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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.

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July 7, 2019

Whole-genome analysis of Exserohilum rostratum from an outbreak of fungal meningitis and other infections.

Exserohilum rostratum was the cause of most cases of fungal meningitis and other infections associated with the injection of contaminated methylprednisolone acetate produced by the New England Compounding Center (NECC). Until this outbreak, very few human cases of Exserohilum infection had been reported, and very little was known about this dematiaceous fungus, which usually infects plants. Here, we report using whole-genome sequencing (WGS) for the detection of single nucleotide polymorphisms (SNPs) and phylogenetic analysis to investigate the molecular origin of the outbreak using 22 isolates of E. rostratum retrieved from 19 case patients with meningitis or epidural/spinal abscesses, 6 isolates from contaminated NECC vials, and 7 isolates unrelated to the outbreak. Our analysis indicates that all 28 isolates associated with the outbreak had nearly identical genomes of 33.8 Mb. A total of 8 SNPs were detected among the outbreak genomes, with no more than 2 SNPs separating any 2 of the 28 genomes. The outbreak genomes were separated from the next most closely related control strain by ~136,000 SNPs. We also observed significant genomic variability among strains unrelated to the outbreak, which may suggest the possibility of cryptic speciation in E. rostratum. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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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.

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July 7, 2019

The genome of the intracellular bacterium of the coastal bivalve, Solemya velum: a blueprint for thriving in and out of symbiosis

BACKGROUND:Symbioses between chemoautotrophic bacteria and marine invertebrates are rare examples of living systems that are virtually independent of photosynthetic primary production. These associations have evolved multiple times in marine habitats, such as deep-sea hydrothermal vents and reducing sediments, characterized by steep gradients of oxygen and reduced chemicals. Due to difficulties associated with maintaining these symbioses in the laboratory and culturing the symbiotic bacteria, studies of chemosynthetic symbioses rely heavily on culture independent methods. The symbiosis between the coastal bivalve, Solemya velum, and its intracellular symbiont is a model for chemosynthetic symbioses given its accessibility in intertidal environments and the ability to maintain it under laboratory conditions. To better understand this symbiosis, the genome of the S. velum endosymbiont was sequenced.RESULTS:Relative to the genomes of obligate symbiotic bacteria, which commonly undergo erosion and reduction, the S. velum symbiont genome was large (2.7Mb), GC-rich (51%), and contained a large number (78) of mobile genetic elements. Comparative genomics identified sets of genes specific to the chemosynthetic lifestyle and necessary to sustain the symbiosis. In addition, a number of inferred metabolic pathways and cellular processes, including heterotrophy, branched electron transport, and motility, suggested that besides the ability to function as an endosymbiont, the bacterium may have the capacity to live outside the host.CONCLUSIONS:The physiological dexterity indicated by the genome substantially improves our understanding of the genetic and metabolic capabilities of the S. velum symbiont and the breadth of niches the partners may inhabit during their lifecycle.

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July 7, 2019

An evaluation of alternative methods for constructing phylogenies from whole genome sequence data: a case study with Salmonella.

Comparative genomics based on whole genome sequencing (WGS) is increasingly being applied to investigate questions within evolutionary and molecular biology, as well as questions concerning public health (e.g., pathogen outbreaks). Given the impact that conclusions derived from such analyses may have, we have evaluated the robustness of clustering individuals based on WGS data to three key factors: (1) next-generation sequencing (NGS) platform (HiSeq, MiSeq, IonTorrent, 454, and SOLiD), (2) algorithms used to construct a SNP (single nucleotide polymorphism) matrix (reference-based and reference-free), and (3) phylogenetic inference method (FastTreeMP, GARLI, and RAxML). We carried out these analyses on 194 whole genome sequences representing 107 unique Salmonella enterica subsp. enterica ser. Montevideo strains. Reference-based approaches for identifying SNPs produced trees that were significantly more similar to one another than those produced under the reference-free approach. Topologies inferred using a core matrix (i.e., no missing data) were significantly more discordant than those inferred using a non-core matrix that allows for some missing data. However, allowing for too much missing data likely results in a high false discovery rate of SNPs. When analyzing the same SNP matrix, we observed that the more thorough inference methods implemented in GARLI and RAxML produced more similar topologies than FastTreeMP. Our results also confirm that reproducibility varies among NGS platforms where the MiSeq had the lowest number of pairwise differences among replicate runs. Our investigation into the robustness of clustering patterns illustrates the importance of carefully considering how data from different platforms are combined and analyzed. We found clear differences in the topologies inferred, and certain methods performed significantly better than others for discriminating between the highly clonal organisms investigated here. The methods supported by our results represent a preliminary set of guidelines and a step towards developing validated standards for clustering based on whole genome sequence data.

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July 7, 2019

Seeking the source of Pseudomonas aeruginosa infections in a recently opened hospital: an observational study using whole-genome sequencing.

Pseudomonas aeruginosa is a common nosocomial pathogen responsible for significant morbidity and mortality internationally. Patients may become colonised or infected with P. aeruginosa after exposure to contaminated sources within the hospital environment. The aim of this study was to determine whether whole-genome sequencing (WGS) can be used to determine the source in a cohort of burns patients at high risk of P. aeruginosa acquisition.An observational prospective cohort study.Burns care ward and critical care ward in the UK.Patients with >7% total burns by surface area were recruited into the study.All patients were screened for P. aeruginosa on admission and samples taken from their immediate environment, including water. Screening patients who subsequently developed a positive P. aeruginosa microbiology result were subject to enhanced environmental surveillance. All isolates of P. aeruginosa were genome sequenced. Sequence analysis looked at similarity and relatedness between isolates.WGS for 141 P. aeruginosa isolates were obtained from patients, hospital water and the ward environment. Phylogenetic analysis revealed eight distinct clades, with a single clade representing the majority of environmental isolates in the burns unit. Isolates from three patients had identical genotypes compared with water isolates from the same room. There was clear clustering of water isolates by room and outlet, allowing the source of acquisitions to be unambiguously identified. Whole-genome shotgun sequencing of biofilm DNA extracted from a thermostatic mixer valve revealed this was the source of a P. aeruginosa subpopulation previously detected in water. In the remaining two cases there was no clear link to the hospital environment.This study reveals that WGS can be used for source tracking of P. aeruginosa in a hospital setting, and that acquisitions can be traced to a specific source within a hospital ward. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

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July 7, 2019

The odd one out: Bacillus ACT bacteriophage CP-51 exhibits unusual properties compared to related Spounavirinae W.Ph. and Bastille.

The Bacillus ACT group includes three important pathogenic species of Bacillus: anthracis, cereus and thuringiensis. We characterized three virulent bacteriophages, Bastille, W.Ph. and CP-51, that infect various strains of these three species. We have determined the complete genome sequences of CP-51, W.Ph. and Bastille, and their physical genome structures. The CP-51 genome sequence could only be obtained using a combination of conventional and second and third next generation sequencing technologies – illustrating the problems associated with sequencing highly modified DNA. We present evidence that the generalized transduction facilitated by CP-51 is independent of a specific genome structure, but likely due to sporadic packaging errors of the terminase. There is clear correlation of the genetic and morphological features of these phages validating their placement in the Spounavirinae subfamily (SPO1-related phages) of the Myoviridae. This study also provides tools for the development of phage-based diagnostics/therapeutics for this group of pathogens. Copyright © 2014 Elsevier Inc. All rights reserved.

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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.

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July 7, 2019

The Harvest suite for rapid core-genome alignment and visualization of thousands of intraspecific microbial genomes.

Whole-genome sequences are now available for many microbial species and clades, however, existing whole-genome alignment methods are limited in their ability to perform sequence comparisons of multiple sequences simultaneously. Here we present the Harvest suite of core-genome alignment and visualization tools for the rapid and simultaneous analysis of thousands of intraspecific microbial strains. Harvest includes Parsnp, a fast core-genome multi-aligner, and Gingr, a dynamic visual platform. Together they provide interactive core-genome alignments, variant calls, recombination detection, and phylogenetic trees. Using simulated and real data we demonstrate that our approach exhibits unrivaled speed while maintaining the accuracy of existing methods. The Harvest suite is open-source and freely available from: http://github.com/marbl/harvest.

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July 7, 2019

Quality scores for 32,000 genomes.

More than 80% of the microbial genomes in GenBank are of ‘draft’ quality (12,553 draft vs. 2,679 finished, as of October, 2013). We have examined all the microbial DNA sequences available for complete, draft, and Sequence Read Archive genomes in GenBank as well as three other major public databases, and assigned quality scores for more than 30,000 prokaryotic genome sequences.Scores were assigned using four categories: the completeness of the assembly, the presence of full-length rRNA genes, tRNA composition and the presence of a set of 102 conserved genes in prokaryotes. Most (~88%) of the genomes had quality scores of 0.8 or better and can be safely used for standard comparative genomics analysis. We compared genomes across factors that may influence the score. We found that although sequencing depth coverage of over 100x did not ensure a better score, sequencing read length was a better indicator of sequencing quality. With few exceptions, most of the 30,000 genomes have nearly all the 102 essential genes.The score can be used to set thresholds for screening data when analyzing “all published genomes” and reference data is either not available or not applicable. The scores highlighted organisms for which commonly used tools do not perform well. This information can be used to improve tools and to serve a broad group of users as more diverse organisms are sequenced. Unexpectedly, the comparison of predicted tRNAs across 15,000 high quality genomes showed that anticodons beginning with an ‘A’ (codons ending with a ‘U’) are almost non-existent, with the exception of one arginine codon (CGU); this has been noted previously in the literature for a few genomes, but not with the depth found here.

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July 7, 2019

Genome sequence of the moderately halophilic bacterium Salinicoccus carnicancri type strain Crm(T) (= DSM 23852(T)).

Salinicoccus carnicancri Jung et al. 2010 belongs to the genus Salinicoccus in the family Staphylococcaceae. Members of the Salinicoccus are moderately halophilic and originate from various salty environments. The halophilic features of the Salinicoccus suggest their possible uses in biotechnological applications, such as biodegradation and fermented food production. However, the genus Salinicoccus is poorly characterized at the genome level, despite its potential importance. This study presents the draft genome sequence of S. carnicancri strain Crm(T) and its annotation. The 2,673,309 base pair genome contained 2,700 protein-coding genes and 78 RNA genes with an average G+C content of 47.93 mol%. It was notable that the strain carried 72 predicted genes associated with osmoregulation, which suggests the presence of beneficial functions that facilitate growth in high-salt environments.

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July 7, 2019

Complete genome sequence of Staphylococcus aureus Z172, a vancomycin-intermediate and daptomycin-nonsusceptible methicillin-resistant strain isolated in Taiwan.

We report the complete genome sequence of Z172, a representative strain of sequence type 239-staphylococcal cassette chromosome mec type III (ST239-SCCmec type III) hospital-associated methicillin-resistant Staphylococcus aureus in Taiwan. Strain Z172 also exhibits a vancomycin-intermediate and daptomycin-nonsusceptible phenotype.

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July 7, 2019

Mutation in the C-di-AMP cyclase dacA affects fitness and resistance of methicillin resistant Staphylococcus aureus.

Faster growing and more virulent strains of methicillin resistant Staphylococcus aureus (MRSA) are increasingly displacing highly resistant MRSA. Elevated fitness in these MRSA is often accompanied by decreased and heterogeneous levels of methicillin resistance; however, the mechanisms for this phenomenon are not yet fully understood. Whole genome sequencing was used to investigate the genetic basis of this apparent correlation, in an isogenic MRSA strain pair that differed in methicillin resistance levels and fitness, with respect to growth rate. Sequencing revealed only one single nucleotide polymorphism (SNP) in the diadenylate cyclase gene dacA in the faster growing but less resistant strain. Diadenylate cyclases were recently discovered to synthesize the new second messenger cyclic diadenosine monophosphate (c-di-AMP). Introduction of this mutation into the highly resistant but slower growing strain reduced resistance and increased its growth rate, suggesting a direct connection between the dacA mutation and the phenotypic differences of these strains. Quantification of cellular c-di-AMP revealed that the dacA mutation decreased c-di-AMP levels resulting in reduced autolysis, increased salt tolerance and a reduction in the basal expression of the cell wall stress stimulon. These results indicate that c-di-AMP affects cell envelope-related signalling in S. aureus. The influence of c-di-AMP on growth rate and methicillin resistance in MRSA indicate that altering c-di-AMP levels could be a mechanism by which MRSA strains can increase their fitness levels by reducing their methicillin resistance levels.

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