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July 7, 2019

Complete genome sequence of Salmonella enterica subsp. enterica serovar Minnesota strain

Mango has been implicated as food vehicle in several Salmonella-causing foodborne outbreaks. Here, Salmonella enterica subsp. enterica serovar Minnesota was isolated from fresh mango fruit imported from Mexico in 2014. The complete genome sequence of S. Minnesota CFSAN017963 was sequenced using single-molecule real-time DNA sequencing. Distinct prophage regions, Salmonella pathogenicity islands, and fimbrial gene clusters were observed in comparative genomic analysis on S. Minnesota CFSAN017963 with other phylogenetically closely related Salmonella serovars. Core genome multilocus sequencing typing analysis of all the S. Minnesota isolates in the Genbank and Enterobase also revealed a high genomic diversity among the genomes analyzed.


July 7, 2019

Integrated genomic and proteomic analyses of high-level chloramphenicol resistance in Campylobacter jejuni.

Campylobacter jejuni is a major zoonotic pathogen, and its resistance to antibiotics is of great concern for public health. However, few studies have investigated the global changes of the entire organism with respect to antibiotic resistance. Here, we provide mechanistic insights into high-level resistance to chloramphenicol in C. jejuni, using integrated genomic and proteomic analyses. We identified 27 single nucleotide polymorphisms (SNPs) as well as an efflux pump cmeB mutation that conferred modest resistance. We determined two radical S-adenosylmethionine (SAM) enzymes, one each from an SNP gene and a differentially expressed protein. Validation of major metabolic pathways demonstrated alterations in oxidative phosphorylation and ABC transporters, suggesting energy accumulation and increase in methionine import. Collectively, our data revealed a novel rRNA methylation mechanism by a radical SAM superfamily enzyme, indicating that two resistance mechanisms existed in Campylobacter. This work provided a systems biology perspective on understanding the antibiotic resistance mechanisms in bacteria.


July 7, 2019

HISEA: HIerarchical SEed Aligner for PacBio data.

The next generation sequencing (NGS) techniques have been around for over a decade. Many of their fundamental applications rely on the ability to compute good genome assemblies. As the technology evolves, the assembly algorithms and tools have to continuously adjust and improve. The currently dominant technology of Illumina produces reads that are too short to bridge many repeats, setting limits on what can be successfully assembled. The emerging SMRT (Single Molecule, Real-Time) sequencing technique from Pacific Biosciences produces uniform coverage and long reads of length up to sixty thousand base pairs, enabling significantly better genome assemblies. However, SMRT reads are much more expensive and have a much higher error rate than Illumina’s – around 10-15% – mostly due to indels. New algorithms are very much needed to take advantage of the long reads while mitigating the effect of high error rate and lowering the required coverage.An essential step in assembling SMRT data is the detection of alignments, or overlaps, between reads. High error rate and very long reads make this a much more challenging problem than for Illumina data. We present a new pairwise read aligner, or overlapper, HISEA (Hierarchical SEed Aligner) for SMRT sequencing data. HISEA uses a novel two-step k-mer search, employing consistent clustering, k-mer filtering, and read alignment extension.We compare HISEA against several state-of-the-art programs – BLASR, DALIGNER, GraphMap, MHAP, and Minimap – on real datasets from five organisms. We compare their sensitivity, precision, specificity, F1-score, as well as time and memory usage. We also introduce a new, more precise, evaluation method. Finally, we compare the two leading programs, MHAP and HISEA, for their genome assembly performance in the Canu pipeline.Our algorithm has the best alignment detection sensitivity among all programs for SMRT data, significantly higher than the current best. The currently best assembler for SMRT data is the Canu program which uses the MHAP aligner in its pipeline. We have incorporated our new HISEA aligner in the Canu pipeline and benchmarked it against the best pipeline for multiple datasets at two relevant coverage levels: 30x and 50x. Our assemblies are better than those using MHAP for both coverage levels. Moreover, Canu+HISEA assemblies for 30x coverage are comparable with Canu+MHAP assemblies for 50x coverage, while being faster and cheaper.The HISEA algorithm produces alignments with highest sensitivity compared with the current state-of-the-art algorithms. Integrated in the Canu pipeline, currently the best for assembling PacBio data, it produces better assemblies than Canu+MHAP.


July 7, 2019

Trajectories and drivers of genome evolution in surface-associated marine Phaeobacter.

The extent of genome divergence and the evolutionary events leading to speciation of marine bacteria have mostly been studied for (locally) abundant, free-living groups. The genus Phaeobacter is found on different marine surfaces, seems to occupy geographically disjunct habitats, and is involved in different biotic interactions, and was therefore targeted in the present study. The analysis of the chromosomes of 32 closely related but geographically spread Phaeobacter strains revealed an exceptionally large, highly syntenic core genome. The flexible gene pool is constantly but slightly expanding across all Phaeobacter lineages. The horizontally transferred genes mostly originated from bacteria of the Roseobacter group and horizontal transfer most likely was mediated by gene transfer agents. No evidence for geographic isolation and habitat specificity of the different phylogenomic Phaeobacter clades was detected based on the sources of isolation. In contrast, the functional gene repertoire and physiological traits of different phylogenomic Phaeobacter clades were sufficiently distinct to suggest an adaptation to an associated lifestyle with algae, to additional nutrient sources, or toxic heavy metals. Our study reveals that the evolutionary trajectories of surface-associated marine bacteria can differ significantly from free-living marine bacteria or marine generalists.© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.


July 7, 2019

The complete mitochondrial genome of Wonwhang (Pyrus pyrifolia)

This is a de novo assembly and annotation of a complete mitochondrial genome from Pyrus pyrifolia in the family Rosaceae. The complete mitochondrial genome of P. pyrifolia was assembled from PacBio RSII P6-C4 sequencing reads. The circular genome was 458,873?bp in length, containing 39 protein-coding genes, 23 tRNA genes and three rRNA genes. The nucleotide composition was A (27.5%), T (27.3%), G (22.6%) and C (22.6%) with GC content of 45.2%. Most of protein-coding genes use the canonical start codon ATG, whereas nad1, cox1, matR and rps4 use ACG, mttB uses ATT, rpl16 and rps19 uses GTG. The stop codon is also common in all mitochondrial genes. The phylogenetic analysis showed that P. pyrifolia was clustered with the Malus of Rosaceae family. Maximum-likelihood analysis suggests a clear relationship of Rosids and Asterids, which support the traditional classification.


July 7, 2019

First Azospirillum genome from aquatic environments: Whole-genome sequence of Azospirillum thiophilum BV-S(T), a novel diazotroph harboring a capacity of sulfur-chemolithotrophy from a sulfide spring.

Azospirillum thiophilum BV-S(T), isolated from a sulfide spring, is a novel nitrogen-fixing bacterium harboring sulfur-lithotrophy. In order to identify genetic characteristics with habitat- and metabolic features contrasting to those from terrestrial Azospirillum species, we present here the genome sequence of a novel species A. thiophilum BV-S(T), with a significance of first genome report in the aquatic Azospirillum species. The genome of strain BV-S(T) is comprised of 7.6Mb chromosome with a GC content of 68.2%. This information will contribute to expand understandings of sulfur-oxidizer microbes that preserve inherencies as a diazotroph, and further it will provide insights into genome plasticity of the genus Azospirillum for niche specific adaptations. Copyright © 2015 Elsevier B.V. All rights reserved.


July 7, 2019

OxyR-dependent formation of DNA methylation patterns in OpvABOFF and OpvABON cell lineages of Salmonella enterica.

Phase variation of the Salmonella enterica opvAB operon generates a bacterial lineage with standard lipopolysaccharide structure (OpvAB(OFF)) and a lineage with shorter O-antigen chains (OpvAB(ON)). Regulation of OpvAB lineage formation is transcriptional, and is controlled by the LysR-type factor OxyR and by DNA adenine methylation. The opvAB regulatory region contains four sites for OxyR binding (OBSA-D), and four methylatable GATC motifs (GATC1-4). OpvAB(OFF) and OpvAB(ON) cell lineages display opposite DNA methylation patterns in the opvAB regulatory region: (i) in the OpvAB(OFF) state, GATC1 and GATC3 are non-methylated, whereas GATC2 and GATC4 are methylated; (ii) in the OpvAB(ON) state, GATC2 and GATC4 are non-methylated, whereas GATC1 and GATC3 are methylated. We provide evidence that such DNA methylation patterns are generated by OxyR binding. The higher stability of the OpvAB(OFF) lineage may be caused by binding of OxyR to sites that are identical to the consensus (OBSA and OBSc), while the sites bound by OxyR in OpvAB(ON) cells (OBSB and OBSD) are not. In support of this view, amelioration of either OBSB or OBSD locks the system in the ON state. We also show that the GATC-binding protein SeqA and the nucleoid protein HU are ancillary factors in opvAB control.© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.


July 7, 2019

Analysis of hepatitis C NS5A resistance associated polymorphisms using ultra deep single molecule real time (SMRT) sequencing.

Development of Hepatitis C virus (HCV) resistance against direct-acting antivirals (DAAs), including NS5A inhibitors, is an obstacle to successful treatment of HCV when DAAs are used in sub-optimal combinations. Furthermore, it has been shown that baseline (pre-existing) resistance against DAAs is present in treatment naïve-patients and this will potentially complicate future treatment strategies in different HCV genotypes (GTs). Thus the aim was to detect low levels of NS5A resistant associated variants (RAVs) in a limited sample set of treatment-naïve patients of HCV GT1a and 3a, since such polymorphisms can display in vitro resistance as high as 60000 fold. Ultra-deep single molecule real time (SMRT) sequencing with the Pacific Biosciences (PacBio) RSII instrument was used to detect these RAVs. The SMRT sequencing was conducted on ten samples; three of them positive with Sanger sequencing (GT1a Q30H and Y93N, and GT3a Y93H), five GT1a samples, and two GT3a non-positive samples. The same methods were applied to the HCV GT1a H77-plasmid in a dilution series, in order to determine the error rates of replication, which in turn was used to determine the limit of detection (LOD), as defined by mean + 3SD, of minority variants down to 0.24%. We found important baseline NS5A RAVs at levels between 0.24 and 0.5%, which could potentially have clinical relevance. This new method with low level detection of baseline RAVs could be useful in predicting the most cost-efficient combination of DAA treatment, and reduce the treatment duration for an HCV infected individual. Copyright © 2015 Elsevier B.V. All rights reserved.


July 7, 2019

Oral phage therapy of acute bacterial diarrhea with two coliphage preparations: A randomized trial in children from Bangladesh

Background Antibiotic resistance is rising in important bacterial pathogens. Phage therapy (PT), the use of bacterial viruses infecting the pathogen in a species-specific way, is a potential alternative. Method T4-like coliphages or a commercial Russian coliphage product or placebo was orally given over 4 days to Bangladeshi children hospitalized with acute bacterial diarrhea. Safety of oral phage was assessed clinically and by functional tests; coliphage and Escherichia coli titers and enteropathogens were determined in stool and quantitative diarrhea parameters (stool output, stool frequency) were measured. Stool microbiota was studied by 16S rRNA gene sequencing; the genomes of four fecal Streptococcus isolates were sequenced. Findings No adverse events attributable to oral phage application were observed (primary safety outcome). Fecal coliphage was increased in treated over control children, but the titers did not show substantial intestinal phage replication (secondary microbiology outcome). 60% of the children suffered from a microbiologically proven E. coli diarrhea; the most frequent diagnosis was ETEC infections. Bacterial co-pathogens were also detected. Half of the patients contained phage-susceptible E. coli colonies in the stool. E. coli represented less than 5% of fecal bacteria. Stool ETEC titers showed only a short-lived peak and were otherwise close to the replication threshold determined for T4 phage in vitro. An interim analysis after the enrollment of 120 patients showed no amelioration in quantitative diarrhea parameter by PT over standard care (tertiary clinical outcome). Stool microbiota was characterized by an overgrowth with Streptococcus belonging to the Streptococcus gallolyticus and Streptococcus salivarius species groups, their abundance correlated with quantitative diarrhea outcome, but genome sequencing did not identify virulence genes. Interpretation Oral coliphages showed a safe gut transit in children, but failed to achieve intestinal amplification and to improve diarrhea outcome, possibly due to insufficient phage coverage and too low E. coli pathogen titers requiring higher oral phage doses. More knowledge is needed on in vivo phage–bacterium interaction and the role of E. coli in childhood diarrhea for successful PT. Funding The study was supported by a grant from Nestlé Nutrition and Nestlé Health Science. The trial was registered with Identifier NCT00937274 at ClinicalTrials.gov.


July 7, 2019

Comparative genomics of early-diverging mushroom-forming fungi provides insights into the origins of lignocellulose decay capabilities.

Evolution of lignocellulose decomposition was one of the most ecologically important innovations in fungi. White-rot fungi in the Agaricomycetes (mushrooms and relatives) are the most effective microorganisms in degrading both cellulose and lignin components of woody plant cell walls (PCW). However, the precise evolutionary origins of lignocellulose decomposition are poorly understood, largely because certain early-diverging clades of Agaricomycetes and its sister group, the Dacrymycetes, have yet to be sampled, or have been undersampled, in comparative genomic studies. Here, we present new genome sequences of ten saprotrophic fungi, including members of the Dacrymycetes and early-diverging clades of Agaricomycetes (Cantharellales, Sebacinales, Auriculariales, and Trechisporales), which we use to refine the origins and evolutionary history of the enzymatic toolkit of lignocellulose decomposition. We reconstructed the origin of ligninolytic enzymes, focusing on class II peroxidases (AA2), as well as enzymes that attack crystalline cellulose. Despite previous reports of white rot appearing as early as the Dacrymycetes, our results suggest that white-rot fungi evolved later in the Agaricomycetes, with the first class II peroxidases reconstructed in the ancestor of the Auriculariales and residual Agaricomycetes. The exemplars of the most ancient clades of Agaricomycetes that we sampled all lack class II peroxidases, and are thus concluded to use a combination of plesiomorphic and derived PCW degrading enzymes that predate the evolution of white rot.


July 7, 2019

Complete genome sequence of Serratia marcescens SmUNAM836, a nonpigmented multidrug-resistant strain isolated from a Mexican Patient with obstructive pulmonary disease.

Serratia marcescens SmUNAM836 is a multidrug-resistant clinical strain isolated in Mexico City from a patient with chronic obstructive pulmonary disease. Its complete genome sequence was determined using PacBio RS II SMRT technology, consisting of a 5.2-Mb chromosome and a 26.3-kb plasmid, encoding multiple resistance determinants and virulence factors. Copyright © 2016 Sandner-Miranda et al.


July 7, 2019

Complete genome sequences of two interactive moderate thermophiles, Paenibacillus napthalenovorans 32O-Y and Paenibacillus sp. 32O-W.

Microorganisms with the capability to desulfurize petroleum are in high demand with escalating restrictions currently placed on fuel purity. Thermophilic desulfurizers are particularly valuable in high-temperature industrial applications. We report the whole-genome sequences of Paenibacillus napthalenovorans 32O-Y and Paenibacillus sp. 32O-W, which can and cannot, respectively, metabolize dibenzothiophene. Copyright © 2016 Butler et al.


July 7, 2019

Complete closed genome sequences of Salmonella enterica subsp. enterica serotypes Anatum, Montevideo, Typhimurium, and Newport, isolated from beef, cattle, and humans.

Salmonella enterica spp. are a diverse group of bacteria with a wide range of virulence potential. To facilitate genome comparisons across this virulence spectrum, we present eight complete closed genome sequences of four S. enterica serotypes (Anatum, Montevideo, Typhimurium, and Newport), isolated from various cattle samples and from humans. Copyright © 2016 Harhay et al.


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