Prokaryotic DNA Research Articles

Aspects of protein-DNA interactions: a review of quantitative thermodynamic theory for modelling synthetic circuits utilising LacI and CI repressors, IPTG and the reporter gene lacZ.

Protein-DNA interactions are central to the control of gene expression across all forms of life. The development of approaches to rigorously model such interactions has often been hindered both by a lack of quantitative binding data and by the difficulty in accounting for parameters relevant to the intracellular situation, such as DNA looping and thermodynamic non-ideality. Here, we review these considerations by developing a thermodynamically based mathematical model that attempts to simulate the functioning of an Escherichia coli expression system incorporating two of the best characterised prokaryotic DNA binding proteins, Lac repressor and lambda CI repressor. The key aim was to reproduce experimentally observed reporter gene activities arising from the expression of either wild-type CI repressor or one of three positive-control CI mutants. The model considers the role of several potentially important, but sometimes neglected, biochemical features, including DNA looping, macromolecular crowding and non-specific binding, and allowed us to obtain association constants for the binding of CI and its variants to a specific operator sequence.

Replacement of 2'-Deoxycytidine by 2'-Deoxycytidine Analogues in the E. coli Genome.

Several modified bases have been observed in the genomic DNA of bacteriophages, prokaryotes, and eukaryotes that play a role in restriction systems and/or epigenetic regulation. In our efforts to understand the consequences of replacing a large fraction of a canonical nucleoside with a modified nucleoside, we previously replaced around 75% of thymidine (T) with 5'-hydroxymethyl-2'-deoxyuridine (5hmU) in the Escherichia coli genome. In this study, we engineered the pyrimidine nucleotide biosynthetic pathway using T4 bacteriophage genes to achieve approximately 63% replacement of 2'-deoxycytidine (dC) with 5-hydroxymethyl-2'-deoxycytidine (5hmC) in the E. coli genome and approximately 71% replacement in plasmids. We further engineered the glucose metabolic pathway to transform the 5hmC into glucosyl-5-hydroxymethyl-2'-deoxycytidine (5-gmC) and achieved 20% 5-gmC in the genome and 45% 5-gmC in plasmid DNA.

Abundant DNA 6mA methylation during early embryogenesis of zebrafish and pig.

DNA N6-methyldeoxyadenosine (6mA) is a well-known prokaryotic DNA modification that has been shown to exist and play epigenetic roles in eukaryotic DNA. Here we report that 6mA accumulates up to ∼0.1–0.2% of total deoxyadenosine during early embryogenesis of vertebrates, but diminishes to the background level with the progression of the embryo development. During this process a large fraction of 6mAs locate in repetitive regions of the genome.

Foreign DNA acquisition by invertebrate genomes.

Recent studies have highlighted that the accidental acquisition of DNA from other species by invertebrate genomes is much more common than originally thought. The transferred DNAs are of bacterial or eukaryote origin and in both cases the receiver species may end up utilising the transferred genes for its own benefit. Frequent contact with prokaryotic DNA from symbiotic endocellular bacteria may predispose invertebrates to incorporate this genetic material into their genomes. Increasing evidence also points to viruses as major players in transferring genes and mobile elements between the species they infect. Unexpectedly a gene flux between Hymenoptera and Lepidoptera mediated by endogenous viruses of parasitic wasps has been recently unravelled, suggesting we are probably just seeing the tip of the iceberg concerning horizontal gene transfers in invertebrates. In the context of insect for feed and food, if the new technology of insect genome editing (such as Crisper/Cas9) were used to modify the genome of reared insects it is important to take into account the risk that an introduced gene can be transferred. More generally, although insects are traditionally consumed in Asia and Africa, knowledge on insect viruses is still limited rendering it difficult to predict the impact they might have in the context of insect rearing at an industrial scale.

Synthetic evolutionary origin of a proofreading reverse transcriptase.

Most reverse transcriptase (RT) enzymes belong to a single protein family of ancient evolutionary origin. These polymerases are inherently error prone, owing to their lack of a proofreading (3'- 5' exonuclease) domain. To determine if the lack of proofreading is a historical coincidence or a functional limitation of reverse transcription, we attempted to evolve a high-fidelity, thermostable DNA polymerase to use RNA templates efficiently. The evolutionarily distinct reverse transcription xenopolymerase (RTX) actively proofreads on DNA and RNA templates, which greatly improves RT fidelity. In addition, RTX enables applications such as single-enzyme reverse transcription-polymerase chain reaction and direct RNA sequencing without complementary DNA isolation. The creation of RTX confirms that proofreading is compatible with reverse transcription.

Replacement of Thymidine by a Modified Base in the Escherichia coli Genome.

Prokaryotic and eukaryotic genomic DNA is comprised of the four building blocks A, G, C, and T. We have begun to explore the consequences of replacing a large fraction or all of a nucleoside in genomic DNA with a modified nucleoside. As a first step we have investigated the possibility of replacement of T by 2'-deoxy-5-(hydroxymethyl)uridine (5hmU) in the genomic DNA of Escherichia coli. Metabolic engineering with phage genes followed by random mutagenesis enabled us to achieve approximately 75% replacement of T by 5hmU in the E. coli genome and in plasmids.

RepeatAnalyzer: a tool for analysing and managing short-sequence repeat data.

BackgroundShort-sequence repeats (SSRs) occur in both prokaryotic and eukaryotic DNA, inter- and intragenically, and may be exact or inexact copies. When heterogeneous SSRs are present in a given locus, we can take advantage of the pattern of different repeats to genotype strains based on the SSRs. Cataloguing and tracking these repeats can be difficult as diverse groups of researchers are involved in the identification of the repeats. Additionally, the task is error-prone when done manually.ResultsWe developed RepeatAnalyzer, a new software tool capable of tracking, managing, analysing and cataloguing SSRs and genotypes using Anaplasma marginale as a model species. RepeatAnalyzer’s analysis capability includes novel metrics for measuring regional genetic diversity (corresponding to variety and regularity of SSR occurrence). As a part of its visualization capabilities, RepeatAnalyzer produces high quality maps of the geographic distribution of genotypes or SSRs over a region of interest. RepeatAnalyzer’s repeat identification functionality was validated for all SSRs and genotypes reported in 21 publications, using 380 A. marginale isolates gathered from the five publications within that list that provided access to their isolates. The tool produced accurate genotyping results in every case. In addition, it uncovered a number of errors in the published literature: 11 cases where SSRs were misreported, 5 cases where two different SSRs had been given the same name, and 16 cases where two or more names had been given to a single SSR. The analysis and visualization functionalities of the tool are demonstrated using several examples.ConclusionsRepeatAnalyzer is a robust software tool that can be used for storing, managing, and analysing short-sequence repeats for the purpose of strain identification. The tool can be used for any set of SSRs regardless of species. When applied to A. marginale, our test case, we show that genotype lengths for a given region follow a normal distribution, while SSR frequencies follow a power-law-like distribution. Further, we find that over 90 % of repeats are 28 to 29 amino acids long, which is in agreement with conventional wisdom. Lastly, our analysis reveals that the most common edit distance is five or six, which is counter-intuitive since we expected that result to be closer to one, resulting from the simplest change from one repeat to another.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2686-2) contains supplementary material, which is available to authorized users.

Metagenomic analysis between free-living and cultured Epinephelus fuscoguttatus under different environmental conditions in Indonesian waters

In this study, we analyzed and compared feces of free-living and cultivated fish species, Epinephelus fuscoguttatus under different environmental conditions in Indonesian waters. Metagenome analysis was performed using Illumina MiSeq sequencing of the whole metagenomic DNA isolated from fish feces samples. The analysis covered both prokaryotic and eukaryotic DNA. Feces samples from mariculture fish revealed a highly stable distribution of several orders of bacteria when compared to samples from free-living fish, which were highly diverse and dominated by Vibrionales, Pseudomonales, Rhizobiales and non-classifiable Alphaproteobacteria. The eukaryotic content of the samples was dominated by residues of the host and nine additional fish species that formed a portion of the diet. Investigations on functional annotations for predominant bacterial taxa, using Gene Ontology enrichment, revealed a number of functions related to DNA metabolic processes, especially DNA repair, as well as antibiotic response in the free-living fish species.

Conserved motif VIII of murine DNA methyltransferase Dnmt3a is essential for methylation activity.

BackgroundDnmt3a is a DNA methyltransferase that establishes de novo DNA methylation in mammals. The structure of the Dnmt3a C-terminal domain is similar to the bacterial M. HhaI enzyme, a well-studied prokaryotic DNA methyltransferase. No X-ray structure is available for the complex of Dnmt3a with DNA and the mechanistic details of DNA recognition and catalysis by mammalian Dnmts are not completely understood.ResultsMutant variants of the catalytic domain of the murine Dnmt3a carrying substitutions of highly conserved N167, R200, and R202 have been generated by site directed mutagenesis and purified. Their methylation activity, DNA binding affinity, ability to flip the target cytosine out of the DNA double helix and covalent complex formation with DNA have been examined. Substitutions of N167 lead to reduced catalytic activity and reduced base flipping. Catalytic activity, base flipping, and covalent conjugate formation were almost completely abolished for the mutant enzymes with substitutions of R200 or R202.ConclusionsWe conclude that R202 plays a similar role in catalysis in Dnmt3a-CD as R232 in M.SssI and R165 in M.HhaI, which could be positioning of the cytosine for nucleophilic attack by a conserved Cys. R200 of Dnmt3a-CD is important in both catalysis and cytosine flipping. Both conserved R200 and R202 are involved in creating and stabilizing of the transient covalent intermediate of the methylation reaction. N167 might contribute to the positioning of the residues from the motif VI, but does not play a direct role in catalysis.

Nanosized Fe3O4 an efficient PCR yield enhancer—Comparative study with Au, Ag nanoparticles

Nanomaterials-assisted PCR is a promising field of nanobiotechnology that amalgamates nanomaterials into the conventional PCR system to achieve better amplification of desired product. With literature documenting the variable effects of these nanomaterials on the PCR yield and amplification; it was thought worthwhile to compare the PCR enhancing efficiency of three transition metal nanoparticles in form of stable colloidal suspensions at varying concentrations.The nanoparticles(NPs) of silver, gold and magnetite were chemically synthesized by reducing their respective salts and characterized using UV–vis spectroscopy. Their morphology was assessed using nanoparticle tracking system and AFM. The effect of these nanofluids on amplification of 800bp prokaryotic DNA template with 30% GC content was studied using conventional thermal cycler. The reaction kinetics for all the three nanofluids yielded a Gaussian curve of amplification with varying concentrations. The ammonium salt of oleic acid coated magnetite (Fe3O4) nanoparticles at a concentration of 0.72×10−2nM and average size of 33nm demonstrated highest amplification efficiency of 190% as compared to the citrate stabilized AgNP-25nm (45%) and AuNP-15.19nm (134%) using a conventional PCR system. The major reasons that allow Fe3O4 NPs outperform the other 2 transition metal NP’s seem to be attributed to its heat conduction property as well as effective adsorption of PCR components onto the ammonium salt of oleic acid coated magnetite nanofluids. The data from our study offers valuable information for the application of ferrofluids as economically, efficient and effective alternative for nanomaterial-assisted PCR yield enhancers.

Directly interrogating single quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe.

In this study we describe a new methodology to physically probe individual complexes formed between proteins and DNA. By combining nanoscale, high speed physical force measurement with sensitive fluorescence imaging we investigate the complex formed between the prokaryotic DNA repair protein UvrA2 and DNA. This approach uses a triangular, optically-trapped “nanoprobe” with a nanometer scale tip protruding from one vertex. By scanning this tip along a single DNA strand suspended between surface-bound micron-scale beads, quantum-dot tagged UvrA2 molecules bound to these ‘”DNA tightropes” can be mechanically interrogated. Encounters with UvrA2 led to deflections of the whole nanoprobe structure, which were converted to resistive force. A force histogram from all 144 detected interactions generated a bimodal distribution centered on 2.6 and 8.1 pN, possibly reflecting the asymmetry of UvrA2’s binding to DNA. These observations successfully demonstrate the use of a highly controllable purpose-designed and built synthetic nanoprobe combined with fluorescence imaging to study protein-DNA interactions at the single molecule level.

Changes in 16s RNA Gene Microbial Community Profiling by Concentration of Prokaryotic DNA

Microbial metagenomics are hindered in clinical tissue samples as a result of the large relative amount of human DNA in relation to microbial DNA acting as competitive inhibitors of downstream applications. We evaluated the LOOXSTER® Enrichment Kit to separate eukaryotic and prokaryotic DNA in submucosal intestinal tissue samples having a low microbial biomass and to determine the effects of enrichment on 16s rRNA microbiota sequencing. The enrichment kit reduced the amount of human DNA in the samples 40–70% resulting in a 3.5-fold increase in the number of 16s bacterial gene sequences detected on the Illumina MiSeq platform. This increase was accompanied by the detection of 41 additional bacterial genera and 94 tentative species. The additional bacterial taxa detected accounted for as much as 25% of the total bacterial population that significantly altered the relative prevalence and composition of the intestinal microbiota. The ability to reduce the competitive inhibition created by human DNA and the concentration of bacterial DNA may allow metagenomics to be performed on complex tissues containing a low bacterial biomass.

Synthesis of New Alkylated Mononucleotide Analogues and Their Repair Proficiency by the Prokaryotic DNA Repair Protein AlkB

We describe the synthesis of a variety of new alkylated mononucleotides and examine their repair proficiency with the prokaryotic direct DNA repair protein AlkB. A comparison of rates of repair among the different substrates supports the currently accepted radical mechanism.

Timeline: Sending Out an SOS

Walked out this morning, don't believe what I saw/Hundred billion bottles washed up on the shore –Sting. When looking at the myriad changes in the transcriptome and proteome during the response to DNA damage, it seems like hundreds of billions of bottles might be washing across your computer screen. Focusing on just the proteomics datasets describing the dynamics of protein modifications that occur in response to DNA damage, there are tens of thousands of post-translational modifications, which you can read about in many papers, including several from Stephen Elledge, one of the recipients of the 2015 Albert Lasker Basic Medical Research Award. Although prokaryotic and eukaryotic DNA damage responses differ in many ways, post-translational modifications are a key feature of both responses. In bacteria, cleavage of the transcriptional repressor LexA activates a gene expression program known as the SOS response, which brings us to the other recipient of the award, Evelyn Witkin. This Timeline highlights when some of the key concepts in the DNA damage response were reported, and you can learn more about the contributions of Witkin, Elledge, and many others to the development of these concepts in the Benchmark Essay by Jim Haber.

Yeast homologous recombination-based promoter engineering for the activation of silent natural product biosynthetic gene clusters

Large-scale sequencing of prokaryotic (meta)genomic DNA suggests that most bacterial natural product gene clusters are not expressed under common laboratory culture conditions. Silent gene clusters represent a promising resource for natural product discovery and the development of a new generation of therapeutics. Unfortunately, the characterization of molecules encoded by these clusters is hampered owing to our inability to express these gene clusters in the laboratory. To address this bottleneck, we have developed a promoter-engineering platform to transcriptionally activate silent gene clusters in a model heterologous host. Our approach uses yeast homologous recombination, an auxotrophy complementation-based yeast selection system and sequence orthogonal promoter cassettes to exchange all native promoters in silent gene clusters with constitutively active promoters. As part of this platform, we constructed and validated a set of bidirectional promoter cassettes consisting of orthogonal promoter sequences, Streptomyces ribosome binding sites, and yeast selectable marker genes. Using these tools we demonstrate the ability to simultaneously insert multiple promoter cassettes into a gene cluster, thereby expediting the reengineering process. We apply this method to model active and silent gene clusters (rebeccamycin and tetarimycin) and to the silent, cryptic pseudogene-containing, environmental DNA-derived Lzr gene cluster. Complete promoter refactoring and targeted gene exchange in this "dead" cluster led to the discovery of potent indolotryptoline antiproliferative agents, lazarimides A and B. This potentially scalable and cost-effective promoter reengineering platform should streamline the discovery of natural products from silent natural product biosynthetic gene clusters.

PWE-190 Passive transmission of helicobacter felis occurs in a specific pathogen free (SPF) laboratory environment

<h3>Introduction</h3> <i>Helicobacter felis</i>infection of C57BL/6 mice is an established model of human gastric carcinogenesis. Mice infected with <i>H. felis</i>by orogastric gavage show acute gastritis, and subsequently develop pre-neoplastic pathology. The acute component of this model mimics the pathology that has been identified in cases of acute <i>Helicobacter pylori </i>infection in humans, however most of these reports describe the consequences of ingestion of large <i>H. pylori</i>inocula. It is not clear how this reflects the natural acquisition of <i>H. pylori</i>which is usually a subclinical event. Previous studies have reported that co-housing <i>H. felis</i>infected mice with uninfected littermates did not lead to <i>H. felis</i>transmission, but these experiments were performed before molecular diagnostic tests for bacterial colonisation were available. <h3>Method</h3> A C57BL/6 mouse, conventionally infected with<i>H. felis</i>, was co-housed with 9 uninfected littermates. Prokaryotic DNA was extracted from stool samples collected from individual mice at regular intervals. Mice were culled at week 7.Gastric mucosal samples were collected for nucleic acid and histological assessment. <i>H. felis</i>transmission was assessed by H+E, modified Giemsa and Warthin-Starry stains and by quantitative PCR (qPCR) for the <i>H. felis</i>gene <i>flaA</i>. Longitudinal changes in faecal microbiota were assessed by qPCR for selected bacterial phyla. <h3>Results</h3> <i>FlaA </i>was identified by qPCR in gastric and stool samples from the actively infected mouse. All 3 histological stains identified <i>H. felis</i>in the gastric antrum of this mouse, its gastric corpus exhibited early atrophy and inflammation. qPCR of stool samples from the other 9 mice did not identify <i>flaA </i>DNA. In keeping with previous studies, <i>H. felis </i>was not detected histologically in the gastric antral mucosa of these mice and the gastric corpus mucosa wasmorphologically normal. However <i>flaA </i>template DNA was identified in gastric mucosal samples from 2 of the 9 mice.Selected bacterial phyla were quantified from stool samples taken longitudinally from each mouse. PCA analysis demonstrated that the colonic microbiota of mice converged over time. A particular shift in faecal microbiota was observed in the actively infected mouse. No differences were identified in the mice that had been passively colonised with <i>H. felis</i>compared to uninfected mice. <h3>Conclusion</h3> <i>H. felis</i>was transmitted between co-housed mice in SPF conditions. Passive colonisation led to little inflammation and no gross shift in faecal microbiotal composition was seen. This may represent a model of asymptomatic human <i>H. pylori</i>acquisition. Further investigation using larger cohorts of animals maintained for longer times will address whether the mode of acquisition of <i>H. felis </i>influences eventual pathological outcome. <h3>Disclosure of interest</h3> None Declared.

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways

Oxidatively generated guanine radical cations in DNA can undergo various nucleophilic reactions including the formation of C8-guanine cross-links with adjacent or nearby N3-thymines in DNA in the presence of O2. The G*[C8-N3]T* lesions have been identified in the DNA of human cells exposed to oxidative stress, and are most likely genotoxic if not removed by cellular defense mechanisms. It has been shown that the G*[C8-N3]T* lesions are substrates of nucleotide excision repair in human cell extracts. Cleavage at the sites of the lesions was also observed but not further investigated (Ding et al. (2012) Nucleic Acids Res. 40, 2506-2517). Using a panel of eukaryotic and prokaryotic bifunctional DNA glycosylases/lyases (NEIL1, Nei, Fpg, Nth, and NTH1) and apurinic/apyrimidinic (AP) endonucleases (Apn1, APE1, and Nfo), the analysis of cleavage fragments by PAGE and MALDI-TOF/MS show that the G*[C8-N3]T* lesions in 17-mer duplexes are incised on either side of G*, that none of the recovered cleavage fragments contain G*, and that T* is converted to a normal T in the 3'-fragment cleavage products. The abilities of the DNA glycosylases to incise the DNA strand adjacent to G*, while this base is initially cross-linked with T*, is a surprising observation and an indication of the versatility of these base excision repair proteins.

DNA specific fluorescent symmetric dimeric bisbenzimidazoles DBP(n): the synthesis, spectral properties, and biological activity.

A series of new fluorescent symmetric dimeric bisbenzimidazoles DBP(n) bearing bisbenzimidazole fragments joined by oligomethylene linkers with a central 1,4-piperazine residue were synthesized. The complex formation of DBP(n) in the DNA minor groove was demonstrated. The DBP(n) at micromolar concentrations inhibit in vitro eukaryotic DNA topoisomerase I and prokaryotic DNA methyltransferase (MTase) M.SssI. The DBP(n) were soluble well in aqueous solutions and could penetrate cell and nuclear membranes and stain DNA in live cells. The DBP(n) displayed a moderate effect on the reactivation of gene expression.

A New Tool for in vivo Manipulation of Brain microRNA Levels: The Work of Smalheiser et al. (2014)

A New Tool for in vivo Manipulation of Brain microRNA Levels: The Work of Smalheiser et al. (2014)

Su1927 Passive Transmission of Helicobacter felis Occurs in a Specific Pathogen Free (SPF) Laboratory Environment

Introduction Helicobacter felisinfection of C57BL/6 mice is an established model of human gastric carcinogenesis. Mice infected with H. felisby orogastric gavage show acute gastritis, and subsequently develop pre-neoplastic pathology. The acute component of this model mimics the pathology that has been identified in cases of acute Helicobacter pylori infection in humans, however most of these reports describe the consequences of ingestion of large H. pyloriinocula. It is not clear how this reflects the natural acquisition of H. pyloriwhich is usually a subclinical event. Previous studies have reported that co-housing H. felisinfected mice with uninfected littermates did not lead to H. felistransmission, but these experiments were performed before molecular diagnostic tests for bacterial colonisation were available. Method A C57BL/6 mouse, conventionally infected withH. felis, was co-housed with 9 uninfected littermates. Prokaryotic DNA was extracted from stool samples collected from individual mice at regular intervals. Mice were culled at week 7.Gastric mucosal samples were collected for nucleic acid and histological assessment. H. felistransmission was assessed by H+E, modified Giemsa and Warthin-Starry stains and by quantitative PCR (qPCR) for the H. felisgene flaA. Longitudinal changes in faecal microbiota were assessed by qPCR for selected bacterial phyla. Results FlaA was identified by qPCR in gastric and stool samples from the actively infected mouse. All 3 histological stains identified H. felisin the gastric antrum of this mouse, its gastric corpus exhibited early atrophy and inflammation. qPCR of stool samples from the other 9 mice did not identify flaA DNA. In keeping with previous studies, H. felis was not detected histologically in the gastric antral mucosa of these mice and the gastric corpus mucosa wasmorphologically normal. However flaA template DNA was identified in gastric mucosal samples from 2 of the 9 mice.Selected bacterial phyla were quantified from stool samples taken longitudinally from each mouse. PCA analysis demonstrated that the colonic microbiota of mice converged over time. A particular shift in faecal microbiota was observed in the actively infected mouse. No differences were identified in the mice that had been passively colonised with H. feliscompared to uninfected mice. Conclusion H. feliswas transmitted between co-housed mice in SPF conditions. Passive colonisation led to little inflammation and no gross shift in faecal microbiotal composition was seen. This may represent a model of asymptomatic human H. pyloriacquisition. Further investigation using larger cohorts of animals maintained for longer times will address whether the mode of acquisition of H. felis influences eventual pathological outcome. Disclosure of interest None Declared.