July 19, 2019  |  

Genome-wide mapping of methylated adenine residues in pathogenic Escherichia coli using single-molecule real-time sequencing.

Single-molecule real-time (SMRT) DNA sequencing allows the systematic detection of chemical modifications such as methylation but has not previously been applied on a genome-wide scale. We used this approach to detect 49,311 putative 6-methyladenine (m6A) residues and 1,407 putative 5-methylcytosine (m5C) residues in the genome of a pathogenic Escherichia coli strain. We obtained strand-specific information for methylation sites and a quantitative assessment of the frequency of methylation at each modified position. We deduced the sequence motifs recognized by the methyltransferase enzymes present in this strain without prior knowledge of their specificity. Furthermore, we found that deletion of a phage-encoded methyltransferase-endonuclease (restriction-modification; RM) system induced global transcriptional changes and led to gene amplification, suggesting that the role of RM systems extends beyond protecting host genomes from foreign DNA.


July 19, 2019  |  

The complete genome sequence of Escherichia coli EC958: a high quality reference sequence for the globally disseminated multidrug resistant E. coli O25b:H4-ST131 clone.

Escherichia coli ST131 is now recognised as a leading contributor to urinary tract and bloodstream infections in both community and clinical settings. Here we present the complete, annotated genome of E. coli EC958, which was isolated from the urine of a patient presenting with a urinary tract infection in the Northwest region of England and represents the most well characterised ST131 strain. Sequencing was carried out using the Pacific Biosciences platform, which provided sufficient depth and read-length to produce a complete genome without the need for other technologies. The discovery of spurious contigs within the assembly that correspond to site-specific inversions in the tail fibre regions of prophages demonstrates the potential for this technology to reveal dynamic evolutionary mechanisms. E. coli EC958 belongs to the major subgroup of ST131 strains that produce the CTX-M-15 extended spectrum ß-lactamase, are fluoroquinolone resistant and encode the fimH30 type 1 fimbrial adhesin. This subgroup includes the Indian strain NA114 and the North American strain JJ1886. A comparison of the genomes of EC958, JJ1886 and NA114 revealed that differences in the arrangement of genomic islands, prophages and other repetitive elements in the NA114 genome are not biologically relevant and are due to misassembly. The availability of a high quality uropathogenic E. coli ST131 genome provides a reference for understanding this multidrug resistant pathogen and will facilitate novel functional, comparative and clinical studies of the E. coli ST131 clonal lineage.


July 19, 2019  |  

Bacteriophage orphan DNA methyltransferases: insights from their bacterial origin, function, and occurrence.

Type II DNA methyltransferases (MTases) are enzymes found ubiquitously in the prokaryotic world, where they play important roles in several cellular processes, such as host protection and epigenetic regulation. Three classes of type II MTases have been identified thus far in bacteria which function in transferring a methyl group from S-adenosyl-l-methionine (SAM) to a target nucleotide base, forming N-6-methyladenine (class I), N-4-methylcytosine (class II), or C-5-methylcytosine (class III). Often, these MTases are associated with a cognate restriction endonuclease (REase) to form a restriction-modification (R-M) system protecting bacterial cells from invasion by foreign DNA. When MTases exist alone, which are then termed orphan MTases, they are believed to be mainly involved in regulatory activities in the bacterial cell. Genomes of various lytic and lysogenic phages have been shown to encode multi- and mono-specific orphan MTases that have the ability to confer protection from restriction endonucleases of their bacterial host(s). The ability of a phage to overcome R-M and other phage-targeting resistance systems can be detrimental to particular biotechnological processes such as dairy fermentations. Conversely, as phages may also be beneficial in certain areas such as phage therapy, phages with additional resistance to host defenses may prolong the effectiveness of the therapy. This minireview will focus on bacteriophage-encoded MTases, their prevalence and diversity, as well as their potential origin and function.


July 19, 2019  |  

Identification of restriction-modification systems of Bifidobacterium animalis subsp. lactis CNCM I-2494 by SMRT Sequencing and associated methylome analysis.

Bifidobacterium animalis subsp. lactis CNCM I-2494 is a component of a commercialized fermented dairy product for which beneficial effects on health has been studied by clinical and preclinical trials. To date little is known about the molecular mechanisms that could explain the beneficial effects that bifidobacteria impart to the host. Restriction-modification (R-M) systems have been identified as key obstacles in the genetic accessibility of bifidobacteria, and circumventing these is a prerequisite to attaining a fundamental understanding of bifidobacterial attributes, including the genes that are responsible for health-promoting properties of this clinically and industrially important group of bacteria. The complete genome sequence of B. animalis subsp. lactis CNCM I-2494 is predicted to harbour the genetic determinants for two type II R-M systems, designated BanLI and BanLII. In order to investigate the functionality and specificity of these two putative R-M systems in B. animalis subsp. lactis CNCM I-2494, we employed PacBio SMRT sequencing with associated methylome analysis. In addition, the contribution of the identified R-M systems to the genetic accessibility of this strain was assessed.


July 19, 2019  |  

Comparative genomic analysis and virulence differences in closely related Salmonella enterica serotype Heidelberg isolates from humans, retail meats, and animals.

Salmonella enterica subsp. enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. Recently, an antibiotic-resistant strain of this serovar was implicated in a large 2011 multistate outbreak resulting from consumption of contaminated ground turkey that involved 136 confirmed cases, with one death. In this study, we assessed the evolutionary diversity of 44 S. Heidelberg isolates using whole-genome sequencing (WGS) generated by the 454 GS FLX (Roche) platform. The isolates, including 30 with nearly indistinguishable (one band difference) Xbal pulsed-field gel electrophoresis patterns (JF6X01.0032, JF6X01.0058), were collected from various sources between 1982 and 2011 and included nine isolates associated with the 2011 outbreak. Additionally, we determined the complete sequence for the chromosome and three plasmids from a clinical isolate associated with the 2011 outbreak using the Pacific Biosciences (PacBio) system. Using single-nucleotide polymorphism (SNP) analyses, we were able to distinguish highly clonal isolates, including strains isolated at different times in the same year. The isolates from the recent 2011 outbreak clustered together with a mean SNP variation of only 17 SNPs. The S. Heidelberg isolates carried a variety of phages, such as prophage P22, P4, lambda-like prophage Gifsy-2, and the P2-like phage which carries the sopE1 gene, virulence genes including 62 pathogenicity, and 13 fimbrial markers and resistance plasmids of the incompatibility (Inc)I1, IncA/C, and IncHI2 groups. Twenty-one strains contained an IncX plasmid carrying a type IV secretion system. On the basis of the recent and historical isolates used in this study, our results demonstrated that, in addition to providing detailed genetic information for the isolates, WGS can identify SNP targets that can be utilized for differentiating highly clonal S. Heidelberg isolates.


July 19, 2019  |  

Unlocking the mystery of the hard-to-sequence phage genome: PaP1 methylome and bacterial immunity.

Whole-genome sequencing is an important method to understand the genetic information, gene function, biological characteristics and survival mechanisms of organisms. Sequencing large genomes is very simple at present. However, we encountered a hard-to-sequence genome of Pseudomonas aeruginosa phage PaP1. Shotgun sequencing method failed to complete the sequence of this genome.After persevering for 10 years and going over three generations of sequencing techniques, we successfully completed the sequence of the PaP1 genome with a length of 91,715 bp. Single-molecule real-time sequencing results revealed that this genome contains 51?N-6-methyladenines and 152?N-4-methylcytosines. Three significant modified sequence motifs were predicted, but not all of the sites found in the genome were methylated in these motifs. Further investigations revealed a novel immune mechanism of bacteria, in which host bacteria can recognise and repel modified bases containing inserts in a large scale. This mechanism could be accounted for the failure of the shotgun method in PaP1 genome sequencing. This problem was resolved using the nfi- mutant of Escherichia coli DH5a as a host bacterium to construct a shotgun library.This work provided insights into the hard-to-sequence phage PaP1 genome and discovered a new mechanism of bacterial immunity. The methylome of phage PaP1 is responsible for the failure of shotgun sequencing and for bacterial immunity mediated by enzyme Endo V activity; this methylome also provides a valuable resource for future studies on PaP1 genome replication and modification, as well as on gene regulation and host interaction.


July 19, 2019  |  

BREX is a novel phage resistance system widespread in microbial genomes.

The perpetual arms race between bacteria and phage has resulted in the evolution of efficient resistance systems that protect bacteria from phage infection. Such systems, which include the CRISPR-Cas and restriction-modification systems, have proven to be invaluable in the biotechnology and dairy industries. Here, we report on a six-gene cassette in Bacillus cereus which, when integrated into the Bacillus subtilis genome, confers resistance to a broad range of phages, including both virulent and temperate ones. This cassette includes a putative Lon-like protease, an alkaline phosphatase domain protein, a putative RNA-binding protein, a DNA methylase, an ATPase-domain protein, and a protein of unknown function. We denote this novel defense system BREX (Bacteriophage Exclusion) and show that it allows phage adsorption but blocks phage DNA replication. Furthermore, our results suggest that methylation on non-palindromic TAGGAG motifs in the bacterial genome guides self/non-self discrimination and is essential for the defensive function of the BREX system. However, unlike restriction-modification systems, phage DNA does not appear to be cleaved or degraded by BREX, suggesting a novel mechanism of defense. Pan genomic analysis revealed that BREX and BREX-like systems, including the distantly related Pgl system described in Streptomyces coelicolor, are widely distributed in ~10% of all sequenced microbial genomes and can be divided into six coherent subtypes in which the gene composition and order is conserved. Finally, we detected a phage family that evades the BREX defense, implying that anti-BREX mechanisms may have evolved in some phages as part of their arms race with bacteria.© 2014 The Authors.


July 19, 2019  |  

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage f-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage.The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.


July 19, 2019  |  

Continuous evolution of Bacillus thuringiensis toxins overcomes insect resistance.

The Bacillus thuringiensis d-endotoxins (Bt toxins) are widely used insecticidal proteins in engineered crops that provide agricultural, economic, and environmental benefits. The development of insect resistance to Bt toxins endangers their long-term effectiveness. Here we have developed a phage-assisted continuous evolution selection that rapidly evolves high-affinity protein-protein interactions, and applied this system to evolve variants of the Bt toxin Cry1Ac that bind a cadherin-like receptor from the insect pest Trichoplusia ni (TnCAD) that is not natively bound by wild-type Cry1Ac. The resulting evolved Cry1Ac variants bind TnCAD with high affinity (dissociation constant Kd?=?11-41?nM), kill TnCAD-expressing insect cells that are not susceptible to wild-type Cry1Ac, and kill Cry1Ac-resistant T. ni insects up to 335-fold more potently than wild-type Cry1Ac. Our findings establish that the evolution of Bt toxins with novel insect cell receptor affinity can overcome insect Bt toxin resistance and confer lethality approaching that of the wild-type Bt toxin against non-resistant insects.


July 19, 2019  |  

Host genome integration and giant virus-induced reactivation of the virophage mavirus.

Endogenous viral elements are increasingly found in eukaryotic genomes, yet little is known about their origins, dynamics, or function. Here we provide a compelling example of a DNA virus that readily integrates into a eukaryotic genome where it acts as an inducible antiviral defence system. We found that the virophage mavirus, a parasite of the giant Cafeteria roenbergensis virus (CroV), integrates at multiple sites within the nuclear genome of the marine protozoan Cafeteria roenbergensis. The endogenous mavirus is structurally and genetically similar to eukaryotic DNA transposons and endogenous viruses of the Maverick/Polinton family. Provirophage genes are not constitutively expressed, but are specifically activated by superinfection with CroV, which induces the production of infectious mavirus particles. Virophages can inhibit the replication of mimivirus-like giant viruses and an anti-viral protective effect of provirophages on their hosts has been hypothesized. We find that provirophage-carrying cells are not directly protected from CroV; however, lysis of these cells releases infectious mavirus particles that are then able to suppress CroV replication and enhance host survival during subsequent rounds of infection. The microbial host-parasite interaction described here involves an altruistic aspect and suggests that giant-virus-induced activation of provirophages might be ecologically relevant in natural protist populations.


July 7, 2019  |  

Comparative genome analysis of Pseudomonas knackmussii B13, the first bacterium known to degrade chloroaromatic compounds.

Pseudomonas knackmussii B13 was the first strain to be isolated in 1974 that could degrade chlorinated aromatic hydrocarbons. This discovery was the prologue for subsequent characterization of numerous bacterial metabolic pathways, for genetic and biochemical studies, and which spurred ideas for pollutant bioremediation. In this study, we determined the complete genome sequence of B13 using next generation sequencing technologies and optical mapping. Genome annotation indicated that B13 has a variety of metabolic pathways for degrading monoaromatic hydrocarbons including chlorobenzoate, aminophenol, anthranilate and hydroxyquinol, but not polyaromatic compounds. Comparative genome analysis revealed that B13 is closest to Pseudomonas denitrificans and Pseudomonas aeruginosa. The B13 genome contains at least eight genomic islands [prophages and integrative conjugative elements (ICEs)], which were absent in closely related pseudomonads. We confirm that two ICEs are identical copies of the 103?kb self-transmissible element ICEclc that carries the genes for chlorocatechol metabolism. Comparison of ICEclc showed that it is composed of a variable and a ‘core’ region, which is very conserved among proteobacterial genomes, suggesting a widely distributed family of so far uncharacterized ICE. Resequencing of two spontaneous B13 mutants revealed a number of single nucleotide substitutions, as well as excision of a large 220?kb region and a prophage that drastically change the host metabolic capacity and survivability. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.


July 7, 2019  |  

Draft genome sequence of Erwinia tracheiphila, an economically important bacterial pathogen of cucurbits.

Erwinia tracheiphila is one of the most economically important pathogens of cucumbers, melons, squashes, pumpkins, and gourds in the northeastern and midwestern United States, yet its molecular pathology remains uninvestigated. Here, we report the first draft genome sequence of an E. tracheiphila strain isolated from an infected wild gourd (Cucurbita pepo subsp. texana) plant. The genome assembly consists of 7 contigs and includes a putative plasmid and at least 20 phage and prophage elements. Copyright © 2015 Shapiro et al.


July 7, 2019  |  

Paenibacillus larvae-directed bacteriophage HB10c2 and its application in American Foulbrood-affected honey bee larvae.

Paenibacillus larvae is the causative agent of American foulbrood (AFB), the most serious honey bee brood bacterial disease. We isolated and characterized P. larvae-directed bacteriophages and developed criteria for safe phage therapy. Whole-genome analysis of a highly lytic virus of the family Siphoviridae (HB10c2) provided a detailed safety profile and uncovered its lysogenic nature and a putative beta-lactamase-like protein. To rate its antagonistic activity against the pathogens targeted and to specify potentially harmful effects on the bee population and the environment, P. larvae genotypes ERIC I to IV, representatives of the bee gut microbiota, and a broad panel of members of the order Bacillales were analyzed for phage HB10c2-induced lysis. Breeding assays with infected bee larvae revealed that the in vitro phage activity observed was not predictive of the real-life scenario and therapeutic efficacy. On the basis of the disclosed P. larvae-bacteriophage coevolution, we discuss the future prospects of AFB phage therapy. Copyright © 2015, American Society for Microbiology. All Rights Reserved.


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