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July 19, 2019

Single haplotype assembly of the human genome from a hydatidiform mole.

A complete reference assembly is essential for accurately interpreting individual genomes and associating variation with phenotypes. While the current human reference genome sequence is of very high quality, gaps and misassemblies remain due to biological and technical complexities. Large repetitive sequences and complex allelic diversity are the two main drivers of assembly error. Although increasing the length of sequence reads and library fragments can improve assembly, even the longest available reads do not resolve all regions. In order to overcome the issue of allelic diversity, we used genomic DNA from an essentially haploid hydatidiform mole, CHM1. We utilized several resources from this DNA including a set of end-sequenced and indexed BAC clones and 100× Illumina whole-genome shotgun (WGS) sequence coverage. We used the WGS sequence and the GRCh37 reference assembly to create an assembly of the CHM1 genome. We subsequently incorporated 382 finished BAC clone sequences to generate a draft assembly, CHM1_1.1 (NCBI AssemblyDB GCA_000306695.2). Analysis of gene, repetitive element, and segmental duplication content show this assembly to be of excellent quality and contiguity. However, comparison to assembly-independent resources, such as BAC clone end sequences and PacBio long reads, indicate misassembled regions. Most of these regions are enriched for structural variation and segmental duplication, and can be resolved in the future. This publicly available assembly will be integrated into the Genome Reference Consortium curation framework for further improvement, with the ultimate goal being a completely finished gap-free assembly. © 2014 Steinberg et al.; Published by Cold Spring Harbor Laboratory Press.

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July 19, 2019

ModM DNA methyltransferase methylome analysis reveals a potential role for Moraxella catarrhalis phasevarions in otitis media.

Moraxella catarrhalis is a significant cause of otitis media and exacerbations of chronic obstructive pulmonary disease. Here, we characterize a phase-variable DNA methyltransferase (ModM), which contains 5′-CAAC-3′ repeats in its open reading frame that mediate high-frequency mutation resulting in reversible on/off switching of ModM expression. Three modM alleles have been identified (modM1-3), with modM2 being the most commonly found allele. Using single-molecule, real-time (SMRT) genome sequencing and methylome analysis, we have determined that the ModM2 methylation target is 5′-GAR(m6)AC-3′, and 100% of these sites are methylated in the genome of the M. catarrhalis 25239 ModM2 on strain. Proteomic analysis of ModM2 on and off variants revealed that ModM2 regulates expression of multiple genes that have potential roles in colonization, infection, and protection against host defenses. Investigation of the distribution of modM alleles in a panel of M. catarrhalis strains, isolated from the nasopharynx of healthy children or middle ear effusions from patients with otitis media, revealed a statistically significant association of modM3 with otitis media isolates. The modulation of gene expression via the ModM phase-variable regulon (phasevarion), and the significant association of the modM3 allele with otitis media, suggests a key role for ModM phasevarions in the pathogenesis of this organism.-Blakeway, L. V., Power, P. M., Jen, F. E.-C., Worboys, S. R., Boitano, M., Clark, T. A., Korlach, J., Bakaletz, L. O., Jennings, M. P., Peak, I. R., Seib, K. L. ModM DNA methyltransferase methylome analysis reveals a potential role for Moraxella catarrhalis phasevarions in otitis media. © FASEB.

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July 19, 2019

Analysis of the Campylobacter jejuni genome by SMRT DNA Sequencing identifies restriction-modification motifs.

Campylobacter jejuni is a leading bacterial cause of human gastroenteritis. The goal of this study was to analyze the C. jejuni F38011 strain, recovered from an individual with severe enteritis, at a genomic and proteomic level to gain insight into microbial processes. The C. jejuni F38011 genome is comprised of 1,691,939 bp, with a mol.% (G+C) content of 30.5%. PacBio sequencing coupled with REBASE analysis was used to predict C. jejuni F38011 genomic sites and enzymes that may be involved in DNA restriction-modification. A total of five putative methylation motifs were identified as well as the C. jejuni enzymes that could be responsible for the modifications. Peptides corresponding to the deduced amino acid sequence of the C. jejuni enzymes were identified using proteomics. This work sets the stage for studies to dissect the precise functions of the C. jejuni putative restriction-modification enzymes. Taken together, the data generated in this study contributes to our knowledge of the genomic content, methylation profile, and encoding capacity of C. jejuni.

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July 19, 2019

Genome sequencing and comparative genomics provides insights on the evolutionary dynamics and pathogenic potential of different H-serotypes of Shiga toxin-producing Escherichia coli O104.

Various H-serotypes of the Shiga toxin-producing Escherichia coli (STEC) O104, including H4, H7, H21, and H¯, have been associated with sporadic cases of illness and have caused food-borne outbreaks globally. In the U.S., STEC O104:H21 caused an outbreak associated with milk in 1994. However, there is little known on the evolutionary origins of STEC O104 strains, and how genotypic diversity contributes to pathogenic potential of various O104 H-antigen serotypes isolated from different ecological niches and/or geographical regions.Two STEC O104:H21 (milk outbreak strain) and O104:H7 (cattle isolate) strains were shot-gun sequenced, and the genomes were closed. The intimin (eae) gene, involved in the attaching-effacing phenotype of diarrheagenic E. coli, was not found in either strain. Examining various O104 genome sequences, we found that two “complete” left and right end portions of the locus of enterocyte effacement (LEE) pathogenicity island were present in 13 O104 strains; however, the central portion of LEE was missing, where the eae gene is located. In O104:H4 strains, the missing central portion of the LEE locus was replaced by a pathogenicity island carrying the aidA (adhesin involved in diffuse adherence) gene and antibiotic resistance genes commonly carried on plasmids. Enteroaggregative E. coli-specific virulence genes and European outbreak O104:H4-specific stx2-encoding Escherichia P13374 or Escherichia TL-2011c bacteriophages were missing in some of the O104:H4 genome sequences available from public databases. Most of the genomic variations in the strains examined were due to the presence of different mobile genetic elements, including prophages and genomic island regions. The presence of plasmids carrying virulence-associated genes may play a role in the pathogenic potential of O104 strains.The two strains sequenced in this study (O104:H21 and O104:H7) are genetically more similar to each other than to the O104:H4 strains that caused an outbreak in Germany in 2011 and strains found in Central Africa. A hypothesis on strain evolution and pathogenic potential of various H-serotypes of E. coli O104 strains is proposed.

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July 19, 2019

Complete genome sequence and analysis of Lactobacillus hokkaidonensis LOOC260(T), a psychrotrophic lactic acid bacterium isolated from silage.

Lactobacillus hokkaidonensis is an obligate heterofermentative lactic acid bacterium, which is isolated from Timothy grass silage in Hokkaido, a subarctic region of Japan. This bacterium is expected to be useful as a silage starter culture in cold regions because of its remarkable psychrotolerance; it can grow at temperatures as low as 4°C. To elucidate its genetic background, particularly in relation to the source of psychrotolerance, we constructed the complete genome sequence of L. hokkaidonensis LOOC260(T) using PacBio single-molecule real-time sequencing technology.The genome of LOOC260(T) comprises one circular chromosome (2.28 Mbp) and two circular plasmids: pLOOC260-1 (81.6 kbp) and pLOOC260-2 (41.0 kbp). We identified diverse mobile genetic elements, such as prophages, integrated and conjugative elements, and conjugative plasmids, which may reflect adaptation to plant-associated niches. Comparative genome analysis also detected unique genomic features, such as genes involved in pentose assimilation and NADPH generation.This is the first complete genome in the L. vaccinostercus group, which is poorly characterized, so the genomic information obtained in this study provides insight into the genetics and evolution of this group. We also found several factors that may contribute to the ability of L. hokkaidonensis to grow at cold temperatures. The results of this study will facilitate further investigation for the cold-tolerance mechanism of L. hokkaidonensis.

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July 19, 2019

Genome-wide DNA methylation analysis of Haloferax volcanii H26 and identification of DNA methyltransferase related PD-(D/E)XK nuclease family protein HVO_A0006.

Restriction-modification (RM) systems have evolved to protect the cell from invading DNAs and are composed of two enzymes: a DNA methyltransferase and a restriction endonuclease. Although RM systems are present in both archaeal and bacterial genomes, DNA methylation in archaea has not been well defined. In order to characterize the function of RM systems in archaeal species, we have made use of the model haloarchaeon Haloferax volcanii. A genomic DNA methylation analysis of H. volcanii strain H26 was performed using PacBio single molecule real-time (SMRT) sequencing. This analysis was also performed on a strain of H. volcanii in which an annotated DNA methyltransferase gene HVO_A0006 was deleted from the genome. Sequence analysis of H26 revealed two motifs which are modified in the genome: C(m4)TAG and GCA(m6)BN6VTGC. Analysis of the ?HVO_A0006 strain indicated that it exhibited reduced adenine methylation compared to the parental strain and altered the detected adenine motif. However, protein domain architecture analysis and amino acid alignments revealed that HVO_A0006 is homologous only to the N-terminal endonuclease region of Type IIG RM proteins and contains a PD-(D/E)XK nuclease motif, suggesting that HVO_A0006 is a PD-(D/E)XK nuclease family protein. Further bioinformatic analysis of the HVO_A0006 gene demonstrated that the gene is rare among the Halobacteria. It is surrounded by two transposition genes suggesting that HVO_A0006 is a fragment of a Type IIG RM gene, which has likely been acquired through gene transfer, and affects restriction-modification activity by interacting with another RM system component(s). Here, we present the first genome-wide characterization of DNA methylation in an archaeal species and examine the function of a DNA methyltransferase related gene HVO_A0006.

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July 19, 2019

Molecular analysis of asymptomatic bacteriuria Escherichia coli strain VR50 reveals adaptation to the urinary tract by gene acquisition.

Urinary tract infections (UTIs) are among the most common infectious diseases of humans, with Escherichia coli responsible for >80% of all cases. One extreme of UTI is asymptomatic bacteriuria (ABU), which occurs as an asymptomatic carrier state that resembles commensalism. To understand the evolution and molecular mechanisms that underpin ABU, the genome of the ABU E. coli strain VR50 was sequenced. Analysis of the complete genome indicated that it most resembles E. coli K-12, with the addition of a 94-kb genomic island (GI-VR50-pheV), eight prophages, and multiple plasmids. GI-VR50-pheV has a mosaic structure and contains genes encoding a number of UTI-associated virulence factors, namely, Afa (afimbrial adhesin), two autotransporter proteins (Ag43 and Sat), and aerobactin. We demonstrated that the presence of this island in VR50 confers its ability to colonize the murine bladder, as a VR50 mutant with GI-VR50-pheV deleted was attenuated in a mouse model of UTI in vivo. We established that Afa is the island-encoded factor responsible for this phenotype using two independent deletion (Afa operon and AfaE adhesin) mutants. E. coli VR50afa and VR50afaE displayed significantly decreased ability to adhere to human bladder epithelial cells. In the mouse model of UTI, VR50afa and VR50afaE displayed reduced bladder colonization compared to wild-type VR50, similar to the colonization level of the GI-VR50-pheV mutant. Our study suggests that E. coli VR50 is a commensal-like strain that has acquired fitness factors that facilitate colonization of the human bladder. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 19, 2019

Complete nucleotide sequences of bla(CTX-M)-harboring IncF plasmids from community-associated Escherichia coli strains in the United States.

Community-associated infections due to Escherichia coli producing CTX-M-type extended-spectrum ß-lactamases are increasingly recognized in the United States. The bla(CTX-M) genes are frequently carried on IncF group plasmids. In this study, bla(CTX-M-15)-harboring plasmids pCA14 (sequence type 131 [ST131]) and pCA28 (ST44) and bla(CTX-M-14)-harboring plasmid pCA08 (ST131) were sequenced and characterized. The three plasmids were closely related to other IncFII plasmids from continents outside the United States in the conserved backbone region and multiresistance regions (MRRs). Each of the bla(CTX-M-15)-carrying plasmids pCA14 and pCA28 belonged to F31:A4:B1 (FAB [FII, FIA, FIB] formula) and showed a high level of similarity (92% coverage of pCA14 and 99% to 100% nucleotide identity), suggesting a possible common origin. The blaC(TX-M-14)-carrying plasmid pCA08 belonged to F2:A2:B20 and was highly similar to pKF3-140 from China (88% coverage of pCA08 and 99% to 100% nucleotide identity). All three plasmids carried multiple antimicrobial resistance genes and modules associated with virulence and biochemical pathways, which likely confer selective advantages for their host strains. The bla(CTX-M)-carrying IncFII-IA-IB plasmids implicated in community-associated infections in the United States shared key structural features with those identified from other continents, underscoring the global nature of this plasmid epidemic. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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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.

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July 19, 2019

The complete methylome of Helicobacter pylori UM032.

The genome of the human gastric pathogen Helicobacter pylori encodes a large number of DNA methyltransferases (MTases), some of which are shared among many strains, and others of which are unique to a given strain. The MTases have potential roles in the survival of the bacterium. In this study, we sequenced a Malaysian H. pylori clinical strain, designated UM032, by using a combination of PacBio Single Molecule, Real-Time (SMRT) and Illumina MiSeq next generation sequencing platforms, and used the SMRT data to characterize the set of methylated bases (the methylome).The N4-methylcytosine and N6-methyladenine modifications detected at single-base resolution using SMRT technology revealed 17 methylated sequence motifs corresponding to one Type I and 16 Type II restriction-modification (R-M) systems. Previously unassigned methylation motifs were now assigned to their respective MTases-coding genes. Furthermore, one gene that appears to be inactive in the H. pylori UM032 genome during normal growth was characterized by cloning.Consistent with previously-studied H. pylori strains, we show that strain UM032 contains a relatively large number of R-M systems, including some MTase activities with novel specificities. Additional studies are underway to further elucidating the biological significance of the R-M systems in the physiology and pathogenesis of H. pylori.

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July 19, 2019

Chaos of rearrangements in the mating-type chromosomes of the anther-smut fungus Microbotryum lychnidis-dioicae.

Sex chromosomes in plants and animals and fungal mating-type chromosomes often show exceptional genome features, with extensive suppression of homologous recombination and cytological differentiation between members of the diploid chromosome pair. Despite strong interest in the genetics of these chromosomes, their large regions of suppressed recombination often are enriched in transposable elements and therefore can be challenging to assemble. Here we show that the latest improvements of the PacBio sequencing yield assembly of the whole genome of the anther-smut fungus, Microbotryum lychnidis-dioicae (the pathogenic fungus causing anther-smut disease of Silene latifolia), into finished chromosomes or chromosome arms, even for the repeat-rich mating-type chromosomes and centromeres. Suppressed recombination of the mating-type chromosomes is revealed to span nearly 90% of their lengths, with extreme levels of rearrangements, transposable element accumulation, and differentiation between the two mating types. We observed no correlation between allelic divergence and physical position in the nonrecombining regions of the mating-type chromosomes. This may result from gene conversion or from rearrangements of ancient evolutionary strata, i.e., successive steps of suppressed recombination. Centromeres were found to be composed mainly of copia-like transposable elements and to possess specific minisatellite repeats identical between the different chromosomes. We also identified subtelomeric motifs. In addition, extensive signs of degeneration were detected in the nonrecombining regions in the form of transposable element accumulation and of hundreds of gene losses on each mating-type chromosome. Furthermore, our study highlights the potential of the latest breakthrough PacBio chemistry to resolve complex genome architectures. Copyright © 2015 by the Genetics Society of America.

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July 19, 2019

Insertion sequence IS26 reorganizes plasmids in clinically isolated multidrug-resistant bacteria by replicative transposition.

Carbapenemase-producing Enterobacteriaceae (CPE), which are resistant to most or all known antibiotics, constitute a global threat to public health. Transposable elements are often associated with antibiotic resistance determinants, suggesting a role in the emergence of resistance. One insertion sequence, IS26, is frequently associated with resistance determinants, but its role remains unclear. We have analyzed the genomic contexts of 70 IS26 copies in several clinical and surveillance CPE isolates from the National Institutes of Health Clinical Center. We used target site duplications and their patterns as guides and found that a large fraction of plasmid reorganizations result from IS26 replicative transpositions, including replicon fusions, DNA inversions, and deletions. Replicative transposition could also be inferred for transposon Tn4401, which harbors the carbapenemase blaKPC gene. Thus, replicative transposition is important in the ongoing reorganization of plasmids carrying multidrug-resistant determinants, an observation that carries substantial clinical and epidemiological implications for understanding how such extreme drug resistance phenotypes evolve.Although IS26 is frequently reported to reside in resistance plasmids of clinical isolates, the characteristic hallmark of transposition, target site duplication (TSD), is generally not observed, raising questions about the mode of transposition for IS26. The previous observation of cointegrate formation during transposition implies that IS26 transposes via a replicative mechanism. The other possible outcome of replicative transposition is DNA inversion or deletion, when transposition occurs intramolecularly, and this would also generate a specific TSD pattern that might also serve as supporting evidence for the transposition mechanism. The numerous examples we present here demonstrate that replicative transposition, used by many mobile elements (including IS26 and Tn4401), is prevalent in the plasmids of clinical isolates and results in significant plasmid reorganization. This study also provides a method to trace the evolution of resistance plasmids based on TSD patterns. Copyright © 2015 He et al.

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July 19, 2019

Complete genome sequence of Sporisorium scitamineum and biotrophic interaction transcriptome with sugarcane.

Sporisorium scitamineum is a biotrophic fungus responsible for the sugarcane smut, a worldwide spread disease. This study provides the complete sequence of individual chromosomes of S. scitamineum from telomere to telomere achieved by a combination of PacBio long reads and Illumina short reads sequence data, as well as a draft sequence of a second fungal strain. Comparative analysis to previous available sequences of another strain detected few polymorphisms among the three genomes. The novel complete sequence described herein allowed us to identify and annotate extended subtelomeric regions, repetitive elements and the mitochondrial DNA sequence. The genome comprises 19,979,571 bases, 6,677 genes encoding proteins, 111 tRNAs and 3 assembled copies of rDNA, out of our estimated number of copies as 130. Chromosomal reorganizations were detected when comparing to sequences of S. reilianum, the closest smut relative, potentially influenced by repeats of transposable elements. Repetitive elements may have also directed the linkage of the two mating-type loci. The fungal transcriptome profiling from in vitro and from interaction with sugarcane at two time points (early infection and whip emergence) revealed that 13.5% of the genes were differentially expressed in planta and particular to each developmental stage. Among them are plant cell wall degrading enzymes, proteases, lipases, chitin modification and lignin degradation enzymes, sugar transporters and transcriptional factors. The fungus also modulates transcription of genes related to surviving against reactive oxygen species and other toxic metabolites produced by the plant. Previously described effectors in smut/plant interactions were detected but some new candidates are proposed. Ten genomic islands harboring some of the candidate genes unique to S. scitamineum were expressed only in planta. RNAseq data was also used to reassure gene predictions.

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July 19, 2019

TAL effectors and activation of predicted host targets distinguish Asian from African strains of the rice pathogen Xanthomonas oryzae pv. oryzicola while strict conservation suggests universal importance of five TAL effectors.

Xanthomonas oryzae pv. oryzicola (Xoc) causes the increasingly important disease bacterial leaf streak of rice (BLS) in part by type III delivery of repeat-rich transcription activator-like (TAL) effectors to upregulate host susceptibility genes. By pathogen whole genome, single molecule, real-time sequencing and host RNA sequencing, we compared TAL effector content and rice transcriptional responses across 10 geographically diverse Xoc strains. TAL effector content is surprisingly conserved overall, yet distinguishes Asian from African isolates. Five TAL effectors are conserved across all strains. In a prior laboratory assay in rice cv. Nipponbare, only two contributed to virulence in strain BLS256 but the strict conservation indicates all five may be important, in different rice genotypes or in the field. Concatenated and aligned, TAL effector content across strains largely reflects relationships based on housekeeping genes, suggesting predominantly vertical transmission. Rice transcriptional responses did not reflect these relationships, and on average, only 28% of genes upregulated and 22% of genes downregulated by a strain are up- and down- regulated (respectively) by all strains. However, when only known TAL effector targets were considered, the relationships resembled those of the TAL effectors. Toward identifying new targets, we used the TAL effector-DNA recognition code to predict effector binding elements in promoters of genes upregulated by each strain, but found that for every strain, all upregulated genes had at least one. Filtering with a classifier we developed previously decreases the number of predicted binding elements across the genome, suggesting that it may reduce false positives among upregulated genes. Applying this filter and eliminating genes for which upregulation did not strictly correlate with presence of the corresponding TAL effector, we generated testable numbers of candidate targets for four of the five strictly conserved TAL effectors.

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July 19, 2019

Biosynthesis of the novel macrolide antibiotic anthracimycin.

We report the identification of the biosynthetic gene cluster for the unusual antibiotic anthracimycin (atc) from the marine derived producer strain Streptomyces sp. T676 isolated off St. John’s Island, Singapore. The 53?253 bps atc locus includes a trans-acyltransferase (trans-AT) polyketide synthase (PKS), and heterologous expression in Streptomyces coelicolor resulted in anthracimycin production. Analysis of the atc cluster revealed that anthracimycin is likely generated by four PKS gene products AtcC-AtcF without involvement of post-PKS tailoring enzymes, and a biosynthetic pathway is proposed. The availability of the atc cluster provides a basis for investigating the biosynthesis of anthracimycin and its subsequent bioengineering to provide novel analogues with improved pharmacological properties.

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