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Auto Tag: Bacterial genome

April 21, 2020  |  

Whole genome sequencing of a novel, dichloromethane-fermenting Peptococcaceae from an enrichment culture

Bacteria capable of dechlorinating the toxic environmental contaminant dichloromethane (DCM, CHt2Cl2) are of great interest for potential bioremediation applications. A novel, strictly anaerobic, DCM-fermenting bacterium, “DCMF”, was enriched from organochlorine-contaminated groundwater near Botany Bay, Australia. The enrichment culture was maintained in minimal, mineral salt medium amended with dichloromethane as the sole energy source. PacBio whole genome SMRTtextsuperscriptTM sequencing of DCMF allowed textitde novo, gap-free assembly despite the presence of cohabiting organisms in the culture. Illumina sequencing reads were utilised to correct minor indels. The single, circularised 6.44 Mb chromosome was annotated with the IMG pipeline and contains 5,773 predicted protein-coding genes. Based on 16S rRNA gene and predicted proteome phylogeny, the organism appears to be a novel member of the textitPeptococcaceae family. The DCMF genome is large in comparison to known DCM-fermenting bacteria and includes 96 predicted methylamine methyltransferases, which may provide clues to the basis of its DCM metabolism. Full annotation has been provided in a custom genome browser and search tool, in addition to multiple sequence alignments and phylogenetic trees for every predicted protein, available at http://www.slimsuite.unsw.edu.au/research/dcmf/.

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April 21, 2020  |  

Long-read amplicon denoising.

Long-read next-generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies from PacBio reads. Called ‘amplicon denoising’, this problem has been extensively studied for short-read sequencing technologies, but current solutions do not always successfully generalize to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower. On two Mock Virus Community datasets with ground truth, each sequenced on a different PacBio instrument, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method, successfully discriminating templates that differ by a just single nucleotide. Julia implementations of Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD), and a webserver interface, are freely available. © The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.

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April 21, 2020  |  

Genome-Wide Screening for Enteric Colonization Factors in Carbapenem-Resistant ST258 Klebsiella pneumoniae.

A diverse, antibiotic-naive microbiota prevents highly antibiotic-resistant microbes, including carbapenem-resistant Klebsiella pneumoniae (CR-Kp), from achieving dense colonization of the intestinal lumen. Antibiotic-mediated destruction of the microbiota leads to expansion of CR-Kp in the gut, markedly increasing the risk of bacteremia in vulnerable patients. While preventing dense colonization represents a rational approach to reduce intra- and interpatient dissemination of CR-Kp, little is known about pathogen-associated factors that enable dense growth and persistence in the intestinal lumen. To identify genetic factors essential for dense colonization of the gut by CR-Kp, we constructed a highly saturated transposon mutant library with >150,000 unique mutations in an ST258 strain of CR-Kp and screened for in vitro growth and in vivo intestinal colonization in antibiotic-treated mice. Stochastic and partially reversible fluctuations in the representation of different mutations during dense colonization revealed the dynamic nature of intestinal microbial populations. We identified genes that are crucial for early and late stages of dense gut colonization and confirmed their role by testing isogenic mutants in in vivo competition assays with wild-type CR-Kp Screening of the transposon library also identified mutations that enhanced in vivo CR-Kp growth. These newly identified colonization factors may provide novel therapeutic opportunities to reduce intestinal colonization by CR-KpIMPORTANCEKlebsiella pneumoniae is a common cause of bloodstream infections in immunocompromised and hospitalized patients, and over the last 2 decades, some strains have acquired resistance to nearly all available antibiotics, including broad-spectrum carbapenems. The U.S. Centers for Disease Control and Prevention has listed carbapenem-resistant K. pneumoniae (CR-Kp) as an urgent public health threat. Dense colonization of the intestine by CR-Kp and other antibiotic-resistant bacteria is associated with an increased risk of bacteremia. Reducing the density of gut colonization by CR-Kp is likely to reduce their transmission from patient to patient in health care facilities as well as systemic infections. How CR-Kp expands and persists in the gut lumen, however, is poorly understood. Herein, we generated a highly saturated mutant library in a multidrug-resistant K. pneumoniae strain and identified genetic factors that are associated with dense gut colonization by K. pneumoniae This study sheds light on host colonization by K. pneumoniae and identifies potential colonization factors that contribute to high-density persistence of K. pneumoniae in the intestine. Copyright © 2019 Jung et al.

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April 21, 2020  |  

Trophic specialization results in genomic reduction in free-living marine idiomarina bacteria.

The streamlining hypothesis is generally used to explain the genomic reduction events related to the small genome size of free-living bacteria like marine bacteria SAR11. However, our current understanding of the correlation between bacterial genome size and environmental adaptation relies on too few species. It is still unclear whether there are other paths leading to genomic reduction in free-living bacteria. The genome size of marine free-living bacteria of the genus Idiomarina belonging to the order Alteromonadales (Gammaproteobacteria) is much smaller than the size of related genomes from bacteria in the same order. Comparative genomic and physiological analyses showed that the genomic reduction pattern in this genus is different from that of the classical SAR11 lineage. Genomic reduction reconstruction and substrate utilization profile showed that Idiomarina spp. lost a large number of genes related to carbohydrate utilization, and instead they specialized on using proteinaceous resources. Here we propose a new hypothesis to explain genomic reduction in this genus; we propose that trophic specialization increasing the metabolic efficiency for using one kind of substrate but reducing the substrate utilization spectrum could result in bacterial genomic reduction, which would be not uncommon in nature. This hypothesis was further tested in another free-living genus, Kangiella, which also shows dramatic genomic reduction. These findings highlight that trophic specialization is potentially an important path leading to genomic reduction in some marine free-living bacteria, which is distinct from the classical lineages like SAR11.IMPORTANCE The streamlining hypothesis is usually used to explain the genomic reduction events in free-living bacteria like SAR11. However, we find that the genomic reduction phenomenon in the bacterial genus Idiomarina is different from that in SAR11. Therefore, we propose a new hypothesis to explain genomic reduction in this genus based on trophic specialization that could result in genomic reduction, which would be not uncommon in nature. Not only can the trophic specialization hypothesis explain the genomic reduction in the genus Idiomarina, but it also sheds new light on our understanding of the genomic reduction processes in other free-living bacterial lineages. Copyright © 2019 Qin et al.

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April 21, 2020  |  

Decreased biofilm formation ability of Acinetobacter baumannii after spaceflight on China’s Shenzhou 11 spacecraft.

China has prepared for construction of a space station by the early 2020s. The mission will require astronauts to stay on the space station for at least 180 days. Microbes isolated from the International Space Station (ISS) have shown profound resistance to clinical antibiotics and environmental stresses. Previous studies have demonstrated that the space environment could affect microbial survival, growth, virulence, biofilms, metabolism, as well as their antibiotic-resistant phenotypes. Furthermore, several studies have reported that astronauts experience a decline in their immunity during long-duration spaceflights. Monitoring microbiomes in the ISS or the spacecraft will be beneficial for the prevention of infection among the astronauts during spaceflight. The development of a manned space program worldwide not only provides an opportunity to investigate the impact of this extreme environment on opportunistic pathogenic microbes, but also offers a unique platform to detect mutations in pathogenic bacteria. Various microorganisms have been carried on a spacecraft for academic purposes. Acinetobacter baumannii is a common multidrug-resistant bacterium often prevalent in hospitals. Variations in the ability to cope with environmental hazards increase the chances of microbial survival. Our study aimed to compare phenotypic variations and analyze genomic and transcriptomic variations in A. baumannii among three different groups: SS1 (33 days on the Shenzhou 11 spacecraft), GS1 (ground control), and Aba (reference strain). Consequently, the biofilm formation ability of the SS1 strain decreased after 33 days of spaceflight. Furthermore, high-throughput sequencing revealed that some differentially expressed genes were downregulated in the SS1 strain compared with those in the GS1 strain. In conclusion, this present study provides insights into the environmental adaptation of A. baumannii and might be useful for understanding changes in the opportunistic pathogenic microbes on our spacecraft and on China’s future ISS. © 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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April 21, 2020  |  

Reconstruction of the genomes of drug-resistant pathogens for outbreak investigation through metagenomic sequencing

Culture-independent methods that target genome fragments have shown promise in identifying certain pathogens, but the holy grail of comprehensive pathogen genome detection from microbiologically complex samples for subsequent forensic analyses remains a challenge. In the context of an investigation of a nosocomial outbreak, we used shotgun metagenomic sequencing of a human fecal sample and a neural network algorithm based on tetranucleotide frequency profiling to reconstruct microbial genomes and tested the same approach using rectal swabs from a second patient. The approach rapidly and readily detected the genome of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae in the patient fecal specimen and in the rectal swab sample, achieving a level of strain resolution that was sufficient for confident transmission inference during a highly clonal outbreak. The analysis also detected previously unrecognized colonization of the patient by vancomycin-resistant Enterococcus faecium, another multidrug-resistant bacterium.IMPORTANCE The study results reported here perfectly demonstrate the power and promise of clinical metagenomics to recover genome sequences of important drug-resistant bacteria and to rapidly provide rich data that inform outbreak investigations and treatment decisions, independently of the need to culture the organisms.

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April 21, 2020  |  

Complete Genome Sequence of “Candidatus Thioglobus sp.” Strain NP1, an Open-Ocean Isolate from the SUP05 Clade of Marine Gammaproteobacteria

Candidatus Thioglobus sp.textquotedblright strain NP1 is an open-ocean isolate from the SUP05 clade of Gammaproteobacteria. Whole-genome comparisons of strain NP1 to other sequenced isolates from the SUP05 clade indicate that it represents a new species of SUP05 that lacks the ability to fix inorganic carbon using the Calvin-Benson-Bassham cycle.

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April 21, 2020  |  

Genome mining identifies cepacin as a plant-protective metabolite of the biopesticidal bacterium Burkholderia ambifaria.

Beneficial microorganisms are widely used in agriculture for control of plant pathogens, but a lack of efficacy and safety information has limited the exploitation of multiple promising biopesticides. We applied phylogeny-led genome mining, metabolite analyses and biological control assays to define the efficacy of Burkholderia ambifaria, a naturally beneficial bacterium with proven biocontrol properties but potential pathogenic risk. A panel of 64 B.?ambifaria strains demonstrated significant antimicrobial activity against priority plant pathogens. Genome sequencing, specialized metabolite biosynthetic gene cluster mining and metabolite analysis revealed an armoury of known and unknown pathways within B.?ambifaria. The biosynthetic gene cluster responsible for the production of the metabolite cepacin was identified and directly shown to mediate protection of germinating crops against Pythium damping-off disease. B.?ambifaria maintained biopesticidal protection and overall fitness in the soil after deletion of its third replicon, a non-essential plasmid associated with virulence in Burkholderia?cepacia complex bacteria. Removal of the third replicon reduced B.?ambifaria persistence in a murine respiratory infection model. Here, we show that by using interdisciplinary phylogenomic, metabolomic and functional approaches, the mode of action of natural biological control agents related to pathogens can be systematically established to facilitate their future exploitation.

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April 21, 2020  |  

Genome Analyses of a New Mycoplasma Species from the Scorpion Centruroides vittatus.

Arthropod Mycoplasma are little known endosymbionts in insects, primarily known as plant disease vectors. Mycoplasma in other arthropods such as arachnids are unknown. We report the first complete Mycoplasma genome sequenced, identified, and annotated from a scorpion, Centruroides vittatus, and designate it as Mycoplasma vittatus We find the genome is at least a 683,827 bp single circular chromosome with a GC content of 42.7% and with 987 protein-coding genes. The putative virulence determinants include 11 genes associated with the virulence operon associated with protein synthesis or DNA transcription and ten genes with antibiotic and toxic compound resistance. Comparative analysis revealed that the M. vittatus genome is smaller than other Mycoplasma genomes and exhibits a higher GC content. Phylogenetic analysis shows M. vittatus as part of the Hominis group of Mycoplasma As arthropod genomes accumulate, further novel Mycoplasma genomes may be identified and characterized. Copyright © 2019 Yamashita et al.

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April 21, 2020  |  

Comparative Genomics Approaches to Understanding Virulence and Antimicrobial Resistance of Salmonella Typhimurium ST1539 Isolated from a Poultry Slaughterhouse in Korea.

Non-typhoidal Salmonella (NTS) is one of the most frequent causes of bacterial foodborne illnesses. Considering that the main reservoir of NTS is the intestinal tract of livestock, foods of animal origin are regarded as the main vehicles of Salmonella infection. In particular, poultry colonized with Salmonella Typhimurium (S. Typhimurium), a dominant serotype responsible for human infections, do not exhibit overt signs and symptoms, thereby posing a potential health risk to humans. In this study, comparative genomics approaches were applied to two S. Typhimurium strains, ST1539 and ST1120, isolated from a duck slaughterhouse and a pig farm, respectively, to characterize their virulence and antimicrobial resistance-associated genomic determinants. ST1539 containing a chromosome (4,905,039 bp; 4,403 CDSs) and a plasmid (93,876 bp; 96 CDSs) was phylogenetically distinct from other S. Typhimurium strains such as ST1120 and LT2. Compared to the ST1120 genome (previously deposited in GenBank; CP021909.1 and CP021910.1), ST1539 possesses more virulence determinants, including ST64B prophage, plasmid spv operon encoding virulence factors, genes encoding SseJ effector, Rck invasin, and biofilm-forming factors (bcf operon and pefAB). In accordance with the in silico prediction, ST1539 exhibited higher cytotoxicity against epithelial cells, better survival inside macrophage cells, and faster mice-killing activity than ST1120. However, ST1539 showed less resistance against antibiotics than ST1120, which may be attributed to the multiple resistanceassociated genes in the ST1120 chromosome. The accumulation of comparative genomics data on S. Typhimurium isolates from livestock would enrich our understanding of strategies Salmonella employs to adapt to diverse host animals.

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April 21, 2020  |  

Human contamination in bacterial genomes has created thousands of spurious proteins.

Contaminant sequences that appear in published genomes can cause numerous problems for downstream analyses, particularly for evolutionary studies and metagenomics projects. Our large-scale scan of complete and draft bacterial and archaeal genomes in the NCBI RefSeq database reveals that 2250 genomes are contaminated by human sequence. The contaminant sequences derive primarily from high-copy human repeat regions, which themselves are not adequately represented in the current human reference genome, GRCh38. The absence of the sequences from the human assembly offers a likely explanation for their presence in bacterial assemblies. In some cases, the contaminating contigs have been erroneously annotated as containing protein-coding sequences, which over time have propagated to create spurious protein “families” across multiple prokaryotic and eukaryotic genomes. As a result, 3437 spurious protein entries are currently present in the widely used nr and TrEMBL protein databases. We report here an extensive list of contaminant sequences in bacterial genome assemblies and the proteins associated with them. We found that nearly all contaminants occurred in small contigs in draft genomes, which suggests that filtering out small contigs from draft genome assemblies may mitigate the issue of contamination while still keeping nearly all of the genuine genomic sequences. © 2019 Breitwieser et al.; Published by Cold Spring Harbor Laboratory Press.

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April 21, 2020  |  

Complete Genome Sequence of Actinosynnema pretiosum X47, An Industrial Strain that Produces the Antibiotic Ansamitocin AP-3.

Ansamitocins are extraordinarily potent antitumor agents. Ansamitocin P-3 (AP-3), which is produced by Actinosynnema pretiosum, has been developed as a cytotoxic drug for breast cancer. Despite its importance, AP-3 is of limited applicability because of the low production yield. A. pretiosum strain X47 was developed from A. pretiosum ATCC 31565 by mutation breeding and shows a relatively high AP-3 yield. Here, we analyzed the A. pretiosum X47 genome, which is ~8.13 Mb in length with 6693 coding sequences, 58 tRNA genes, and 15 rRNA genes. The DNA sequence of the ansamitocin biosynthetic gene cluster is highly similar to that of the corresponding cluster in A. pretiosum ATCC 31565, with 99.9% identity. However, RT-qPCR analysis showed that the expression levels of ansamitocin biosynthetic genes were significantly increased in X47 compared with the levels in the wild-type strain, consistent with the higher yield of AP-3 in X47. The annotated complete genome sequence of this strain will facilitate understanding the molecular mechanisms of ansamitocin biosynthesis and regulation in A. pretiosum and help further genetic engineering studies to enhance the production of AP-3.

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