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July 7, 2019

Complete genome sequence of ER2796, a DNA methyltransferase-deficient strain of Escherichia coli K-12.

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.


July 7, 2019

Role of restriction-modification systems in prokaryotic evolution and ecology

Restriction–modification (R-M) systems are able to methylate or cleave DNA depending on methylation status of their recognition site. It allows them to protect bacterial cells from invasion by foreign DNA. Comparative analysis of a large number of available bacterial genomes and methylomes clearly demonstrates that the role of R-M systems in bacteria is wider than only defense. R-M systems maintain heterogeneity of a bacterial population and are involved in adaptation of bacteria to change in their environmental conditions. R-M systems can be essential for host colonization by pathogenic bacteria. Phase variation and intragenomic recombinations are sources of the fast evolution of the specificity of R-M systems. This review focuses on the influence of R-M systems on evolution and ecology of prokaryotes.


July 7, 2019

SMRT sequencing of the Campylobacter coli BfR-CA-9557 genome sequence reveals unique methylation motifs.

Campylobacter species are the most prevalent bacterial pathogen causing acute enteritis worldwide. In contrast to Campylobacter jejuni, about 5 % of Campylobacter coli strains exhibit susceptibility to restriction endonuclease digestion by DpnI cutting specifically 5′-G(m)ATC-3′ motifs. This indicates significant differences in DNA methylation between both microbial species. The goal of the study was to analyze the methylome of a C. coli strain susceptible to DpnI digestion, to identify its methylation motifs and restriction modification systems (RM-systems), and compare them to related organisms like C. jejuni and Helicobacter pylori. Using one SMRT cell and the PacBio RS sequencing technology followed by PacBio Modification and Motif Analysis the complete genome of the DpnI susceptible strain C. coli BfR-CA-9557 was sequenced to 500-fold coverage and assembled into a single contig of 1.7 Mbp. The genome contains a CJIE1-like element prophage and is phylogenetically closer to C. coli clade 1 isolates than clade 3. 45,881 6-methylated adenines (ca. 2.7 % of genome positions) that are predominantly arranged in eight different methylation motifs and 1,788 4-methylated cytosines (ca. 0.1 %) have been detected. Only two of these motifs correspond to known restriction modification motifs. Characteristic for this methylome was the very high fraction of methylation of motifs with mostly above 99 %.Only five dominant methylation motifs have been identified in C. jejuni, which have been associated with known RM-systems. C. coli BFR-CA-9557 shares one (RAATTY) of these, but four ORFs could be assigned to putative Type I RM-systems, seven ORFs to Type II RM-systems and three ORFs to Type IV RM-systems. In accordance with DpnI prescreening RM-system IIP, methylation of GATC motifs was detected in C. coli BfR-CA-9557. A homologous IIP RM-system has been described for H. pylori. The remaining methylation motifs are specific for C. coli BfR-CA-9557 and have been neither detected in C. jejuni nor in H. pylori. The results of this study give us new insights into epigenetics of Campylobacteraceae and provide the groundwork to resolve the function of RM-systems in C. coli.


July 7, 2019

Methylome diversification through changes in DNA methyltransferase sequence specificity.

Epigenetic modifications such as DNA methylation have large effects on gene expression and genome maintenance. Helicobacter pylori, a human gastric pathogen, has a large number of DNA methyltransferase genes, with different strains having unique repertoires. Previous genome comparisons suggested that these methyltransferases often change DNA sequence specificity through domain movement–the movement between and within genes of coding sequences of target recognition domains. Using single-molecule real-time sequencing technology, which detects N6-methyladenines and N4-methylcytosines with single-base resolution, we studied methylated DNA sites throughout the H. pylori genome for several closely related strains. Overall, the methylome was highly variable among closely related strains. Hypermethylated regions were found, for example, in rpoB gene for RNA polymerase. We identified DNA sequence motifs for methylation and then assigned each of them to a specific homology group of the target recognition domains in the specificity-determining genes for Type I and other restriction-modification systems. These results supported proposed mechanisms for sequence-specificity changes in DNA methyltransferases. Knocking out one of the Type I specificity genes led to transcriptome changes, which suggested its role in gene expression. These results are consistent with the concept of evolution driven by DNA methylation, in which changes in the methylome lead to changes in the transcriptome and potentially to changes in phenotype, providing targets for natural or artificial selection.


July 7, 2019

Type I restriction enzymes and their relatives.

Type I restriction enzymes (REases) are large pentameric proteins with separate restriction (R), methylation (M) and DNA sequence-recognition (S) subunits. They were the first REases to be discovered and purified, but unlike the enormously useful Type II REases, they have yet to find a place in the enzymatic toolbox of molecular biologists. Type I enzymes have been difficult to characterize, but this is changing as genome analysis reveals their genes, and methylome analysis reveals their recognition sequences. Several Type I REases have been studied in detail and what has been learned about them invites greater attention. In this article, we discuss aspects of the biochemistry, biology and regulation of Type I REases, and of the mechanisms that bacteriophages and plasmids have evolved to evade them. Type I REases have a remarkable ability to change sequence specificity by domain shuffling and rearrangements. We summarize the classic experiments and observations that led to this discovery, and we discuss how this ability depends on the modular organizations of the enzymes and of their S subunits. Finally, we describe examples of Type II restriction-modification systems that have features in common with Type I enzymes, with emphasis on the varied Type IIG enzymes.


July 7, 2019

Methyltransferases acquired by lactococcal 936-type phage provide protection against restriction endonuclease activity

BACKGROUND:So-called 936-type phages are among the most frequently isolated phages in dairy facilities utilising Lactococcus lactis starter cultures. Despite extensive efforts to control phage proliferation and decades of research, these phages continue to negatively impact cheese production in terms of the final product quality and consequently, monetary return.RESULTS:Whole genome sequencing and in silico analysis of three 936-type phage genomes identified several putative (orphan) methyltransferase (MTase)-encoding genes located within the packaging and replication regions of the genome. Utilising SMRT sequencing, methylome analysis was performed on all three phages, allowing the identification of adenine modifications consistent with N-6 methyladenine sequence methylation, which in some cases could be attributed to these phage-encoded MTases. Heterologous gene expression revealed that M.Phi145I/M.Phi93I and M.Phi93DAM, encoded by genes located within the packaging module, provide protection against the restriction enzymes HphI and DpnII, respectively, representing the first functional MTases identified in members of 936-type phages.CONCLUSIONS:SMRT sequencing technology enabled the identification of the target motifs of MTases encoded by the genomes of three lytic 936-type phages and these MTases represent the first functional MTases identified in this species of phage. The presence of these MTase-encoding genes on 936-type phage genomes is assumed to represent an adaptive response to circumvent host encoded restriction-modification systems thereby increasing the fitness of the phages in a dynamic dairy environment.


July 7, 2019

Diversification of bacterial genome content through distinct mechanisms over different timescales.

Bacterial populations often consist of multiple co-circulating lineages. Determining how such population structures arise requires understanding what drives bacterial diversification. Using 616 systematically sampled genomes, we show that Streptococcus pneumoniae lineages are typically characterized by combinations of infrequently transferred stable genomic islands: those moving primarily through transformation, along with integrative and conjugative elements and phage-related chromosomal islands. The only lineage containing extensive unique sequence corresponds to a set of atypical unencapsulated isolates that may represent a distinct species. However, prophage content is highly variable even within lineages, suggesting frequent horizontal transmission that would necessitate rapidly diversifying anti-phage mechanisms to prevent these viruses sweeping through populations. Correspondingly, two loci encoding Type I restriction-modification systems able to change their specificity over short timescales through intragenomic recombination are ubiquitous across the collection. Hence short-term pneumococcal variation is characterized by movement of phage and intragenomic rearrangements, with the slower transfer of stable loci distinguishing lineages.


July 7, 2019

Expansion of the genetic toolkit for metabolic engineering of Clostridium pasteurianum: chromosomal gene disruption of the endogenous CpaAI restriction enzyme.

Clostridium pasteurianum is one of the most promising biofuel producers within the genus Clostridium owing to its unique metabolic ability to ferment glycerol into butanol. Although an efficient means is available for introducing foreign DNA to C. pasteurianum, major genetic tools, such as gene knockout, knockdown, or genome editing, are lacking, preventing metabolic engineering of C. pasteurianum.Here we present a methodology for performing chromosomal gene disruption in C. pasteurianum using the programmable lactococcus Ll.ltrB group II intron. Gene disruption was initially found to be impeded by inefficient electrotransformation of Escherichia coli-C. pasteurianum shuttle vectors, presumably due to host restriction. By assessing the ability of various vector deletion derivatives to electrotransform C. pasteurianum and probing the microorganism’s methylome using next-generation sequence data, we identified a new C. pasteurianum Type I restriction-methylation system, CpaAII, with a predicted recognition sequence of 5′-AAGNNNNNCTCC-3′ (N?=?A, C, G, or T). Following rescue of high-level electrotransformation via mutation of the sole CpaAII site within the shuttle vectors, we retargeted the intron to the cpaAIR gene encoding the CpaAI Type II restriction endonuclease (recognition site of 5′-CGCG-3′). Intron insertion was potentially hindered by low retrohoming efficiency, yet this limitation could be overcome by a procedure for enrichment of the intron insertion. The resulting ?cpaAIR mutant strain was efficiently electrotransformed with M.FnuDII-unmethylated plasmid DNA.The markerless and plasmidless ?cpaAIR mutant strain of C. pasteurianum developed in this study can serve as a general host strain for future genetic and metabolic manipulation. Further, the associated gene disruption protocol should not only serve as a guide for chromosomal gene inactivation studies involving mobile group II introns, but also prove invaluable for applying metabolic engineering strategies to C. pasteurianum.


July 7, 2019

Complete genome determination and analysis of Acholeplasma oculi strain 19L, highlighting the loss of basic genetic features in the Acholeplasmataceae.

BACKGROUND: Acholeplasma oculi belongs to the Acholeplasmataceae family, comprising the genera Acholeplasma and ‘Candidatus Phytoplasma’. Acholeplasmas are ubiquitous saprophytic bacteria. Several isolates are derived from plants or animals, whereas phytoplasmas are characterised as intracellular parasitic pathogens of plant phloem and depend on insect vectors for their spread. The complete genome sequences for eight strains of this family have been resolved so far, all of which were determined depending on clone-based sequencing. RESULTS:The A. oculi strain 19L chromosome was sequenced using two independent approaches. The first approach comprised sequencing by synthesis (Illumina) in combination with Sanger sequencing, while single molecule real time sequencing (PacBio) was used in the second. The genome was determined to be 1,587,120bp in size. Sequencing by synthesis resulted in six large genome fragments, while the single molecule real time sequencing approach yielded one circular chromosome sequence. High-quality sequences were obtained by both strategies differing in six positions, which are interpreted as reliable variations present in the culture population. Our genome analysis revealed 1,471 protein-coding genes and highlighted the absence of the F1FO-type Na+ ATPase system and GroEL/ES chaperone. Comparison of the four available Acholeplasma sequences revealed a core-genome encoding 703 proteins and a pan-genome of 2,867 proteins. CONCLUSIONS:The application of two state-of-the-art sequencing technologies highlights the potential of single molecule real time sequencing for complete genome determination. Comparative genome analyses revealed that the process of losing particular basic genetic features during genome reduction occurs in both genera, as indicated for several phytoplasma strains and at least A. oculi. The loss of the F1FO-type Na+ ATPase system may separate Acholeplasmataceae from other Mollicutes, while the loss of those genes encoding the chaperone GroEL/ES is not a rare exception in this bacterial class.


July 7, 2019

Complete genome sequence of Bifidobacterium longum 105-A, a strain with high transformation efficiency.

Bifidobacterium longum 105-A shows high transformation efficiency and allows for the generation of gene knockout mutants through homologous recombination. Here, we report the complete genome sequence of strain 105-A. Genes encoding at least four putative restriction-modification systems were found in this genome, which might contribute to its transformation efficiency. Copyright © 2014 Kanesaki et al.


July 7, 2019

Inconsistency of phenotypic and genomic characteristics of Campylobacter fetus subspecies requires reevaluation of current diagnostics.

Classifications of the Campylobacter fetus subspecies fetus and venerealis were first described in 1959 and were based on the source of isolation (intestinal versus genital) and the ability of the strains to proliferate in the genital tract of cows. Two phenotypic assays (1% glycine tolerance and H2S production) were described to differentiate the subspecies. Multiple molecular assays have been applied to differentiate the C. fetus subspecies, but none of these tests is consistent with the phenotypic identification methods. In this study, we defined the core genome and accessory genes of C. fetus, which are based on the closed genomes of five C. fetus strains. Phylogenetic analysis of the core genomes of 23 C. fetus strains of the two subspecies showed a division into two clusters. The phylogenetic core genome clusters were not consistent with the phenotypic classifications of the C. fetus subspecies. However, they were consistent with the molecular characteristics of the strains, which were determined by multilocus sequence typing, sap typing, and the presence/absence of insertion sequences and a type I restriction modification system. The similarity of the genome characteristics of three of the phenotypically defined C. fetus subsp. fetus strains to C. fetus subsp. venerealis strains, when considering the core genome and accessory genes, requires a critical evaluation of the clinical relevance of C. fetus subspecies identification by phenotypic assays. Copyright © 2014, American Society for Microbiology. All Rights Reserved.


July 7, 2019

Comparing the genomes of Helicobacter pylori clinical strain UM032 and mice-adapted derivatives.

Helicobacter pylori is a Gram-negative bacterium that persistently infects the human stomach inducing chronic inflammation. The exact mechanisms of pathogenesis are still not completely understood. Although not a natural host for H. pylori, mouse infection models play an important role in establishing the immunology and pathogenicity of H. pylori. In this study, for the first time, the genome sequences of clinical H. pylori strain UM032 and mice-adapted derivatives, 298 and 299, were sequenced using the PacBio Single Molecule, Real-Time (SMRT) technology.Here, we described the single contig which was achieved for UM032 (1,599,441 bp), 298 (1,604,216 bp) and 299 (1,601,149 bp). Preliminary analysis suggested that methylation of H. pylori genome through its restriction modification system may be determinative of its host specificity and adaptation.Availability of these genomic sequences will aid in enhancing our current level of understanding the host specificity of H. pylori.


July 7, 2019

Methods for genome-wide methylome profiling of Campylobacter jejuni.

Methylation has a profound role in the regulation of numerous biological processes in bacteria including virulence. The study of methylation in bacteria has greatly advanced thanks to next-generation sequencing technologies. These technologies have expedited the process of uncovering unique features of many bacterial methylomes such as characterizing previously uncharacterized methyltransferases, cataloging genome-wide DNA methylations in bacteria, identifying the frequency of methylation at particular genomic loci, and revealing regulatory roles of methylation in the biology of various bacterial species. For instance, methylation has been cited as a potential source for the pathogenicity differences observed in C. jejuni strains with syntenic genomes as seen in recent publications. Here, we describe the methodology for the use of Pacific Biosciences’ single molecule real-time (SMRT) sequencing for detecting methylation patterns in C. jejuni and bioinformatics tools to profile its methylome.


July 7, 2019

Genomic analysis of ST88 community-acquired methicillin resistant Staphylococcus aureus in Ghana.

The emergence and evolution of community-acquired methicillin resistant Staphylococcus aureus (CA-MRSA) strains in Africa is poorly understood. However, one particular MRSA lineage called ST88, appears to be rapidly establishing itself as an “African” CA-MRSA clone. In this study, we employed whole genome sequencing to provide more information on the genetic background of ST88 CA-MRSA isolates from Ghana and to describe in detail ST88 CA-MRSA isolates in comparison with other MRSA lineages worldwide.We first established a complete ST88 reference genome (AUS0325) using PacBio SMRT sequencing. We then used comparative genomics to assess relatedness among 17 ST88 CA-MRSA isolates recovered from patients attending Buruli ulcer treatment centres in Ghana, three non-African ST88s and 15 other MRSA lineages.We show that Ghanaian ST88 forms a discrete MRSA lineage (harbouring SCCmec-IV [2B]). Gene content analysis identified five distinct genomic regions enriched among ST88 isolates compared with the other S. aureus lineages. The Ghanaian ST88 isolates had only 658 core genome SNPs and there was no correlation between phylogeny and geography, suggesting the recent spread of this clone. The lineage was also resistant to multiple classes of antibiotics including ß-lactams, tetracycline and chloramphenicol.This study reveals that S. aureus ST88-IV is a recently emerging and rapidly spreading CA-MRSA clone in Ghana. The study highlights the capacity of small snapshot genomic studies to provide actionable public health information in resource limited settings. To our knowledge this is the first genomic assessment of the ST88 CA-MRSA clone.


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