Selective recognition of
            N
            4-methylcytosine in DNA by engineered transcription-activator-like effectors

The epigenetic DNA nucleobases 5-methylcytosine (5mC) and N 4-methylcytosine (4mC) coexist in bacterial genomes and have important functions in host defence and transcription regulation. To better understand the individual biological roles of both methylated nucleobases, analytical strategies for distinguishing unmodified cytosine (C) from 4mC and 5mC are required. Transcription-activator-like effectors (TALEs) are programmable DNA-binding repeat proteins, which can be re-engineered for the direct detection of epigenetic nucleobases in user-defined DNA sequences. We here report the natural, cytosine-binding TALE repeat to not strongly differentiate between 5mC and 4mC. To engineer repeats with selectivity in the context of C, 5mC and 4mC, we developed a homogeneous fluorescence assay and screened a library of size-reduced TALE repeats for binding to all three nucleobases. This provided insights into the requirements of size-reduced TALE repeats for 4mC binding and revealed a single mutant repeat as a selective binder of 4mC. Employment of a TALE with this repeat in affinity enrichment enabled the isolation of a user-defined DNA sequence containing a single 4mC but not C or 5mC from the background of a bacterial genome. Comparative enrichments with TALEs bearing this or the natural C-binding repeat provides an approach for the complete, programmable decoding of all cytosine nucleobases found in bacterial genomes. This article is part of a discussion meeting issue ‘Frontiers in epigenetic chemical biology’.

Download Full-text

IslandHunter – A Java-based GI detection software

10.7287/peerj.preprints.716 ◽

2014 ◽

Author(s):

Shakuntala Baichoo ◽

Haswanee Goodur ◽

Vyasanand Ramtohul

Keyword(s):

Dna Sequences ◽

Bacterial Genome ◽

Gc Content ◽

Genomic Islands ◽

Bacterial Genomes ◽

Flexible Display ◽

The Past ◽

Detection Software ◽

Circular Graph ◽

Tree View

Over the past decade, researchers have discovered that apart from the essential genes, bacterial genomes also contain a variable amount of accessory genes acquired by horizontal gene transfer (HGT) that are categorized as genomic islands (GIs). GIs encode adaptive traits, which might be beneficial for the species under certain growth or environmental conditions. It has always been a challenge for biologists to identify GIs within a bacterial genome as they evolve very rapidly. This paper proposes a standalone software, IslanHunter, that has been developed using Java and BioJava and can extract GI regions using GC content, codon usage bias, dinucleotide frequency bias, tetranucleotide frequency bias, k-mer signature analysis (2-mer, 3-mer, 4-mer, 5-mer, and 6-mer) and presence of mobility genes. IslandHunter provides a simple graphical user interface where disclosed GIs are displayed in a tree-view and a circular graph. Users are presented with options to save the GI regions as blocks of DNA sequences in FASTA format. They can later use these predicted GI regions for further analysis. IslandHunter can take as input, files in GenBank, EMBL or FASTA formats. IslandHunter provides flexible display options and save options. The software has been evaluated against exiting tools with good performance. It is available for evaluation at https://github.com/ShakunBaichoo/IslandHunter .

Download Full-text

A Bacterial Genome And Culture Collection of Gut Microbial in Weanling Piglet

10.21203/rs.3.rs-988012/v1 ◽

2021 ◽

Author(s):

Bo Dong ◽

Xiaoqian Lin ◽

Xiaohuan Jing ◽

Tongyuan Hu ◽

Jianwei Zhou ◽

...

Keyword(s):

Gut Microbiome ◽

Bacterial Genome ◽

Culture Collection ◽

Glycoside Hydrolases ◽

Individual Species ◽

Bacterial Genomes ◽

Microbiome Composition ◽

Weanling Piglets ◽

Substantial Bias ◽

The Individual

Abstract Background: The microbiota hosted in the pig gastrointestinal tract are important to health of this biomedical model. However, the individual species and functional repertoires that make up the pig gut microbiome remain largely undefined. Results: Here we comprehensively investigated the genomes and functions of the piglet gut microbiome using culture-based and metagenomics approaches. A collection included 266 cultured genomes and 482 metagenome-assembled genomes (MAGs) that were clustered to 428 species across 10 phyla was established. Among these clustered species, 333 genomes represent potential new species. Less matches between cultured genomes and MAGs revealed a substantial bias for the acquisition of reference genomes by the two strategies. Glycoside hydrolases was the dominant category of carbohydrate-active enzymes. 445 secondary metabolite biosynthetic genes were predicted from 292 genomes with bacteriocin being the most. Pan genome analysis of Limosilactobacillus reuteri uncover the biosynthesis of reuterin was strain-specific and the production was experimentally determined. Conclusions: A total of 266 isolated bacterial genomes and 482 MAGs were obtained and investigated their functional repertoires. This study provides a comprehensive view of the microbiome composition and the function landscape of the gut of weanling piglets and a valuable bacterial resource for further experimentations.

Download Full-text

IslandHunter – A Java-based GI detection software

10.7287/peerj.preprints.716v1 ◽

2014 ◽

Author(s):

Shakuntala Baichoo ◽

Haswanee Goodur ◽

Vyasanand Ramtohul

Keyword(s):

Dna Sequences ◽

Bacterial Genome ◽

Gc Content ◽

Genomic Islands ◽

Bacterial Genomes ◽

Flexible Display ◽

The Past ◽

Detection Software ◽

Circular Graph ◽

Tree View

Download Full-text

Role of Transcriptional Read-Through in PRE Activity in Drosophila melanogaster

Acta Naturae ◽

10.32607/20758251-2016-8-2-79-86 ◽

2016 ◽

Vol 8 (2) ◽

pp. 79-86 ◽

Cited By ~ 3

Author(s):

P. V. Elizar’ev ◽

D. V. Lomaev ◽

D. A. Chetverina ◽

P. G. Georgiev ◽

M. M. Erokhin

Keyword(s):

Dna Sequences ◽

Cell Types ◽

Transcription Terminator ◽

Response Elements ◽

Polycomb Response Elements ◽

The Individual ◽

Multicellular Organisms ◽

Different Cell Types ◽

Main Factor

Maintenance of the individual patterns of gene expression in different cell types is required for the differentiation and development of multicellular organisms. Expression of many genes is controlled by Polycomb (PcG) and Trithorax (TrxG) group proteins that act through association with chromatin. PcG/TrxG are assembled on the DNA sequences termed PREs (Polycomb Response Elements), the activity of which can be modulated and switched from repression to activation. In this study, we analyzed the influence of transcriptional read-through on PRE activity switch mediated by the yeast activator GAL4. We show that a transcription terminator inserted between the promoter and PRE doesnt prevent switching of PRE activity from repression to activation. We demonstrate that, independently of PRE orientation, high levels of transcription fail to dislodge PcG/TrxG proteins from PRE in the absence of a terminator. Thus, transcription is not the main factor required for PRE activity switch.

Download Full-text

Serodiagnosis and Bacterial Genome of Helicobacter pylori Infection

Toxins ◽

10.3390/toxins13070467 ◽

2021 ◽

Vol 13 (7) ◽

pp. 467

Author(s):

Aina Ichihara ◽

Hinako Ojima ◽

Kazuyoshi Gotoh ◽

Osamu Matsushita ◽

Susumu Take ◽

...

Keyword(s):

Helicobacter Pylori ◽

Antibody Titer ◽

Bacterial Genome ◽

Serum Antibody ◽

Gene Mutations ◽

Bacterial Genomes ◽

Western Blots ◽

A Genome ◽

Vaca Gene ◽

H Pylori

The infection caused by Helicobacter pylori is associated with several diseases, including gastric cancer. Several methods for the diagnosis of H. pylori infection exist, including endoscopy, the urea breath test, and the fecal antigen test, which is the serum antibody titer test that is often used since it is a simple and highly sensitive test. In this context, this study aims to find the association between different antibody reactivities and the organization of bacterial genomes. Next-generation sequences were performed to determine the genome sequences of four strains of antigens with different reactivity. The search was performed on the common genes, with the homology analysis conducted using a genome ring and dot plot analysis. The two antigens of the highly reactive strains showed a high gene homology, and Western blots for CagA and VacA also showed high expression levels of proteins. In the poorly responsive antigen strains, it was found that the inversion occurred around the vacA gene in the genome. The structure of bacterial genomes might contribute to the poor reactivity exhibited by the antibodies of patients. In the future, an accurate serodiagnosis could be performed by using a strain with few gene mutations of the antigen used for the antibody titer test of H. pylori.

Download Full-text

VARIABILITY OF EVOLUTIONARY RATES OF DNA

Genetics ◽

10.1093/genetics/113.4.1077 ◽

1986 ◽

Vol 113 (4) ◽

pp. 1077-1091

Author(s):

John H Gillespie

Keyword(s):

Dna Sequences ◽

Significant Excess ◽

Ergodic Markov Chain ◽

The Third ◽

Markov Chain Models ◽

Rate Of Molecular Evolution ◽

The Mean ◽

Nuclear Loci ◽

The Individual ◽

Evolution Of Proteins

ABSTRACT A statistical analysis of DNA sequences from four nuclear loci and five mitochondrial loci from different orders of mammals is described. A major aim of the study is to describe the variation in the rate of molecular evolution of proteins and DNA. A measure of rate variability is the statistic R, the ratio of the variance in the number of substitutions to the mean number. For proteins, R is found to be in the range 0.16 < R < 35.55, thus extending in both directions the values seen in previous studies. An analysis of codons shows that there is a highly significant excess of double substitutions in the first and second positions, but not in the second and third or first and third positions. The analysis of the dynamics of nucleotide evolution showed that the ergodic Markov chain models that are the basis of most published formulas for correcting for multiple substitutions are incompatible with the data. A bootstrap procedure was used to show that the evolution of the individual nucleotides, even the third positions, show the same variation in rates as seen in the proteins. It is argued that protein and silent DNA evolution are uncoupled, with the evolution at both levels showing patterns that are better explained by the action of natural selection than by neutrality. This conclusion is based primarily on a comparison of the nuclear and mitochondrial results.

Download Full-text

Consistent Metagenome-Derived Metrics Verify and Delineate Bacterial Species Boundaries

mSystems ◽

10.1128/msystems.00731-19 ◽

2020 ◽

Vol 5 (1) ◽

Cited By ~ 14

Author(s):

Matthew R. Olm ◽

Alexander Crits-Christoph ◽

Spencer Diamond ◽

Adi Lavy ◽

Paula B. Matheus Carnevali ◽

...

Keyword(s):

Bacterial Diversity ◽

Ribosomal Proteins ◽

Large Scale ◽

Bacterial Species ◽

Bacterial Genome ◽

16S Rrna Genes ◽

Rrna Genes ◽

Species Discrimination ◽

Bacterial Genomes ◽

Discrimination Power

ABSTRACT Longstanding questions relate to the existence of naturally distinct bacterial species and genetic approaches to distinguish them. Bacterial genomes in public databases form distinct groups, but these databases are subject to isolation and deposition biases. To avoid these biases, we compared 5,203 bacterial genomes from 1,457 environmental metagenomic samples to test for distinct clouds of diversity and evaluated metrics that could be used to define the species boundary. Bacterial genomes from the human gut, soil, and the ocean all exhibited gaps in whole-genome average nucleotide identities (ANI) near the previously suggested species threshold of 95% ANI. While genome-wide ratios of nonsynonymous and synonymous nucleotide differences (dN/dS) decrease until ANI values approach ∼98%, two methods for estimating homologous recombination approached zero at ∼95% ANI, supporting breakdown of recombination due to sequence divergence as a species-forming force. We evaluated 107 genome-based metrics for their ability to distinguish species when full genomes are not recovered. Full-length 16S rRNA genes were least useful, in part because they were underrecovered from metagenomes. However, many ribosomal proteins displayed both high metagenomic recoverability and species discrimination power. Taken together, our results verify the existence of sequence-discrete microbial species in metagenome-derived genomes and highlight the usefulness of ribosomal genes for gene-level species discrimination. IMPORTANCE There is controversy about whether bacterial diversity is clustered into distinct species groups or exists as a continuum. To address this issue, we analyzed bacterial genome databases and reports from several previous large-scale environment studies and identified clear discrete groups of species-level bacterial diversity in all cases. Genetic analysis further revealed that quasi-sexual reproduction via horizontal gene transfer is likely a key evolutionary force that maintains bacterial species integrity. We next benchmarked over 100 metrics to distinguish these bacterial species from each other and identified several genes encoding ribosomal proteins with high species discrimination power. Overall, the results from this study provide best practices for bacterial species delineation based on genome content and insight into the nature of bacterial species population genetics.

Download Full-text

Direct Detection of the Hybridization of Synthetic Homo-Oligomer DNA Sequences by Field Effect

The Journal of Physical Chemistry B ◽

10.1021/jp963056h ◽

1997 ◽

Vol 101 (15) ◽

pp. 2980-2985 ◽

Cited By ~ 242

Author(s):

E. Souteyrand ◽

J. P. Cloarec ◽

J. R. Martin ◽

C. Wilson ◽

I. Lawrence ◽

...

Keyword(s):

Dna Sequences ◽

Field Effect ◽

Direct Detection

Download Full-text

Modeling colicin operons as genetic arms in plasmid-genome conflicts

10.1101/2021.05.15.444200 ◽

2021 ◽

Author(s):

Pavithra Anantharaman Sudhakari ◽

Bhaskar Chandra Mohan Ramisetty

Keyword(s):

Negative Selection ◽

Bacterial Genome ◽

Evolutionary Perspective ◽

Bacterial Genomes ◽

Sequence Analyses ◽

Agent Based ◽

Physiological Relevance ◽

Maintenance Systems ◽

Selection Of ◽

Lethal Genes

Plasmids are acellular propagating entities that depend, as molecular parasites, on bacteria for propagation. The conflict between the bacterial genome and the parasitic plasmids allows the emergence of genetic arms such as Colicin (Col) operons. Endonuclease Col operons encode three proteins; an endonuclease colicin (cleaves nucleic acids), an immunity protein (inactivates its cognate colicin), and lysis protein (aids in colicin release via host cell lysis). Col operons are efficient plasmid-maintenance systems; (i) the plasmid cured cells are killed by the colicins; (ii) damaged cells lyse and releases the colicins that eliminate the competitors; and (iii) the released plasmids invade new bacteria. Surprisingly, some bacterial genomes have Col operons. The eco-evolutionary drive and physiological relevance of genomic Col operons are unknown. We investigated plasmidic and genomic Col operons using sequence analyses from an eco-evolutionary perspective. We found 1,248 genomic and plasmidic colicins across 30 bacterial genera. Although 51% of the genomes harbor colicins, the majority of the genomic colicins lacked a functional lysis gene, suggesting the negative selection of lethal genes. The immunity gene of the Col operon protects the cured host thereby eliminating the metabolic burden due to plasmid. We show mutual exclusivity of col operons on genomes and plasmids. We propose anti-addiction hypothesis for genomic colicins. Using a stochastic agent-based model, we show that the genomic colicins confer an advantage to the host genome in terms of immunity to the toxin and elimination of plasmid burden. Col operons are genetic arms that regulate the ecological interplay of bacterial genomes and plasmids.

Download Full-text

Bactopia: a flexible pipeline for complete analysis of bacterial genomes

10.1101/2020.02.28.969394 ◽

2020 ◽

Author(s):

Robert A. Petit ◽

Timothy D. Read

Keyword(s):

Standard Procedure ◽

Bacterial Species ◽

Bacterial Genome ◽

Complete Analysis ◽

Comparative Genomic ◽

Bacterial Genomes ◽

Analysis Pipeline ◽

Genomic Analyses ◽

Conserved Genes ◽

Downstream Analysis

AbstractSequencing of bacterial genomes using Illumina technology has become such a standard procedure that often data are generated faster than can be conveniently analyzed. We created a new series of pipelines called Bactopia, built using Nextflow workflow software, to provide efficient comparative genomic analyses for bacterial species or genera. Bactopia consists of a dataset setup step (Bactopia Datasets; BaDs) where a series of customizable datasets are created for the species of interest; the Bactopia Analysis Pipeline (BaAP), which performs quality control, genome assembly and several other functions based on the available datasets and outputs the processed data to a structured directory format; and a series of Bactopia Tools (BaTs) that perform specific post-processing on some or all of the processed data. BaTs include pan-genome analysis, computing average nucleotide identity between samples, extracting and profiling the 16S genes and taxonomic classification using highly conserved genes. It is expected that the number of BaTs will increase to fill specific applications in the future. As a demonstration, we performed an analysis of 1,664 public Lactobacillus genomes, focusing on L. crispatus, a species that is a common part of the human vaginal microbiome. Bactopia is an open source system that can scale from projects as small as one bacterial genome to thousands that allows for great flexibility in choosing comparison datasets and options for downstream analysis. Bactopia code can be accessed at https://www.github.com/bactopia/bactopia.

Download Full-text