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Auto Tag: Hierarchical Genome Assembly

September 22, 2019

A metabolic and genomic assessment of sugar fermentation profiles of the thermophilic Thermotogales, Fervidobacterium pennivorans.

A metabolic, genomic and proteomic assessment of Fervidobacterium pennivorans strains was undertaken to clarify the metabolic and genetic capabilities of this Thermotogales species. The type strain Ven5 originally isolated from a hot mud spa in Italy, and a newly isolated strain (DYC) from a hot spring at Ngatamariki, New Zealand, were compared for metabolic and genomic differences. The fermentation profiles of both strains on cellobiose generated similar major end products (acetate, alanine, glutamate, H2, and CO2). The vast majority of end products produced were redox neutral, and carbon balances were in the range of 95-115%. Each strain showed distinct fermentation profiles on sugar substrates. The genome of strain DYC was sequenced and shown to have high sequence similarity and synteny with F. pennivorans Ven5 genome, suggesting they are the same species. The unique genome regions in Ven5, corresponded to genes involved in the Entner-Doudoroff pathway confirming our observation of DYC’s inability to utilize gluconate. Genome analysis was able to elucidate pathways involved in production of the observed end-products with the exception of alanine and glutamate synthesis which were resolved with less clarity due to poor sequence identity and missing critical enzymes within the pathway, respectively.

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September 22, 2019

A complete Leishmania donovani reference genome identifies novel genetic variations associated with virulence.

Leishmania donovani is responsible for visceral leishmaniasis, a neglected and lethal parasitic disease with limited treatment options and no vaccine. The study of L. donovani has been hindered by the lack of a high-quality reference genome and this can impact experimental outcomes including the identification of virulence genes, drug targets and vaccine development. We therefore generated a complete genome assembly by deep sequencing using a combination of second generation (Illumina) and third generation (PacBio) sequencing technologies. Compared to the current L. donovani assembly, the genome assembly reported within resulted in the closure over 2,000 gaps, the extension of several chromosomes up to telomeric repeats and the re-annotation of close to 15% of protein coding genes and the annotation of hundreds of non-coding RNA genes. It was possible to correctly assemble the highly repetitive A2 and Amastin virulence gene clusters. A comparative sequence analysis using the improved reference genome confirmed 70 published and identified 15 novel genomic differences between closely related visceral and atypical cutaneous disease-causing L. donovani strains providing a more complete map of genes associated with virulence and visceral organ tropism. Bioinformatic tools including protein variation effect analyzer and basic local alignment search tool were used to prioritize a list of potential virulence genes based on mutation severity, gene conservation and function. This complete genome assembly and novel information on virulence factors will support the identification of new drug targets and the development of a vaccine for L. donovani.

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September 22, 2019

Complete genome sequencing of Comamonas kerstersii 8943, a causative agent for peritonitis.

Because of poor differentiation among the members of genus Comamonas using phenotypic methods, human infections caused by C. kerstersii are sporadically reported in the literature. Here, we represent the first complete genome sequence of C. kerstersii 8943, which caused peritonitis in a patient with continuous ambulatory peritoneal dialysis (CAPD). The complete genome with no gaps was obtained using third-generation Pacific Biosciences (PacBio) RSII sequencing system with single-molecule real-time (SMRT) analysis. Protein-coding genes, rRNAs and tRNAs were predicted. Functional annotations of the genome using different databases revealed several genes related to pathogenicity including antibiotic resistance genes and prophages. Our work demonstrates that whole genome sequencing can enhance the resolution of clinical investigations and our data can be used as a reference genome during the rapid diagnosis of C. kerstersii infections in the future.

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September 22, 2019

Characterization of Streptococcus pluranimalium from a cattle with mastitis by whole genome sequencing and functional validation.

Streptococcus pluranimalium is a new member of the Streptococcus genus isolated from multiple different animal hosts. It has been identified as a pathogen associated with subclinical mastitis, valvular endocarditis and septicaemia in animals. Moreover, this bacterium has emerged as a new pathogen for human infective endocarditis and brain abscess. However, the patho-biological properties of S. pluranimalium remain virtually unknown. The aim of this study was to determine the complete genome sequence of S. pluranimalium strain TH11417 isolated from a cattle with mastitis, and to characterize its antimicrobial resistance, virulence, and carbon catabolism.The genome of S. pluranimalium TH11417, determined by single-molecule real-time (SMRT) sequencing, consists of 2,065,522 base pair (bp) with a G?+?C content of 38.65%, 2,007 predicted coding sequence (CDS), 58 transfer RNA (tRNA) genes and five ribosome RNA (rRNA) operons. It contains a novel ISSpl1 element (a memeber of the IS3 family) and a ?11417.1 prophage that carries the mef(A), msr(D) and lnu(C) genes. Consistently, our antimicrobial susceptibility test confirmed that S. pluranimalium TH11417 was resistant to erythromycin and lincomycin. However, this strain did not show virulence in murine pneumonia (intranasal inoculation, 107 colony forming unit – CFU) and sepsis (intraperitoneal inoculation, 107 CFU) models. Additionally, this strain is able to grow with glucose, lactose or galactose as the sole carbon source, and possesses a lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS).We reported the first whole genome sequence of S. pluranimalium isolated from a cattle with mastitis. It harbors a prophage carrying the mef(A), msr(D) and lnu(C) genes, and is avirulent in the murine infection model.

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September 22, 2019

Phenotypic and genomic comparison of Photorhabdus luminescens subsp. laumondii TT01 and a widely used rifampicin-resistant Photorhabdus luminescens laboratory strain.

Photorhabdus luminescens is an enteric bacterium, which lives in mutualistic association with soil nematodes and is highly pathogenic for a broad spectrum of insects. A complete genome sequence for the type strain P. luminescens subsp. laumondii TT01, which was originally isolated in Trinidad and Tobago, has been described earlier. Subsequently, a rifampicin resistant P. luminescens strain has been generated with superior possibilities for experimental characterization. This strain, which is widely used in research, was described as a spontaneous rifampicin resistant mutant of TT01 and is known as TT01-RifR.Unexpectedly, upon phenotypic comparison between the rifampicin resistant strain and its presumed parent TT01, major differences were found with respect to bioluminescence, pigmentation, biofilm formation, haemolysis as well as growth. Therefore, we renamed the strain TT01-RifR to DJC. To unravel the genomic basis of the observed differences, we generated a complete genome sequence for strain DJC using the PacBio long read technology. As strain DJC was supposed to be a spontaneous mutant, only few sequence differences were expected. In order to distinguish these from potential sequencing errors in the published TT01 genome, we re-sequenced a derivative of strain TT01 in parallel, also using the PacBio technology. The two TT01 genomes differed at only 30 positions. In contrast, the genome of strain DJC varied extensively from TT01, showing 13,000 point mutations, 330 frameshifts, and 220 strain-specific regions with a total length of more than 300 kb in each of the compared genomes.According to the major phenotypic and genotypic differences, the rifampicin resistant P. luminescens strain, now named strain DJC, has to be considered as an independent isolate rather than a derivative of strain TT01. Strains TT01 and DJC both belong to P. luminescens subsp. laumondii.

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September 22, 2019

Complete Genome Sequence of Massilia oculi sp. nov. CCUG 43427T (=DSM 26321T), the Type Strain of M. oculi, and Comparison with Genome Sequences of Other Massilia Strains.

Massilia oculi sp. nov. of type strain CCUG 43427T is a Gram-negative, rod-shaped, nonspore-forming bacterium, which was recently isolated from the eye of a patient suffering from endophthalmitis and was described as novel species in Massilia genus. In this study, we present the complete genome sequence of this strain by using Pacbio SMRT cell platform and compare this sequence with the genomes of 30 Massilia representative strains. Also, a comprehensive search was conducted for genes and proteins involved in antibiotic resistance and pathogenicity. The genome of CCUG 43427T is 5,844,653 bp with 65.55% GC content. This genome contains four prophages and four genomic islands (GIs). The cobalt/zinc/cadmium transporter locus CzcABCD is included in these GIs. This GI was predicted to play important role in bacterial heavy-metal tolerance. The in silico genome analysis also revealed that this strain contains a lot of antibiotic resistance and pathogenicity related genes. This result suggested that this strain may has evolved a wide arsenal of weapons for pathogenicity and survival. Genome comparison among CCUG 43427T and other 30 Massilia strains revealed that more than 400 genes are unique in CCUG 43427T. Among these, one gene cluster, which was annotated to be important for LOS biosynthesis, catalytic mechanism and the substrate specificity of the enzyme, was predicted to be horizontally transferred by using phylogenies and biased GC content.

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September 22, 2019

Identification of DNA base modifications by means of Pacific Biosciences RS Sequencing technology.

Whole phage genomes can be sequenced readily using one or a combination of next generation sequencing (NGS) technologies. One of the most recently developed NGS platforms, the so-called Single-Molecule Real-Time (SMRT) sequencing approach provided by the PacBio RS platform, is particularly useful in providing complete (i.e., un-gapped) genome sequences, but differs from other technologies in that the platform also allows for downstream analysis to identify nucleotides that have been modified by DNA methylation. Here, we describe the methodological approach for the detection of genomic methylation motifs by means of SMRT sequencing.

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September 22, 2019

Construction of stable fluorescent laboratory control strains for several food safety relevant Enterobacteriaceae.

Using naturally-occurring bacterial strains as positive controls in testing protocols is typically feared due to the risk of cross-contaminating samples. We have developed a collection of strains which express Green Fluorescent Protein (GFP) at high-level, permitting rapid screening of the following species on selective or non-selective plates: Escherichia coli O157:H7, Shigella sonnei, S. flexneri, Salmonella enterica subsp. Enterica serovar Gaminera, S. Mbandaka, S. Tennesse, S. Minnesota, S. Senftenberg and S. Typhimurium. These new strains fluoresce when irradiated with UV light and maintain this phenotype in absence of antibiotic selection. Recombinants were phenotypically equivalent to the parent strain, except for S. Tennessee Sal66 that appeared Lac- on Xylose Lysine Deoxycholate (XLD) agar plates and Lac+ on Mac Conkey and Hektoen Enteric agar plates. Analysis of closed whole genome sequences revealed that Sal66 had lost one lactose operon; slower rates of lactose metabolism may affect lactose fermentation on XLD agar. These fluorescent enteric control strains were challenging to develop and should provide an easy and effective means of identifying cross-contamination. Published by Elsevier Ltd.

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September 22, 2019

Genomic and metatranscriptomic analyses of Weissella koreensis reveal its metabolic and fermentative features during kimchi fermentation

The genomic and metabolic features of Weissella koreensis, one of the major lactic acid bacteria in kimchi, were investigated through genomic, metabolic, and transcriptomic analyses for the genomes of strains KCTC 3621T, KACC 15510, and WiKim0080. W. koreensis strains were intrinsically vancomycin-resistant and harbored potential hemolysin genes that were actively transcribed although no hemolysin activity was detected. KEGG and reconstructed fermentative metabolic pathways displayed that W. koreensis strains commonly employ the heterolactic pathway to produce d-lactate, ethanol, acetate, CO2, d-sorbitol, thiamine, and folate from various carbohydrates including d-glucose, d-mannose, d-lactose, l-malate, d-xylose, l-arabinose, d-ribose, N-acetyl-glucosamine, and gluconate, and strains KCTC 3621T and WiKim0080 additionally have metabolic pathways of d-galacturonate and d-glucoronate. Phenotypic analyses showed that all strains did not ferment d-galactose, probably due to the lack of d-galactose transporting system, and strains KCTC 3621T and WiKim0080 fermented d-fructose, indicating the presence of d-fructose transporting system. Fermentative features of W. koreensis were investigated through kimchi transcriptional analysis, suggesting that W. koreensis is mainly responsible for kimchi fermentation with the production of various fermentative metabolites during late fermentation period. This was the first study to investigate the genomic and metabolic features of W. koreensis, which may provide better understandings on kimchi fermentation.

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September 22, 2019

Genome-scale analysis of Acetobacterium bakii reveals the cold adaptation of psychrotolerant acetogens by post-transcriptional regulation.

Acetogens synthesize acetyl-CoA via CO2 or CO fixation, producing organic compounds. Despite their ecological and industrial importance, their transcriptional and post-transcriptional regulation has not been systematically studied. With completion of the genome sequence of Acetobacterium bakii (4.28-Mb), we measured changes in the transcriptome of this psychrotolerant acetogen in response to temperature variations under autotrophic and heterotrophic growth conditions. Unexpectedly, acetogenesis genes were highly up-regulated at low temperatures under heterotrophic, as well as autotrophic, growth conditions. To mechanistically understand the transcriptional regulation of acetogenesis genes via changes in RNA secondary structures of 5′-untranslated regions (5′-UTR), the primary transcriptome was experimentally determined, and 1379 transcription start sites (TSS) and 1100 5′-UTR were found. Interestingly, acetogenesis genes contained longer 5′-UTR with lower RNA-folding free energy than other genes, revealing that the 5′-UTRs control the RNA abundance of the acetogenesis genes under low temperature conditions. Our findings suggest that post-transcriptional regulation via RNA conformational changes of 5′-UTRs is necessary for cold-adaptive acetogenesis.© 2018 Shin et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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September 22, 2019

Genomic characterization of carbapenemase-producing Klebsiella pneumoniae with chromosomally encoded blaNDM-1.

We report here Klebsiella pneumoniae strains carrying chromosomal blaNDM-1 in Thailand. The genomes of these two isolates include a 160-kbp insertion containing blaNDM-1, which is almost identical to that in the IncHI1B-like plasmid. Further analysis indicated that IS5-mediated intermolecular transposition and Tn3 transposase-mediated homologous recombination resulted in the integration of blaNDM-1 into the chromosome from an IncHI1B-like plasmid. The spread of this type of carbapenem-resistant Enterobacteriaceae may threaten public health and warrants further monitoring. Copyright © 2018 American Society for Microbiology.

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September 22, 2019

Paenibacillus seodonensis sp. nov., isolated from a plant of the genus Campanula.

Strain DCT-19T, representing a Gram-stain-positive, rodshaped, aerobic bacterium, was isolated from a native plant belonging to the genus Campanula on Dokdo, the Republic of Korea. Comparative analysis of the 16S rRNA gene sequence showed that this strain was closely related to Paenibacillus amylolyticus NRRL NRS-290T (98.6%, 16S rRNA gene sequence similarity), Paenibacillus tundrae A10bT (98.1%), and Paenibacillus xylanexedens NRRL B-51090T (97.6%). DNADNA hybridization indicated that this strain had relatively low levels of DNA-DNA relatedness with P. amylolyticus NRRL NRS-290T (30.0%), P. xylanexedens NRRL B-51090T (29.0%), and P. tundrae A10bT (24.5%). Additionally, the genomic DNA G + C content of DCT-19T was 44.8%. The isolated strain grew at pH 6.0-8.0 (optimum, pH 7.0), 0-4% (w/v) NaCl (optimum, 0%), and a temperature of 15-45°C (optimum 25-30°C). The sole respiratory quinone in the strain was menaquinone-7, and the predominant fatty acids were C15:0 anteiso, C16:0 iso, and C16:0. In addition, the major polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Based on its phenotypic properties, genotypic distinctiveness, and chemotaxonomic features, strain DCT-19T is proposed as a novel species in the genus Paenibacillus, for which the name Paenibacillus seodonensis sp. nov. is proposed (=KCTC 43009T =LMG 30888T). The type strain of Paenibacillus seodonensis is DCT-19T.

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September 22, 2019

Staying alive: growth and survival of Bifidobacterium animalis subsp. animalis under in vitro and in vivo conditions.

Members of the Bifidobacterium genus are widely used as probiotics in fermented milk products. Bifidobacterium animalis subsp. animalis CNCM I-4602 grows and survives poorly in reconstituted skimmed milk (RSM). Availing of genome and transcriptome information, this poor growth and survival phenotype in milk was substantially improved by the addition of certain compounds, such as yeast extract, uric acid, glutathione, cysteine, ferrous sulfate, and a combination of magnesium sulfate and manganese sulfate. Carbohydrate utilization of CNCM I-4602 was also investigated, allowing the identification of several carbohydrate utilization gene clusters, and highlighting this strain’s inability to utilize lactose, unlike the type strain of this subspecies, B. animalis subsp. animalis ATCC25527 and the B. animalis subsp. lactis subspecies. In addition, the ability of B. animalis subsp. animalis CNCM I-4602 to colonize a murine model was investigated, which showed that this strain persists in the murine gut for a period of at least 4 weeks. Associated in vivo transcriptome analysis revealed that, among other genes, a gene cluster encoding a predicted type IVb tight adherence (Tad) pilus was upregulated, indicating that this extracellular structure plays a role in the colonization/adaptation of the murine gastrointestinal tract by this strain.

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September 22, 2019

Genome wide characterization of enterotoxigenic Escherichia coli serogroup O6 isolates from multiple outbreaks and sporadic infections from 1975-2016.

Enterotoxigenic Escherichia coli (ETEC) are an important cause of diarrhea globally, particularly among children under the age of five in developing countries. ETEC O6 is the most common ETEC serogroup, yet the genome wide population structure of isolates of this serogroup is yet to be determined. In this study, we have characterized 40 ETEC O6 isolates collected between 1975-2016 by whole genome sequencing (WGS) and by phenotypic antimicrobial susceptibility testing. To determine the relatedness of isolates, we evaluated two methods-whole genome high-quality single nucleotide polymorphism (whole genome-hqSNP) and core genome SNP analyses using Lyve-SET and Parsnp respectively. All isolates were tested for antimicrobial susceptibility using a panel of 14 antibiotics. ResFinder 2.1 and a custom quinolone resistance determinants workflow were used for resistance determinant detection. VirulenceFinder 1.5 was used for prediction of the virulence genes. Thirty-seven isolates clustered into three major clades (I, II, III) by whole genome-hqSNP and core genome SNP analyses, while three isolates included in the whole genome-hqSNP analysis only did not cluster with clades I-III by both analyses and formed a distantly related outgroup, designated clade IV. Median number of pairwise whole genome-hqSNPs in clonal ETEC O6 outbreaks ranged from 0 to 5. Of the 40 isolates tested for antimicrobial susceptibility, 18 isolates were pansusceptible. Twenty-two isolates were resistant to at least one antibiotic, nine of which were multidrug resistant. Phenotypic antimicrobial resistance (AR) correlated with AR determinants in 22 isolates. Thirty-two isolates harbored both enterotoxin virulence genes while the remaining 8 isolates had only one of the two virulence genes. In summary, whole genome-hqSNP and core genome SNP analyses from this study revealed similar evolutionary relationships and an overall diversity of ETEC O6 isolates independent of time of isolation. Less than 5 pairwise hqSNPs between ETEC O6 isolates is circumstantially indicative of an outbreak cluster. Findings from this study will be a basis for quicker outbreak detection and control by efficient subtyping by WGS.

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September 22, 2019

Comparative genomics of 84 Pectobacterium genomes reveals the variations related to a pathogenic lifestyle.

Pectobacterium spp. are necrotrophic bacterial plant pathogens of the family Pectobacteriaceae, responsible for a wide spectrum of diseases of important crops and ornamental plants including soft rot, blackleg, and stem wilt. P. carotovorum is a genetically heterogeneous species consisting of three valid subspecies, P. carotovorum subsp. brasiliense (Pcb), P. carotovorum subsp. carotovorum (Pcc), and P. carotovorum subsp. odoriferum (Pco).Thirty-two P. carotovorum strains had their whole genomes sequenced, including the first complete genome of Pco and another circular genome of Pcb, as well as the high-coverage genome sequences for 30 additional strains covering Pcc, Pcb, and Pco. In combination with 52 other publicly available genome sequences, the comparative genomics study of P. carotovorum and other four closely related species P. polaris, P. parmentieri, P. atrosepticum, and Candidatus P. maceratum was conducted focusing on CRISPR-Cas defense systems and pathogenicity determinants. Our analysis identified two CRISPR-Cas types (I-F and I-E) in Pectobacterium, as well as another I-C type in Dickeya that is not found in Pectobacterium. The core pathogenicity factors (e.g., plant cell wall-degrading enzymes) were highly conserved, whereas some factors (e.g., flagellin, siderophores, polysaccharides, protein secretion systems, and regulatory factors) were varied among these species and/or subspecies. Notably, a novel type of T6SS as well as the sorbitol metabolizing srl operon was identified to be specific to Pco in Pectobacterium.This study not only advances the available knowledge about the genetic differentiation of individual subspecies of P. carotovorum, but also delineates the general genetic features of P. carotovorum by comparison with its four closely related species, thereby substantially enriching the extent of information now available for functional genomic investigations about Pectobacterium.

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