Project description:Deuterium isotope labelling is important for structural biology methods such as neutron protein crystallography, nuclear magnetic resonance and small angle neutron scattering studies of proteins. Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affect growth of cells. The generation time for Escherichia coli K-12 in deuterated medium is substantially increased compared to cells grown in hydrogenated (protiated) medium. By using a mutagenesis plasmid based approach we have isolated an E. coli strain derived from E. coli K-12 substrain MG1655 that show increased fitness in deuterium based growth media, without general adaptation to media components. By whole-genome sequencing we identified the genomic changes in the obtained strain and show that it can be used for recombinant production of perdeuterated proteins in amounts typically needed for structural biology studies.
Project description:We report the complete sequence of ER2796, a laboratory strain of Escherichia coli K-12 that is completely defective in DNA methylation. Because of its lack of any native methylation, it is extremely useful as a host into which heterologous DNA methyltransferase genes can be cloned and the recognition sequences of their products deduced by Pacific Biosciences Single-Molecule Real Time (SMRT) sequencing. The genome was itself sequenced from a long-insert library using the SMRT platform, resulting in a single closed contig devoid of methylated bases. Comparison with K-12 MG1655, the first E. coli K-12 strain to be sequenced, shows an essentially co-linear relationship with no major rearrangements despite many generations of laboratory manipulation. The comparison revealed a total of 41 insertions and deletions, and 228 single base pair substitutions. In addition, the long-read approach facilitated the surprising discovery of four gene conversion events, three involving rRNA operons and one between two cryptic prophages. Such events thus contribute both to genomic homogenization and to bacteriophage diversification. As one of relatively few laboratory strains of E. coli to be sequenced, the genome also reveals the sequence changes underlying a number of classical mutant alleles including those affecting the various native DNA methylation systems.
Project description:Thermocrinis albus Eder and Huber 2002 is one of three species in the genus Thermocrinis in the family Aquificaceae. Members of this family have become of significant interest because of their involvement in global biogeochemical cycles in high-temperature ecosystems. This interest had already spurred several genome sequencing projects for members of the family. We here report the first completed genome sequence a member of the genus Thermocrinis and the first type strain genome from a member of the family Aquificaceae. The 1,500,577 bp long genome with its 1,603 protein-coding and 47 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
Project description:The Orf virus (ORFV) is the causative agent of orf, a globally-occurring, acute, pustular, contagious disease affecting sheep, goats and humans with a worldwide distribution. Currently, the genomic analysis of four ORFV strains from the Fujian province in southern China and a NA1/11 strain isolated from the Jilin province in northeast China have been reported. However, little is known about the genomic information of ORFV strains from central China.From a recent outbreak in a sheep herd in the Henan province of central China, a novel ORFV strain (HN3/12) was isolated and cultured in ovine fetal turbinate (OFTu) cells. The strain was identified as HN3/12 and verified by PCR based on the DNA sequences of 011 and 059 genes. The whole genomic sequence of this isolate was determined by Next Generation Sequencing technology. To determine the genetic characteristics of the HN3/12 strain, phylogenetic analysis of the 011 and 059 genes and amino acid sequence alignment of the HN3/12 strain were performed and compared with reference parapoxvirus strains.The HN3/12 genome is 136,643 bp in length, contains 63.67% G + C and encodes 132 putative genes. Phylogenetic analysis of the 011 and 059 nucleotide sequences showed that this viral strain was similar to the NA1/11 isolate. The homology analysis indicates that HN3/12 has 93% to 98% identity with published ORFV strains at amino acid level. When open reading frames (ORFs) were aligned among the HN3/12 and four Fujian ORFV strains, most of them have identities greater than 90% and only a few less than 60%. The availability of the whole genomic sequence of HN3/12 aids in our understanding of, and provides new insights into, the genetic diversity of ORFV.
Project description:In vitro reactions are useful to identify putative enzyme substrates, but in vivo validation is required to identify actual enzyme substrates that have biological meaning. To investigate in vivo effects of prolyl endopeptidase (PREP), a serine protease, on alpha melanocyte stimulating hormone (alpha-MSH), we developed a new mass spectrometry based technique to quantitate, in multiplex, the various forms of alpha-MSH.Using Multiple Reaction Monitoring (MRM), we analyzed peptide transitions to quantify three different forms of alpha-MSH. Transitions were first confirmed using standard peptides. Samples were then analyzed by mass spectrometry using a triple quadrupole mass spectrometer, after elution from a reverse phase C18 column by a gradient of acetonitrile.We first demonstrate in vitro that PREP digests biological active alpha melanocyte stimulating hormone (alpha-MSH(1-13)), by cleaving the terminal amidated valine and releasing a truncated alpha melanocyte stimulating hormone (alpha-MSH(1-12)) product--the 12 residues alpha-MSH form. We then use the technique in vivo to analyze the MRM transitions of the three different forms of alpha-MSH: the deacetylated alpha-MSH(1-13), the acetylated alpha-MSH(1-13) and the truncated form alpha-MSH(1-12). For this experiment, we used a mouse model (PREP-GT) in which the serine protease, prolyl endopeptidase, is deficient due to a genetrap insertion. Here we report that the ratio between acetylated alpha-MSH(1-13) and alpha-MSH(1-12) is significantly increased (P-value = 0.015, N = 6) in the pituitaries of PREP-GT mice when compared to wild type littermates. In addition no significant changes were revealed in the relative level of alpha-MSH(1-13) versus the deacetylated alpha-MSH(1-13). These results combined with the demonstration that PREP digests alpha-MSH(1-13) in vitro, strongly suggest that alpha-MSH(1-13) is an in vivo substrate of PREP.The multiplex targeted quantitative peptidomics technique we present in this study will be decidedly useful to monitor several neuropeptide enzymatic reactions in vivo under varying conditions.
Project description:Escherichia coli strain K-12 substrain HMS174 is an engineered descendant of the E. coli K-12 wild-type strain. Like its ancestor, it is an important organism in biotechnological research and is used in fermentation processes for heterologous protein production. Here, we report the complete genome sequence of E. coli HMS174 (ATCC 47011).
Project description:The neurotropic herpesvirus varicella-zoster virus (VZV) establishes a lifelong latent infection in humans following primary infection. The low abundance of VZV nucleic acids in human neurons has hindered an understanding of the mechanisms that regulate viral gene transcription during latency. To overcome this critical barrier, we optimized a targeted capture protocol to enrich VZV DNA and cDNA prior to whole-genome/transcriptome sequence analysis. Since the VZV genome is remarkably stable, it was surprising to detect that VZV32, a VZV laboratory strain with no discernible growth defect in tissue culture, contained a 2,158-bp deletion in open reading frame (ORF) 12. Consequently, ORF 12 and 13 protein expression was abolished and Akt phosphorylation was inhibited. The discovery of the ORF 12 deletion, revealed through targeted genome sequencing analysis, points to the need to authenticate the VZV genome when the virus is propagated in tissue culture.IMPORTANCE Viruses isolated from clinical samples often undergo genetic modifications when cultured in the laboratory. Historically, VZV is among the most genetically stable herpesviruses, a notion supported by more than 60 complete genome sequences from multiple isolates and following multiple in vitro passages. However, application of enrichment protocols to targeted genome sequencing revealed the unexpected deletion of a significant portion of VZV ORF 12 following propagation in cultured human fibroblast cells. While the enrichment protocol did not introduce bias in either the virus genome or transcriptome, the findings indicate the need for authentication of VZV by sequencing when the virus is propagated in tissue culture.
Project description:Several approaches were used to construct a complete NotI restriction enzyme cleavage map of the genome of Escherichia coli MG1655. The approaches included use of transposable element insertions that created auxotrophic mutations and introduced a NotI site into the genome, hybridization of NotI fragments to the ordered lambda library constructed by Kohara et al. (BioTechniques 10:474-477, 1991), Southern blotting of NotI digests with cloned genes as probes, and analysis of the known E. coli DNA sequence for NotI sites. In all, 22 NotI cleavage sites were mapped along with 26 transposon insertions. These sites were localized to clones in the lambda library and, when possible, sequenced genes. The map was compared with that of strain EMG2, a wild-type E. coli K-12 strain, and several differences were found, including a region of about 600 kb with an altered restriction pattern and an additional fragment in MG1655. Comparison of MG1655 with other strains revealed minor differences but indicated that this map was representative of that for many commonly used E. coli K-12 strains.
Project description:Escherichia coli strain K-12 substrain RV308 is an engineered descendant of the K-12 wild-type strain. Like its ancestor, it is an important organism in biotechnological research and is heavily used for the expression of single-chain variable fragments. Here, we report the complete genome sequence of E. coli K-12 RV308 (ATCC 31608).