predicted to involve in the secondary metabolism of A02, and it was richly displayed in two-component signal transduction systems (TCS) in the genome, indicating a complex regulatory systems and high diversity of metabolites. Disruption of the phoP gene of the phoR-phoP TCS and nsdA gene confirmed phosphate sensitivity and global negative regulation of natamycin production. The genome sequence and analyses presented in this study provide an important molecular basis for research on natamycin production in Streptomyces, which could facilitate rational genome modification to improve the industrial use of A02.

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July 7, 2019

Genetic characterization of mcr-1-bearing plasmids to depict molecular mechanisms underlying dissemination of the colistin resistance determinant.

To analyse and compare mcr-1-bearing plasmids from animal Escherichia coli isolates, and to investigate potential mechanisms underlying dissemination of mcr-1.Ninety-seven ESBL-producing E. coli strains isolated from pig farms in China were screened for the mcr-1 gene. Fifteen mcr-1-positive strains were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured.Three major types of mcr-1-bearing plasmids were recovered: IncX4 (~33 kb), IncI2 (~60 kb) and IncHI2 (~216-280 kb), among which the IncX4 and IncI2 plasmids were found to harbour the mcr-1 gene only, whereas multiple resistance elements including blaCTX-M, blaCMY, blaTEM, fosA, qnrS, floR and oqxAB were detected, in various combinations, alongside mcr-1 in the IncHI2 plasmids. The profiles of mcr-1-bearing plasmids in the test strains were highly variable, with coexistence of two mcr-1-bearing plasmids being common. However, the MIC of colistin was not affected by the number of mcr-1-carrying plasmids harboured. Comparative analysis of the plasmids showed that they contained an mcr-1 gene cassette with varied structures (mcr-1-orf, ISApl1-mcr-1-orf and Tn6330), with the IncHI2 type being the most active in acquiring foreign resistance genes. A novel transposon, Tn6330, with the structure ISApl1-mcr-1-orf-ISApl1 was found to be the key element mediating translocation of mcr-1 into various plasmid backbones through formation of a circular intermediate.The mcr-1 gene can be disseminated via multiple mobile elements including Tn6330, its circular intermediate and plasmids harbouring such elements. It is often co-transmitted with other resistance determinants through IncHI2 plasmids. The functional mechanism of Tn6330, a typical composite transposon harbouring mcr-1, should be further investigated.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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July 7, 2019

Post-termination ribosome intermediate acts as the gateway to ribosome recycling.

During termination of translation, the nascent peptide is first released from the ribosome, which must be subsequently disassembled into subunits in a process known as ribosome recycling. In bacteria, termination and recycling are mediated by the translation factors RF, RRF, EF-G, and IF3, but their precise roles have remained unclear. Here, we use single-molecule fluorescence to track the conformation and composition of the ribosome in real time during termination and recycling. Our results show that peptide release by RF induces a rotated ribosomal conformation. RRF binds to this rotated intermediate to form the substrate for EF-G that, in turn, catalyzes GTP-dependent subunit disassembly. After the 50S subunit departs, IF3 releases the deacylated tRNA from the 30S subunit, thus preventing reassembly of the 70S ribosome. Our findings reveal the post-termination rotated state as the crucial intermediate in the transition from termination to recycling. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

Molecular mechanism and genetic determinants of buprofezin degradation.

Buprofezin is a widely used insect growth regulator whose residue has been frequently detected in the environment, posing a threat to aquatic organisms and non-target insects. Microorganisms play an important role in the degradation of buprofezin in the natural environment. However, the relevant catabolic pathway has not been fully characterized, and the molecular mechanism of catabolism is still completely unknown. Rhodococcus qingshengii YL-1 can utilize buprofezin as a sole source of carbon and energy for growth. In this study, the upstream catabolic pathway in strain YL-1 was identified using tandem mass spectrometry. Buprofezin is composed of a benzene ring and a heterocyclic ring. The degradation is initiated by the dihydroxylation of the benzene ring and continues via dehydrogenation, aromatic ring cleavage, breaking of an amide bond and the release of the heterocyclic ring 2-tert-butylimino-3-isopropyl-1,3,5-thiadiazinan-4-one (2-BI). A buprofezin degradation-deficient mutant strain YL-0 was isolated. Comparative genomic analysis combined with gene deletion and complementation experiments revealed that the gene cluster bfzBA3A4A1A2C is responsible for the upstream catabolic pathway of buprofezin. bfzA3A4A1A2 encodes a novel Rieske non-heme iron oxygenase (RHO) system that is responsible for the dihydroxylation of buprofezin at the benzene ring; bfzB is involved in dehydrogenation, and bfzC is in charge of benzene ring cleavage. Furthermore, the products of bfzBA3A4A1A2C can also catalyze dihydroxylation, dehydrogenation and aromatic ring cleavage of biphenyl, flavanone, flavone and bifenthrin. In addition, a transcriptional study revealed that bfzBA3A4A1A2C is organized in one transcriptional unit that is constitutively expressed in strain YL-1.Importance There is an increasing concern about the residue and environmental fate of buprofezin. Microbial metabolism is an important mechanism responsible for the buprofezin degradation in natural environment. However, the molecular mechanism and genetic determinants of microbial degradation of buprofezin has not been well identified. This work revealed that gene cluster bfzBA3A4A1A2C is responsible for the upstream catabolic pathway of buprofezin in R. qingshengii YL-1. The products of bfzBA3A4A1A2C could also degrade bifenthrin, a widely used pyrethroid insecticide. These findings enhance our understanding of the microbial degradation mechanism of buprofezin and benefit the application of strain YL-1 and bfzBA3A4A1A2C in the bioremediation of buprofezin contamination. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Characterization of a PVL-negative community-acquired methicillin-resistant Staphylococcus aureus strain of sequence type 88 in China.

Sequence type 88 community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) strain SR434, isolated from an outpatient with skin and soft tissue infection, was subjected to whole genome sequencing, antimicrobial susceptibility testing, mouse skin infection model and hemolysis analysis to identify its virulence and resistance determinants. MRSA strain SR434 is resistant to clindamycin, erythromycin and fosfomycin. Four plasmids with resistance genes were identified in this strain, including a 20,658bp blaZ-carrying plasmid, a 2473bp ermC-carrying plasmid, a 2622bp fosB7-carrying plasmid (86% identity with plasmid in a ST2590 MRSA strain) and a 4817bp lnuA-carrying plasmid (99% identity with pLNU4 from bovine coagulase-nagetive Staphylococci). This strain contains staphylococcal cassette chromosome mec type IV and does not contain arginine catabolic mobile element or Panton-Valentine-Leukocidin. SR434 harbors genomic islands ?Saa, ?Saß, ?Sa? and FSa3 and pathogenicity islands ?Sa2 that carries genes encoding toxic shock syndrome toxin 1, superantigen enterotoxin C and superantigen enterotoxin L. Mouse skin infection model results show that SR434 had similar virulence potential causing invasive skin infection as a PVL-negative epidemic Korea clone HL1 (ST72). CA-MRSA strain of ST88 lineage might be a great concern for its high virulence. PVL has limited contribution to virulence phenotype among this lineage. Copyright © 2017 Elsevier GmbH. All rights reserved.

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July 7, 2019

MECAT: fast mapping, error correction, and de novo assembly for single-molecule sequencing reads.

We present a tool that combines fast mapping, error correction, and de novo assembly (MECAT; accessible at https://github.com/xiaochuanle/MECAT) for processing single-molecule sequencing (SMS) reads. MECAT’s computing efficiency is superior to that of current tools, while the results MECAT produces are comparable or improved. MECAT enables reference mapping or de novo assembly of large genomes using SMS reads on a single computer.

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July 7, 2019

Detection and genetic features of MCR-1-producing plasmid in human Escherichia coli infection in South Korea.

The plasmid-mediated colistin resistance gene, mcr-1, was identified for the first time from a hospitalized patient in South Korea. The mcr-1 gene was successfully transferred to E. coli J53 recipient and conferred resistance to colistin in the recipient. The mcr-1-harboring plasmid possessed a typical IncI2 group and did not have the mcr-1-associated ISApl1 element. Copyright © 2017 Elsevier Inc. All rights reserved.

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July 7, 2019

IS26-mediated formation of a virulence and resistance plasmid in Salmonella Enteritidis.

To characterize a novel virulence-resistance plasmid pSE380T carried by a Salmonella enterica serotype Enteritidis clinical strain SE380.The plasmid pSE380T was conjugated to Escherichia coli strain J53 and sequenced by PacBio RSII, followed by subsequent annotation and genetic analysis.Sequence analysis of this plasmid revealed that the entire Salmonella Enteritidis-specific virulence plasmid, pSEN, had been incorporated into an IncHI2 MDR plasmid, which comprises the cephalosporin and fosfomycin resistance determinants blaCTX-M-14 and fosA3. Based on BLAST analysis and scrutiny of insertion footprints, the insertion event was found to involve a replicative transposition process mediated by IS26, an IS element frequently detected in various resistance plasmids. The resulting pSE380T plasmid also comprises backbone elements of IncHI2 and IncFIA plasmids, producing a rare fusion product that simultaneously encodes functional features of both, i.e. virulence, resistance and high transmissibility.This is a novel hybrid plasmid mediating MDR and virulence from a clinical Salmonella Enteritidis strain. This plasmid is likely to be transmissible amongst various serotypes of Salmonella and other Enterobacteriaceae species, rendering a wide range of bacterial pathogens resistant to cephalosporins and fosfomycin, and further enhancing their virulence potential. It will be important to monitor the spread and further evolution of this plasmid among the Enterobacteriaceae strains.© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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