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July 19, 2019

The highly heterogeneous methylated genomes and diverse restriction-modification systems of bloom-forming Microcystis.

The occurrence of harmful Microcystis blooms is increasing in frequency in a myriad of freshwater ecosystems. Despite considerable research pertaining to the cause and nature of these blooms, the molecular mechanisms behind the cosmopolitan distribution and phenotypic diversity in Microcystis are still unclear. We compared the patterns and extent of DNA methylation in three strains of Microcystis, PCC 7806SL, NIES-2549 and FACHB-1757, using Single Molecule Real-Time (SMRT) sequencing technology. Intact restriction-modification (R-M) systems were identified from the genomes of these strains, and from two previously sequenced strains of Microcystis, NIES-843 and TAIHU98. A large number of methylation motifs and R-M genes were identified in these strains, which differ substantially among different strains. Of the 35 motifs identified, eighteen had not previously been reported. Strain NIES-843 contains a larger number of total putative methyltransferase genes than have been reported previously from any bacterial genome. Genomic comparisons reveal that methyltransferases (some partial) may have been acquired from the environment through horizontal gene transfer. Copyright © 2018 Elsevier B.V. All rights reserved.

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July 19, 2019

Introduction: The host-associated microbiome: Pattern, process and function.

An explosion of studies in recent years has established the ubiquity of host-associated microbes and their centrality to host biology (McFall-Ngai et al., 2013; Russell, Dubilier, & Rudgers, 2014). Microbes aid in digestion, modulate development, contribute to host immunity, mediate abiotic stress and more. While relationships with host-associated microbes are ubiquitous and important, they are cer- tainly not monolithic. Characterizing the microbial diversity associ- ated with an ever-broadening array of hosts (diverse animals, plants, algae and protists) has shown that essential functions can be per- formed by microbes that are integrated with the host to varying degrees, ranging from embedded endosymbionts to a variable cast of transient microbes acquired from the environment. The maturing host–microbiome field is now developing a mechanistic understand- ing of host/microbe relationships across this spectrum and the cross- talk mediating these interactions. Similarly, studies across systems are illuminating the ecological and evolutionary factors that shape host–microbe interactions today and providing hints into the origins of specific relationships.

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July 19, 2019

A Borrelia burgdorferi mini-vls system that undergoes antigenic switching in mice: investigation of the role of plasmid topology and the long inverted repeat.

Borrelia burgdorferi evades the host immune system by switching the surface antigen. VlsE, in a process known as antigenic variation. The DNA mechanisms and genetic elements present on the vls locus that participate in the switching process remain to be elucidated. Manipulating the vls locus has been difficult due to its instability on Escherichia coli plasmids. In this study, we generated for the first time a mini-vls system composed of a single silent vlsE variable region (silent cassette 2) through the vlsE gene by performing some cloning steps directly in a highly transformable B. burgdorferi strain. Variants of the mini system were constructed with or without the long inverted repeat (IR) located upstream of vlsE and on both circular and linear plasmids to investigate the importance of the IR and plasmid topology on recombinational switching at vlsE. Amplicon sequencing using PacBio long read technology and analysis of the data with our recently reported pipeline and VAST software showed that the system undergoes switching in mice in both linear and circular versions and that the presence of the hairpin does not seem to be crucial in the linear version, however it is required when the topology is circular.© 2018 John Wiley & Sons Ltd.

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July 19, 2019

Comparison between complete genomes of an isolate of Pseudomonas syringae pv. actinidiae from Japan and a New Zealand isolate of the pandemic.

The modern pandemic of the bacterial kiwifruit pathogen Pseudomonas syringae pv actinidiae (Psa) is caused by a particular Psa lineage. To better understand the genetic basis of the virulence of this lineage, we compare the completely assembled genome of a pandemic New Zealand strain with that of the Psa type strain first isolated in Japan in 1983. Aligning the two genomes shows numerous translocations, constrained so as to retain the appropriate orientation of the Architecture Imparting Sequences (AIMs). There are several large horizontally acquired regions, some of which include Type I, Type II or Type III restriction systems. The activity of these systems is reflected in the methylation patterns of the two strains. The pandemic strain carries an Integrative Conjugative Element (ICE) located at a tRNA-Lys site. Two other complex elements are also present at tRNA-Lys sites in the genome. These elements are derived from ICE but have now acquired some alternative secretion function. There are numerous types of mobile element in the two genomes. Analysis of these elements reveals no evidence of recombination between the two Psa lineages.

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July 19, 2019

Deep genome annotation of the opportunistic human pathogen Streptococcus pneumoniae D39.

A precise understanding of the genomic organization into transcriptional units and their regulation is essential for our comprehension of opportunistic human pathogens and how they cause disease. Using single-molecule real-time (PacBio) sequencing we unambiguously determined the genome sequence of Streptococcus pneumoniae strain D39 and revealed several inversions previously undetected by short-read sequencing. Significantly, a chromosomal inversion results in antigenic variation of PhtD, an important surface-exposed virulence factor. We generated a new genome annotation using automated tools, followed by manual curation, reflecting the current knowledge in the field. By combining sequence-driven terminator prediction, deep paired-end transcriptome sequencing and enrichment of primary transcripts by Cappable-Seq, we mapped 1015 transcriptional start sites and 748 termination sites. We show that the pneumococcal transcriptional landscape is complex and includes many secondary, antisense and internal promoters. Using this new genomic map, we identified several new small RNAs (sRNAs), RNA switches (including sixteen previously misidentified as sRNAs), and antisense RNAs. In total, we annotated 89 new protein-encoding genes, 34 sRNAs and 165 pseudogenes, bringing the S. pneumoniae D39 repertoire to 2146 genetic elements. We report operon structures and observed that 9% of operons are leaderless. The genome data are accessible in an online resource called PneumoBrowse (https://veeninglab.com/pneumobrowse) providing one of the most complete inventories of a bacterial genome to date. PneumoBrowse will accelerate pneumococcal research and the development of new prevention and treatment strategies.

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July 19, 2019

Degradation and remobilization of endogenous retroviruses by recombination during the earliest stages of a germ-line invasion.

Endogenous retroviruses (ERVs) are proviral sequences that result from colonization of the host germ line by exogenous retroviruses. The majority of ERVs represent defective retroviral copies. However, for most ERVs, endogenization occurred millions of years ago, obscuring the stages by which ERVs become defective and the changes in both virus and host important to the process. The koala retrovirus, KoRV, only recently began invading the germ line of the koala (Phascolarctos cinereus), permitting analysis of retroviral endogenization on a prospective basis. Here, we report that recombination with host genomic elements disrupts retroviruses during the earliest stages of germ-line invasion. One type of recombinant, designated recKoRV1, was formed by recombination of KoRV with an older degraded retroelement. Many genomic copies of recKoRV1 were detected across koalas. The prevalence of recKoRV1 was higher in northern than in southern Australian koalas, as is the case for KoRV, with differences in recKoRV1 prevalence, but not KoRV prevalence, between inland and coastal New South Wales. At least 15 additional different recombination events between KoRV and the older endogenous retroelement generated distinct recKoRVs with different geographic distributions. All of the identified recombinant viruses appear to have arisen independently and have highly disrupted ORFs, which suggests that recombination with existing degraded endogenous retroelements may be a means by which replication-competent ERVs that enter the germ line are degraded. Copyright © 2018 the Author(s). Published by PNAS.

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July 19, 2019

From short reads to chromosome-scale genome assemblies.

A high-quality, annotated genome assembly is the foundation for many downstream studies. However, obtaining such an assembly is a complex, reiterative process that requires the assimilation of high-quality data and combines different approaches and data types. While some software packages incorporating multiple steps of genome assembly are commercially available, they may not be flexible enough to be routinely applied to all organisms, particularly to nonmodel species such as pathogenic oomycetes and fungi. If researchers understand and apply the most appropriate, currently available tools for each step, it is possible to customize parameters and optimize results for their organism of study. Based on our experience of de novo assembly and annotation of several oomycete species, this chapter provides a modular workflow from processing of raw reads, to initial assembly generation, through optimization, chromosome-scale scaffolding and annotation, outlining input and output data as well as examples and alternative software used for each step. The accompanying Notes provide background information for each step as well as alternative options. The final result of this workflow could be an annotated, high-quality, validated, chromosome-scale assembly or a draft assembly of sufficient quality to meet specific needs of a project.

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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.

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July 7, 2019

Emergence of scarlet fever Streptococcus pyogenes emm12 clones in Hong Kong is associated with toxin acquisition and multidrug resistance.

A scarlet fever outbreak began in mainland China and Hong Kong in 2011 (refs. 1-6). Macrolide- and tetracycline-resistant Streptococcus pyogenes emm12 isolates represent the majority of clinical cases. Recently, we identified two mobile genetic elements that were closely associated with emm12 outbreak isolates: the integrative and conjugative element ICE-emm12, encoding genes for tetracycline and macrolide resistance, and prophage FHKU.vir, encoding the superantigens SSA and SpeC, as well as the DNase Spd1 (ref. 4). Here we sequenced the genomes of 141 emm12 isolates, including 132 isolated in Hong Kong between 2005 and 2011. We found that the introduction of several ICE-emm12 variants, FHKU.vir and a new prophage, FHKU.ssa, occurred in three distinct emm12 lineages late in the twentieth century. Acquisition of ssa and transposable elements encoding multidrug resistance genes triggered the expansion of scarlet fever-associated emm12 lineages in Hong Kong. The occurrence of multidrug-resistant ssa-harboring scarlet fever strains should prompt heightened surveillance within China and abroad for the dissemination of these mobile genetic elements.

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July 7, 2019

Complete annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono).

We report the completely annotated genome sequence of Mycobacterium tuberculosis (Zopf) Lehmann and Neumann (ATCC35812) (Kurono), which is a used for virulence and/or immunization studies. The complete genome sequence of M. tuberculosis Kurono was determined with a length of 4,415,078 bp and a G+C content of 65.60%. The chromosome was shown to contain a total of 4,340 protein-coding genes, 53 tRNA genes, one transfer messenger RNA for all amino acids, and 1 rrn operon. Lineage analysis based on large sequence polymorphisms indicated that M. tuberculosis Kurono belongs to the Euro-American lineage (lineage 4). Phylogenetic analysis using whole genome sequences of M. tuberculosis Kurono in addition to 22 M. tuberculosis complex strains indicated that H37Rv is the closest relative of Kurono based on the results of phylogenetic analysis. These findings provide a basis for research using M. tuberculosis Kurono, especially in animal models. Copyright © 2014 Elsevier Ltd. All rights reserved.

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July 7, 2019

Genome sequence of Serratia nematodiphila DSM 21420T, a symbiotic bacterium from entomopathogenic nematode.

Serratia nematodiphila DSM 21420(T) (=CGMCC 1.6853(T), DZ0503SBS1(T)), isolated from the intestine of Heterorhabditidoides chongmingensis, has been known to have symbiotic-pathogenic life cycle, on the multilateral relationships with entomopathogenic nematode and insect pest. In order to better understanding of this rare feature in Serratia species, we present here the genome sequence of S. nematodiphila DSM 21420(T) with the significance of first genome sequence in this species. Copyright © 2014 Elsevier B.V. All rights reserved.

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July 7, 2019

Prevalence of subtilase cytotoxin-encoding subAB variants among Shiga toxin-producing Escherichia coli strains isolated from wild ruminants and sheep differs from that of cattle and pigs and is predominated by the new allelic variant subAB2-2.

Subtilase cytotoxin (SubAB) is an AB5 toxin produced by Shiga toxin (Stx)-producing Escherichia coli (STEC) strains usually lacking the eae gene product intimin. Three allelic variants of SubAB encoding genes have been described: subAB1, located on a plasmid, subAB2-1, located on the pathogenicity island SE-PAI and subAB2-2 located in an outer membrane efflux protein (OEP) region. SubAB is becoming increasingly recognized as a toxin potentially involved in human pathogenesis. Ruminants and cattle have been identified as reservoirs of subAB-positive STEC. The presence of the three subAB allelic variants was investigated by PCR for 152 STEC strains originating from chamois, ibex, red deer, roe deer, cattle, sheep and pigs. Overall, subAB genes were detected in 45.5% of the strains. Prevalence was highest for STEC originating from ibex (100%), chamois (92%) and sheep (65%). None of the STEC of bovine or of porcine origin tested positive for subAB. None of the strains tested positive for subAB1. The allelic variant subAB2-2 was detected the most commonly, with 51.4% possessing subAb2-1 together with subAB2-2. STEC of ovine origin, serotypes O91:H- and O128:H2, the saa gene, which encodes for the autoagglutinating adhesin and stx2b were significantly associated with subAB-positive STEC. Our results suggest that subAB2-1 and subAB2-2 is widespread among STEC from wild ruminants and sheep and may be important as virulence markers in STEC pathogenic to humans. Copyright © 2014 Elsevier GmbH. All rights reserved.

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July 7, 2019

Burkholderia pseudomallei sequencing identifies genomic clades with distinct recombination, accessory, and epigenetic profiles.

Burkholderia pseudomallei (Bp) is the causative agent of the infectious disease melioidosis. To investigate population diversity, recombination, and horizontal gene transfer in closely related Bp isolates, we performed whole-genome sequencing (WGS) on 106 clinical, animal, and environmental strains from a restricted Asian locale. Whole-genome phylogenies resolved multiple genomic clades of Bp, largely congruent with multilocus sequence typing (MLST). We discovered widespread recombination in the Bp core genome, involving hundreds of regions associated with multiple haplotypes. Highly recombinant regions exhibited functional enrichments that may contribute to virulence. We observed clade-specific patterns of recombination and accessory gene exchange, and provide evidence that this is likely due to ongoing recombination between clade members. Reciprocally, interclade exchanges were rarely observed, suggesting mechanisms restricting gene flow between clades. Interrogation of accessory elements revealed that each clade harbored a distinct complement of restriction-modification (RM) systems, predicted to cause clade-specific patterns of DNA methylation. Using methylome sequencing, we confirmed that representative strains from separate clades indeed exhibit distinct methylation profiles. Finally, using an E. coli system, we demonstrate that Bp RM systems can inhibit uptake of non-self DNA. Our data suggest that RM systems borne on mobile elements, besides preventing foreign DNA invasion, may also contribute to limiting exchanges of genetic material between individuals of the same species. Genomic clades may thus represent functional units of genetic isolation in Bp, modulating intraspecies genetic diversity. © 2015 Nandi et al.; Published by Cold Spring Harbor Laboratory Press.

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July 7, 2019

Accumulation-associated protein enhances Staphylococcus epidermidis biofilm formation under dynamic conditions and is required for infection in a rat catheter model.

Biofilm formation is the primary virulence factor of Staphylococcus epidermidis. S. epidermidis biofilms preferentially form on abiotic surfaces and may contain multiple matrix components, including proteins such as accumulation-associated protein (Aap). Following proteolytic cleavage of the A domain, which has been shown to enhance binding to host cells, B domain homotypic interactions support cell accumulation and biofilm formation. To further define the contribution of Aap to biofilm formation and infection, we constructed an aap allelic replacement mutant and an icaADBC aap double mutant. When subjected to fluid shear, strains deficient in Aap production produced significantly less biofilm than Aap-positive strains. To examine the in vivo relevance of our findings, we modified our previously described rat jugular catheter model and validated the importance of immunosuppression and the presence of a foreign body to the establishment of infection. The use of our allelic replacement mutants in the model revealed a significant decrease in bacterial recovery from the catheter and the blood in the absence of Aap, regardless of the production of polysaccharide intercellular adhesin (PIA), a well-characterized, robust matrix molecule. Complementation of the aap mutant with full-length Aap (containing the A domain), but not the B domain alone, increased initial attachment to microtiter plates, as did in trans expression of the A domain in adhesion-deficient Staphylococcus carnosus. These results demonstrate Aap contributes to S. epidermidis infection, which may in part be due to A domain-mediated attachment to abiotic surfaces. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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