scholarly journals Amplified Intergenic Locus Polymorphism as a Basis for Bacterial Typing of Listeria spp. and Escherichiacoli

2005 ◽  
Vol 71 (6) ◽  
pp. 3144-3152 ◽  
Author(s):  
Lilach Somer ◽  
Yael Danin-Poleg ◽  
Eran Diamant ◽  
Riva Gur-Arie ◽  
Yniv Palti ◽  
...  
Keyword(s):  
Low Cost ◽  
Size Variation ◽  
Pcr Primers ◽  
Intergenic Sequence ◽  
Bacterial Genomes ◽  
Bacterial Typing ◽  
Reading Frame ◽  
E Coli ◽  
Phylogenetic Studies ◽  
Intergenic Regions

ABSTRACT DNA-based methods are increasingly important for bacterial typing. The high number of polymorphic sites present among closely related bacterial genomes is the basis for the presented method. The method identifies multilocus genomic polymorphisms in intergenic regions termed AILP (amplified intergenic locus polymorphism). For each locus, a pair of unique PCR primers was designed to amplify an intergenic sequence from one open reading frame (ORF) to the adjacent ORF. Presence, absence, and size variation of the amplification products were identified and used as genetic markers for rapidly differentiating among strains. Polymorphism was evaluated using 18 AILP sites among 28 strains of Listeria monocytogenes and 6 strains of Listeria spp. and 30 AILP markers among 27 strains of Escherichia coli. Up to four alleles per locus were identified among Listeria strains, and up to six were identified among E. coli strains. In both species, more than half of the AILP sites revealed intraspecies polymorphism. The AILP data were applied to phylogenetic analysis among Listeria and E. coli strains. A clear distinction between L. monocytogenes and Listeria spp. was demonstrated. In addition, the method separated L. monocytogenes into the three known lineages and discriminated the most common virulent serotypic group, 4b. In E. coli, AILP analysis separated the known groups as well as the virulent O157:H7 isolates. These findings for both Listeria and E. coli are in agreement with other phylogenetic studies using molecular markers. The AILP method was found to be rapid, simple, reproducible, and a low-cost method for initial bacterial typing that could serve as a basis for epidemiological investigation.

A Search for Ribonucleic Antiterminator Sites in Bacterial Genomes: Not Only Antitermination

2015 ◽  
Vol 25 (2-3) ◽  
pp. 143-153 ◽  
Author(s):  
Noa Gordon ◽  
Ronen Rosenblum ◽  
Anat Nussbaum-Shochat ◽  
Elad Eliahoo ◽  
Orna Amster-Choder
Keyword(s):  
Rna Stability ◽  
Open Reading Frames ◽  
Structural Constraints ◽  
Bacterial Genomes ◽  
Rna Motifs ◽  
E Coli ◽  
Bioinformatic Tools ◽  
Intergenic Regions

BglG/LicT-like proteins are transcriptional antiterminators that prevent termination of transcription at intrinsic terminators by binding to ribonucleic antiterminator (RAT) sites and stabilizing an RNA conformation which is mutually exclusive with the terminator structure. The known RAT sites, which are located in intergenic regions of sugar utilization operons, show low sequence conservation but significant structural analogy. To assess the prevalence of RATs in bacterial genomes, we employed bioinformatic tools that describe RNA motifs based on both sequence and structural constraints. Using descriptors with different stringency, we searched the genomes of <i>Escherichia</i><i>coli</i> K12, uropathogenic <i>E. coli</i> and <i>Bacillus subtilis</i> for putative RATs. Our search identified all known RATs and additional putative RAT elements. Surprisingly, most putative RATs do not overlap an intrinsic terminator and many reside within open reading frames (ORFs). The ability of one of the putative RATs, which is located within an antiterminator-encoding ORF and does not overlap a terminator, to bind to its cognate antiterminator protein in vitro and in vivo was confirmed experimentally. Our results suggest that the capacity of RAT elements has been exploited during evolution to mediate activities other than antitermination, for example control of transcription elongation or of RNA stability.

scholarly journals Variation in Plastome Sizes Accompanied by Evolutionary History in Monogenomic Triticeae (Poaceae: Triticeae)

2021 ◽  
Vol 12 ◽  
Author(s):  
Ning Chen ◽  
Li-Na Sha ◽  
Yi-Ling Wang ◽  
Ling-Juan Yin ◽  
Yue Zhang ◽  
...  
Keyword(s):  
Chloroplast Genome ◽  
Genome Size ◽  
Divergence Time ◽  
Size Variation ◽  
Intergenic Sequence ◽  
Protein Coding ◽  
Ancestral Genome Reconstruction ◽  
Adaptive Processes ◽  
Intergenic Regions ◽  
Plastid Genome Evolution

To investigate the pattern of chloroplast genome variation in Triticeae, we comprehensively analyzed the indels in protein-coding genes and intergenic sequence, gene loss/pseudonization, intron variation, expansion/contraction in inverted repeat regions, and the relationship between sequence characteristics and chloroplast genome size in 34 monogenomic Triticeae plants. Ancestral genome reconstruction suggests that major length variations occurred in four-stem branches of monogenomic Triticeae followed by independent changes in each genus. It was shown that the chloroplast genome sizes of monogenomic Triticeae were highly variable. The chloroplast genome of Pseudoroegneria, Dasypyrum, Lophopyrum, Thinopyrum, Eremopyrum, Agropyron, Australopyrum, and Henradia in Triticeae had evolved toward size reduction largely because of pseudogenes elimination events and length deletion fragments in intergenic. The Aegilops/Triticum complex, Taeniatherum, Secale, Crithopsis, Herteranthelium, and Hordeum in Triticeae had a larger chloroplast genome size. The large size variation in major lineages and their subclades are most likely consequences of adaptive processes since these variations were significantly correlated with divergence time and historical climatic changes. We also found that several intergenic regions, such as petN–trnC and psbE–petL containing unique genetic information, which can be used as important tools to identify the maternal relationship among Triticeae species. Our results contribute to the novel knowledge of plastid genome evolution in Triticeae.

scholarly journals Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1385
Author(s):  
Giulia Pezzoni ◽  
Lidia Stercoli ◽  
Eleonora Pegoiani ◽  
Emiliana Brocchi
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Recombinant Antigen ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Antigenic Characterization ◽  
Antigenic Properties ◽  
Open Reading Frame 2

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

scholarly journals Cloning and characterization of α-pinene synthase from Chamaecyparis formosensis Matsum

Holzforschung ◽  
2009 ◽  
Vol 63 (1) ◽  
Author(s):  
Fang-Hua Chu ◽  
Pei-Min Kuo ◽  
Yu-Rong Chen ◽  
Sheng-Yang Wang
Keyword(s):  
Major Product ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Geranyl Diphosphate ◽  
Reading Frame ◽  
E Coli ◽  
Phylogenetic Taxonomy ◽  
Chamaecyparis Formosensis ◽  
Pcr Method

AbstractAnalyzing the gene sequences of terpene synthase (TPS) may contribute to a better understanding of terpenes biosynthesis and evolution of phylogenetic taxonomy.Chamaecyparis formosensisis an endemic and precious conifer of Taiwan. To understand the biosynthesis mechanism of terpenes in this tree, a full length of putative mono-TPS, named asCf-Pin(GeneBank accession no. EU099434), was obtained by PCR method and RACE extension. TheCf-Pinhas an 1887-bp open reading frame and encodes 628 amino acids. To identify the function ofCf-Pin,the recombinant protein fromEscherichia coliwas incubated with geranyl diphosphate, produced one major product, the structure of which was elucidated. GC/MS analysis and matching of retention time and mass spectrum with authentic standards revealed that this product isα-pinene. This is the first report of cloning of a mono-TPS and functionally expressed inE. coliand which could be identified asα-pinene synthase from a Cupressaceae conifer.

scholarly journals A New Clone Sweeps Clean: the Enigmatic Emergence of Escherichia coli Sequence Type 131

2014 ◽  
Vol 58 (9) ◽  
pp. 4997-5004 ◽  
Author(s):  
Ritu Banerjee ◽  
James R. Johnson
Keyword(s):  
Escherichia Coli ◽  
Sequence Type ◽  
Rapid Expansion ◽  
Future Research ◽  
Content Type ◽  
Extended Spectrum Beta Lactamase ◽  
E Coli ◽  
Phylogenetic Studies ◽  
Ecological Success

ABSTRACTEscherichia colisequence type 131 (ST131) is an extensively antimicrobial-resistantE. coliclonal group that has spread explosively throughout the world. Recent molecular epidemiologic and whole-genome phylogenetic studies have elucidated the fine clonal structure of ST131, which comprises multiple ST131 subclones with distinctive resistance profiles, including the (nested) H30, H30-R, and H30-Rx subclones. The most prevalent ST131 subclone, H30, arose from a single common fluoroquinolone (FQ)-susceptible ancestor containing allele 30 offimH(type 1 fimbrial adhesin gene). An early H30 subclone member acquired FQ resistance and launched the rapid expansion of the resulting FQ-resistant subclone, H30-R. Subsequently, a member of H30-R acquired the CTX-M-15 extended-spectrum beta-lactamase and launched the rapid expansion of the CTX-M-15-containing subclone within H30-R, H30-Rx. Clonal expansion clearly is now the dominant mechanism for the rising prevalence of both FQ resistance and CTX-M-15 production in ST131 and inE. coligenerally. Reasons for the successful dissemination and expansion of the key ST131 subclones remain undefined but may include increased transmissibility, greater ability to colonize and/or persist in the intestine or urinary tract, enhanced virulence, and more-extensive antimicrobial resistance compared to otherE. coli. Here we discuss the epidemiology and molecular phylogeny of ST131 and its key subclones, possible mechanisms for their ecological success, implications of their widespread dissemination, and future research needs.

scholarly journals Amino acids at the site of V kappa-J kappa recombination not encoded by germline sequences.

1987 ◽  
Vol 166 (3) ◽  
pp. 637-646 ◽  
Author(s):  
M Heller ◽  
J D Owens ◽  
J F Mushinski ◽  
S Rudikoff
Keyword(s):  
Heavy Chain ◽  
De Novo ◽  
Size Variation ◽  
Chain Gene ◽  
Gene Recombination ◽  
Heavy Chain Gene ◽  
Single Nucleotide ◽  
Reading Frame ◽  
Germline Genes ◽  
Unusual Amino Acid

Murine V kappa-J kappa recombination is characterized by a maintenance of size at the site of recombination and the use of nucleic acids found only in germline sequences. This is in contrast to heavy chain VH-D-JH assembly where random nucleotides are added at the recombination sites to produce considerable size variation, even though the heptamer/nonomer recombination sequences are identical in both kappa and heavy chain genes. We have examined the origin of an unusual amino acid, Ile, found at the site of V kappa-J kappa recombination in antigalactan antibodies, by sequence analysis of the corresponding rearranged and germline genes. Results indicate that the Ile codon can be generated by use of a single nucleotide 3' of the V kappa segment in combination with the second and third nucleotides of the first codon of J kappa 5 or J kappa 4. However, several antigalactan antibodies express Ile in combination with J kappa 2. An Ile codon cannot be generated by recombination in any reading frame between germline V kappa and J kappa 2 segments. These results suggest that the origin of the Ile codon in lines using J kappa 2 may represent a novel even in murine light chain assembly, possibly similar to the de novo addition of nucleotides observed in heavy chain gene recombination.

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

scholarly journals Improved CUT&RUN chromatin profiling and analysis tools

2019 ◽  
Author(s):  
Michael P. Meers ◽  
Terri Bryson ◽  
Steven Henikoff
Keyword(s):  
Fusion Protein ◽  
High Efficiency ◽  
Low Cost ◽  
Protein A ◽  
Micrococcal Nuclease ◽  
Permeabilized Cells ◽  
E Coli ◽  
Carry Over ◽  
Chromatin Profiling ◽  
Premature Release

AbstractWe previously described a novel alternative to Chromatin Immunoprecipitation, Cleavage Under Targets & Release Using Nuclease (CUT&RUN), in which unfixed permeabilized cells are incubated with antibody, followed by binding of a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein (1). Upon activation of tethered MNase, the bound complex is excised and released into the supernatant for DNA extraction and sequencing. Here we introduce four enhancements to CUT&RUN: 1) a hybrid Protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification; 2) a modified digestion protocol that inhibits premature release of the nuclease-bound complex; 3) a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein; and 4) a novel peak-calling strategy customized for the low-background profiles obtained using CUT&RUN. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling.

scholarly journals The large-scale blast score ratio (LS-BSR) pipeline: a method to rapidly compare genetic content between bacterial genomes

2014 ◽  
Author(s):  
Jason W Sahl ◽  
Greg Caporaso ◽  
David A Rasko ◽  
Paul S Keim
Keyword(s):  
Large Scale ◽  
Sequence Data ◽  
Parallel Implementation ◽  
Genetic Relationships ◽  
Clinical Diagnostics ◽  
Whole Genome Sequence ◽  
Bacterial Isolates ◽  
Bacterial Genomes ◽  
E Coli ◽  
Blast Score

Background. As whole genome sequence data from bacterial isolates becomes cheaper to generate, computational methods are needed to correlate sequence data with biological observations. Here we present the large-scale BLAST score ratio (LS-BSR) pipeline, which rapidly compares the genetic content of hundreds to thousands of bacterial genomes, and returns a matrix that describes the relatedness of all coding sequences (CDSs) in all genomes surveyed. This matrix can be easily parsed in order to identify genetic relationships between bacterial genomes. Although pipelines have been published that group peptides by sequence similarity, no other software performs the large-scale, flexible, full-genome comparative analyses carried out by LS-BSR. Results. To demonstrate the utility of the method, the LS-BSR pipeline was tested on 96 Escherichia coli and Shigella genomes; the pipeline ran in 163 minutes using 16 processors, which is a greater than 7-fold speedup compared to using a single processor. The BSR values for each CDS, which indicate a relative level of relatedness, were then mapped to each genome on an independent core genome single nucleotide polymorphism (SNP) based phylogeny. Comparisons were then used to identify clade specific CDS markers and validate the LS-BSR pipeline based on molecular markers that delineate between classical E. coli pathogenic variant (pathovar) designations. Scalability tests demonstrated that the LS-BSR pipeline can process 1,000 E. coli genomes in ~60h using 16 processors. Conclusions. LS-BSR is an open-source, parallel implementation of the BSR algorithm, enabling rapid comparison of the genetic content of large numbers of genomes. The results of the pipeline can be used to identify specific markers between user-defined phylogenetic groups, and to identify the loss and/or acquisition of genetic information between bacterial isolates. Taxa-specific genetic markers can then be translated into clinical diagnostics, or can be used to identify broadly conserved putative therapeutic candidates.

scholarly journals Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

2018 ◽  
Author(s):  
Krithika Rajagopalan ◽  
Jonathan Dworkin
Keyword(s):  
Biochemical Characterization ◽  
Regulatory Protein ◽  
Biochemical Properties ◽  
Bacterial Genomes ◽  
Phosphatase Inhibitors ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Genes Encoding ◽  
Number Of Bacteria

AbstractIn bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such asE. coli,which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C phosphatases. These proteins, called eSTPs (eukaryotic-like Ser/Thr phosphatases), have been identified in a number of bacteria, but not inE. coli.Here, we describe a previously unknown eSTP encoded by anE. coliORF,yegK,and characterize its biochemical properties including its kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely relatedE. colistrains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon.ImportanceRegulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria including the model Gram-negative bacteriumE. colidemonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thrphosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

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