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Asset Tag: Bacterial genome

July 19, 2019

Lineage-specific methyltransferases define the methylome of the globally disseminated Escherichia coli ST131 clone.

Escherichia coli sequence type 131 (ST131) is a clone of uropathogenic E. coli that has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome of E. coli EC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered three (m6)A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible for (m6)A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located.DNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistant E. coli sequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-defined E. coli clone. Copyright © 2015 Forde et al.

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July 19, 2019

Lifespan of restriction-modification systems critically affects avoidance of their recognition sites in host genomes.

Avoidance of palindromic recognition sites of Type II restriction-modification (R-M) systems was shown for many R-M systems in dozens of prokaryotic genomes. However the phenomenon has not been investigated systematically for all presently available genomes and annotated R-M systems. We have studied all known recognition sites in thousands of prokaryotic genomes and found factors that influence their avoidance.Only Type II R-M systems consisting of independently acting endonuclease and methyltransferase (called ‘orthodox’ here) cause avoidance of their sites, both palindromic and asymmetric, in corresponding prokaryotic genomes; the avoidance takes place for?~?50 % of 1774 studied cases. It is known that prokaryotes can acquire and lose R-M systems. Thus it is possible to talk about the lifespan of an R-M system in a genome. We have shown that the recognition site avoidance correlates with the lifespan of R-M systems. The sites of orthodox R-M systems that are encoded in host genomes for a long time are avoided more often (up to 100 % in certain cohorts) than the sites of recently acquired ones. We also found cases of site avoidance in absence of the corresponding R-M systems in the genome. An analysis of closely related bacteria shows that such avoidance can be a trace of lost R-M systems. Sites of Type I, II?/G, IIM, III, and IV R-M systems are not avoided in vast majority of cases.The avoidance of orthodox Type II R-M system recognition sites in prokaryotic genomes is a widespread phenomenon. Presence of an R-M system without an underrepresentation of its site may indicate that the R-M system was acquired recently. At the same time, a significant underrepresentation of a site may be a sign of presence of the corresponding R-M system in this organism or in its ancestors for a long time. The drastic difference between site avoidance for orthodox Type II R-M systems and R-M systems of other types can be explained by a higher rate of specificity changes or a less self-toxicity of the latter.

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July 19, 2019

AnnoTALE: bioinformatics tools for identification, annotation, and nomenclature of TALEs from Xanthomonas genomic sequences.

Transcription activator-like effectors (TALEs) are virulence factors, produced by the bacterial plant-pathogen Xanthomonas, that function as gene activators inside plant cells. Although the contribution of individual TALEs to infectivity has been shown, the specific roles of most TALEs, and the overall TALE diversity in Xanthomonas spp. is not known. TALEs possess a highly repetitive DNA-binding domain, which is notoriously difficult to sequence. Here, we describe an improved method for characterizing TALE genes by the use of PacBio sequencing. We present ‘AnnoTALE’, a suite of applications for the analysis and annotation of TALE genes from Xanthomonas genomes, and for grouping similar TALEs into classes. Based on these classes, we propose a unified nomenclature for Xanthomonas TALEs that reveals similarities pointing to related functionalities. This new classification enables us to compare related TALEs and to identify base substitutions responsible for the evolution of TALE specificities.

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July 19, 2019

The epigenomic landscape of prokaryotes.

DNA methylation acts in concert with restriction enzymes to protect the integrity of prokaryotic genomes. Studies in a limited number of organisms suggest that methylation also contributes to prokaryotic genome regulation, but the prevalence and properties of such non-restriction-associated methylation systems remain poorly understood. Here, we used single molecule, real-time sequencing to map DNA modifications including m6A, m4C, and m5C across the genomes of 230 diverse bacterial and archaeal species. We observed DNA methylation in nearly all (93%) organisms examined, and identified a total of 834 distinct reproducibly methylated motifs. This data enabled annotation of the DNA binding specificities of 620 DNA Methyltransferases (MTases), doubling known specificities for previously hard to study Type I, IIG and III MTases, and revealing their extraordinary diversity. Strikingly, 48% of organisms harbor active Type II MTases with no apparent cognate restriction enzyme. These active ‘orphan’ MTases are present in diverse bacterial and archaeal phyla and show motif specificities and methylation patterns consistent with functions in gene regulation and DNA replication. Our results reveal the pervasive presence of DNA methylation throughout the prokaryotic kingdoms, as well as the diversity of sequence specificities and potential functions of DNA methylation systems.

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July 19, 2019

Phase variation of a Type IIG restriction-modification enzyme alters site-specific methylation patterns and gene expression in Campylobacter jejuni strain NCTC11168.

Phase-variable restriction-modification systems are a feature of a diverse range of bacterial species. Stochastic, reversible switches in expression of the methyltransferase produces variation in methylation of specific sequences. Phase-variable methylation by both Type I and Type III methyltransferases is associated with altered gene expression and phenotypic variation. One phase-variable gene of Campylobacter jejuni encodes a homologue of an unusual Type IIG restriction-modification system in which the endonuclease and methyltransferase are encoded by a single gene. Using both inhibition of restriction and PacBio-derived methylome analyses of mutants and phase-variants, the cj0031c allele in C. jejuni strain NCTC11168 was demonstrated to specifically methylate adenine in 5’CCCGA and 5’CCTGA sequences. Alterations in the levels of specific transcripts were detected using RNA-Seq in phase-variants and mutants of cj0031c but these changes did not correlate with observed differences in phenotypic behaviour. Alterations in restriction of phage growth were also associated with phase variation (PV) of cj0031c and correlated with presence of sites in the genomes of these phages. We conclude that PV of a Type IIG restriction-modification system causes changes in site-specific methylation patterns and gene expression patterns that may indirectly change adaptive traits.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 19, 2019

Large genomic differences between Moraxella bovoculi isolates acquired from the eyes of cattle with infectious bovine keratoconjunctivitis versus the deep nasopharynx of asymptomatic cattle.

Moraxella bovoculi is a recently described bacterium that is associated with infectious bovine keratoconjunctivitis (IBK) or “pinkeye” in cattle. In this study, closed circularized genomes were generated for seven M. bovoculi isolates: three that originated from the eyes of clinical IBK bovine cases and four from the deep nasopharynx of asymptomatic cattle. Isolates that originated from the eyes of IBK cases profoundly differed from those that originated from the nasopharynx of asymptomatic cattle in genome structure, gene content and polymorphism diversity and consequently placed into two distinct phylogenetic groups. These results suggest that there are genetically distinct strains of M. bovoculi that may not associate with IBK.

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July 19, 2019

Detection and whole genome sequencing of carbapenemase-producing Aeromonas hydrophila isolated from routine perirectal surveillance culture.

Perirectal surveillance cultures and a stool culture grew Aeromonas species from three patients over a six-week period without epidemiological links. Detection of the blaKPC-2 gene in one isolate prompted inclusion of non-Enterobacteriaceae in our surveillance culture workup. Whole genome sequencing confirmed isolates were unrelated, and provided data for Aeromonas reference genomes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 19, 2019

Genome Update. Let the consumer beware: Streptomyces genome sequence quality.

A genome sequence assembly represents a model of a genome. This article explores some tools and methods for assessing the quality of an assembly, using publicly available data for Streptomyces species as the example. There is great variability in quality of assemblies deposited in GenBank. Only in a small minority of these assemblies are the raw data available, enabling full appraisal of the assembly quality. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

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July 19, 2019

A role for the bacterial GATC methylome in antibiotic stress survival.

Antibiotic resistance is an increasingly serious public health threat. Understanding pathways allowing bacteria to survive antibiotic stress may unveil new therapeutic targets. We explore the role of the bacterial epigenome in antibiotic stress survival using classical genetic tools and single-molecule real-time sequencing to characterize genomic methylation kinetics. We find that Escherichia coli survival under antibiotic pressure is severely compromised without adenine methylation at GATC sites. Although the adenine methylome remains stable during drug stress, without GATC methylation, methyl-dependent mismatch repair (MMR) is deleterious and, fueled by the drug-induced error-prone polymerase Pol IV, overwhelms cells with toxic DNA breaks. In multiple E. coli strains, including pathogenic and drug-resistant clinical isolates, DNA adenine methyltransferase deficiency potentiates antibiotics from the ß-lactam and quinolone classes. This work indicates that the GATC methylome provides structural support for bacterial survival during antibiotic stress and suggests targeting bacterial DNA methylation as a viable approach to enhancing antibiotic activity.

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July 19, 2019

Comprehensive mutagenesis of the fimS promoter regulatory switch reveals novel regulation of type 1 pili in uropathogenic Escherichia coli.

Type 1 pili (T1P) are major virulence factors for uropathogenic Escherichia coli (UPEC), which cause both acute and recurrent urinary tract infections. T1P expression therefore is of direct relevance for disease. T1P are phase variable (both piliated and nonpiliated bacteria exist in a clonal population) and are controlled by an invertible DNA switch (fimS), which contains the promoter for the fim operon encoding T1P. Inversion of fimS is stochastic but may be biased by environmental conditions and other signals that ultimately converge at fimS itself. Previous studies of fimS sequences important for T1P phase variation have focused on laboratory-adapted E. coli strains and have been limited in the number of mutations or by alteration of the fimS genomic context. We surmounted these limitations by using saturating genomic mutagenesis of fimS coupled with accurate sequencing to detect both mutations and phase status simultaneously. In addition to the sequences known to be important for biasing fimS inversion, our method also identifies a previously unknown pair of 5′ UTR inverted repeats that act by altering the relative fimA levels to control phase variation. Thus we have uncovered an additional layer of T1P regulation potentially impacting virulence and the coordinate expression of multiple pilus systems.

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July 19, 2019

PacBio SMRT assembly of a complex multi-replicon genome reveals chlorocatechol degradative operon in a region of genome plasticity.

We have sequenced a Burkholderia genome that contains multiple replicons and large repetitive elements that would make it inherently difficult to assemble by short read sequencing technologies. We illustrate how the integrated long read correction algorithms implemented through the PacBio Single Molecule Real-Time (SMRT) sequencing technology successfully provided a de novo assembly that is a reasonable estimate of both the gene content and genome organization without making any further modifications. This assembly is comparable to related organisms assembled by more labour intensive methods. Our assembled genome revealed regions of genome plasticity for further investigation, one of which harbours a chlorocatechol degradative operon highly homologous to those previously identified on globally ubiquitous plasmids. In an ideal world, this assembly would still require experimental validation to confirm gene order and copy number of repeated elements. However, we submit that particularly in instances where a polished genome is not the primary goal of the sequencing project, PacBio SMRT sequencing provides a financially viable option for generating a biologically relevant genome estimate that can be utilized by other researchers for comparative studies. Copyright © 2016. Published by Elsevier B.V.

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July 19, 2019

Next generation sequencing of Actinobacteria for the discovery of novel natural products.

Like many fields of the biosciences, actinomycete natural products research has been revolutionised by next-generation DNA sequencing (NGS). Hundreds of new genome sequences from actinobacteria are made public every year, many of them as a result of projects aimed at identifying new natural products and their biosynthetic pathways through genome mining. Advances in these technologies in the last five years have meant not only a reduction in the cost of whole genome sequencing, but also a substantial increase in the quality of the data, having moved from obtaining a draft genome sequence comprised of several hundred short contigs, sometimes of doubtful reliability, to the possibility of obtaining an almost complete and accurate chromosome sequence in a single contig, allowing a detailed study of gene clusters and the design of strategies for refactoring and full gene cluster synthesis. The impact that these technologies are having in the discovery and study of natural products from actinobacteria, including those from the marine environment, is only starting to be realised. In this review we provide a historical perspective of the field, analyse the strengths and limitations of the most relevant technologies, and share the insights acquired during our genome mining projects.

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July 19, 2019

Integrating DNA methylation and gene expression data in the development of the soybean-Bradyrhizobium N2-fixing symbiosis.

Very little is known about the role of epigenetics in the differentiation of a bacterium from the free-living to the symbiotic state. Here genome-wide analysis of DNA methylation changes between these states is described using the model of symbiosis between soybean and its root nodule-forming, nitrogen-fixing symbiont, Bradyrhizobium diazoefficiens. PacBio resequencing of the B. diazoefficiens genome from both states revealed 43,061 sites recognized by five motifs with the potential to be methylated genome-wide. Of those sites, 3276 changed methylation states in 2921 genes or 35.5% of all genes in the genome. Over 10% of the methylation changes occurred within the symbiosis island that comprises 7.4% of the genome. The CCTTGAG motif was methylated only during symbiosis with 1361 adenosines methylated among the 1700 possible sites. Another 89 genes within the symbiotic island and 768 genes throughout the genome were found to have methylation and significant expression changes during symbiotic development. Of those, nine known symbiosis genes involved in all phases of symbiotic development including early infection events, nodule development, and nitrogenase production. These associations between methylation and expression changes in many B. diazoefficiens genes suggest an important role of the epigenome in bacterial differentiation to the symbiotic state.

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July 19, 2019

Separate F-type plasmids have shaped the evolution of the H30 subclone of Escherichia coli sequence type 131.

The extraintestinal pathogenic Escherichia coli (ExPEC) H30 subclone of sequence type 131 (ST131-H30) has emerged abruptly as a dominant lineage of ExPEC responsible for human disease. The ST131-H30 lineage has been well described phylogenetically, yet its plasmid complement is not fully understood. Here, single-molecule, real-time sequencing was used to generate the complete plasmid sequences of ST131-H30 isolates and those belonging to other ST131 clades. Comparative analyses revealed separate F-type plasmids that have shaped the evolution of the main fluoroquinolone-resistant ST131-H30 clades. Specifically, an F1:A2:B20 plasmid is strongly associated with the H30R/C1 clade, whereas an F2:A1:B- plasmid is associated with the H30Rx/C2 clade. A series of plasmid gene losses, gains, and rearrangements involving IS26 likely led to the current plasmid complements within each ST131-H30 sublineage, which contain several overlapping gene clusters with putative functions in virulence and fitness, suggesting plasmid-mediated convergent evolution. Evidence suggests that the H30Rx/C2-associated F2:A1:B- plasmid type was present in strains ancestral to the acquisition of fluoroquinolone resistance and prior to the introduction of a multidrug resistance-encoding gene cassette harboring bla CTX-M-15. In vitro experiments indicated a host strain-independent low frequency of plasmid transfer, differential levels of plasmid stability even between closely related ST131-H30 strains, and possible epistasis for carriage of these plasmids within the H30R/Rx lineages. IMPORTANCE A clonal lineage of Escherichia coli known as ST131 has emerged as a dominating strain type causing extraintestinal infections in humans. The evolutionary history of ST131 E. coli is now well understood. However, the role of plasmids in ST131’s evolutionary history is poorly defined. This study utilized real-time, single-molecule sequencing to compare plasmids from various current and historical lineages of ST131. From this work, it was determined that a series of plasmid gains, losses, and recombinational events has led to the currently circulating plasmids of ST131 strains. These plasmids appear to have evolved to acquire similar gene clusters on multiple occasions, suggesting possible plasmid-mediated convergent evolution leading to evolutionary success. These plasmids also appear to be better suited to exist in specific strains of ST131 due to coadaptive mutations. Overall, a series of events has enabled the evolution of ST131 plasmids, possibly contributing to the lineage’s success.

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July 19, 2019

Methylome analysis of two Xanthomonas spp. using Single-Molecule Real-Time Sequencing.

Single-molecule real-time (SMRT) sequencing allows identification of methylated DNA bases and methylation patterns/motifs at the genome level. Using SMRT sequencing, diverse bacterial methylomes including those of Helicobacter pylori, Lactobacillus spp., and Escherichia coli have been determined, and previously unreported DNA methylation motifs have been identified. However, the methylomes of Xanthomonas species, which belong to the most important plant pathogenic bacterial genus, have not been documented. Here, we report the methylomes of Xanthomonas axonopodis pv. glycines (Xag) strain 8ra and X. campestris pv. vesicatoria (Xcv) strain 85-10. We identified N(6)-methyladenine (6mA) and N(4)-methylcytosine (4mC) modification in both genomes. In addition, we assigned putative DNA methylation motifs including previously unreported methylation motifs via REBASE and MotifMaker, and compared methylation patterns in both species. Although Xag and Xcv belong to the same genus, their methylation patterns were dramatically different. The number of 4mC DNA bases in Xag (66,682) was significantly higher (29 fold) than in Xcv (2,321). In contrast, the number of 6mA DNA bases (4,147) in Xag was comparable to the number in Xcv (5,491). Strikingly, there were no common or shared motifs in the 10 most frequently methylated motifs of both strains, indicating they possess unique species- or strain-specific methylation motifs. Among the 20 most frequent motifs from both strains, for 9 motifs at least 1% of the methylated bases were located in putative promoter regions. Methylome analysis by SMRT sequencing technology is the first step toward understanding the biology and functions of DNA methylation in this genus.

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