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July 7, 2019

Twenty years of bacterial genome sequencing.

Twenty years ago, the publication of the first bacterial genome sequence, from Haemophilus influenzae, shook the world of bacteriology. In this Timeline, we review the first two decades of bacterial genome sequencing, which have been marked by three revolutions: whole-genome shotgun sequencing, high-throughput sequencing and single-molecule long-read sequencing. We summarize the social history of sequencing and its impact on our understanding of the biology, diversity and evolution of bacteria, while also highlighting spin-offs and translational impact in the clinic. We look forward to a ‘sequencing singularity’, where sequencing becomes the method of choice for as-yet unthinkable applications in bacteriology and beyond.

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July 7, 2019

Molecular epidemiology of multidrug-resistant Acinetobacter baumannii isolates in a university hospital in Nepal reveals the emergence of a novel epidemic clonal lineage.

The emergence of multidrug-resistant (MDR) Acinetobacter baumannii has become a serious medical problem worldwide. To clarify the genetic and epidemiological properties of MDR A. baumannii strains isolated from a medical setting in Nepal, 246 Acinetobacter spp. isolates obtained from different patients were screened for MDR A. baumannii by antimicrobial disk susceptibility testing. Whole genomes of the MDR A. baumannii isolates were sequenced by MiSeq™ (Illumina), and the complete genome of one isolate (IOMTU433) was sequenced by PacBio RS II. Phylogenetic trees were constructed from single nucleotide polymorphism concatemers. Multilocus sequence types were deduced and drug resistance genes were identified. Of the 246 Acinetobacter spp. isolates, 122 (49.6%) were MDR A. baumannii, with the majority being resistant to aminoglycosides, carbapenems and fluoroquinolones but not to colistin and tigecycline. These isolates harboured the 16S rRNA methylase gene armA as well as bla(NDM-1), bla(OXA-23) or bla(OXA-58). MDR A. baumannii isolates belonging to clonal complex 1 (CC1) and CC2 as well as a novel clonal complex (CC149) have spread throughout a medical setting in Nepal. The MDR isolates harboured genes encoding carbapenemases (OXA and NDM-1) and a 16S rRNA methylase (ArmA). Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

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July 7, 2019

Impact of the omic technologies for understanding the modes of action of biological control agents against plant pathogens

The characterization of microbial biological control agents (MBCAs) is crucial to improve their efficacy and consistency as biopesticides. Powerful approaches to characterize MBCA’s modes of action are provided by modern molecular technologies. This paper reviews improvements achieved in this subject by three “omics” approaches: namely the genomic, the transcriptomic and the proteomic approaches. The paper discusses the advantages and drawbacks of new molecular techniques and ‘discovery driven’ approaches to the study of the biocontrol properties against plant pathogens. Omics technologies are capable of: (i) identifying the genome, transcriptome or proteome features of an MBCA strain, (ii) comparing properties of strains/mutants with different biocontrol efficacy, (iii) identifying and characterizing genes, mRNAs and proteins involved in MBCA modes of action, and (iv) simultaneously studying the transcriptome or proteome of the plant host, the plant pathogen and the MBCAs in relation to their bi- or tri-trophic interactions

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July 7, 2019

Complete genome sequence of Bacillus cereus FORC_005, a food-borne pathogen from the soy sauce braised fish-cake with quail-egg.

Due to abundant contamination in various foods, the pathogenesis of Bacillus cereus has been widely studied in physiological and molecular level. B. cereus FORC_005 was isolated from a Korean side dish, soy sauce braised fish-cake with quail-egg in South Korea. While 21 complete genome sequences of B. cereus has been announced to date, this strain was completely sequenced, analyzed, and compared with other complete genome sequences of B. cereus to elucidate the distinct pathogenic features of a strain isolated in South Korea. The genomic DNA containing a circular chromosome consists of 5,349,617-bp with a GC content of 35.29 %. It was predicted to have 5170 open reading frames, 106 tRNA genes, and 42 rRNA genes. Among the predicted ORFs, 3892 ORFs were annotated to encode functional proteins (75.28 %) and 1278 ORFs were predicted to encode hypothetical proteins (748 conserved and 530 non-conserved hypothetical proteins). This genome information of B. cereus FORC_005 would extend our understanding of its pathogenesis in genomic level for efficient control of its contamination in foods and further food poisoning.

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July 7, 2019

Clonal dissemination of Enterobacter cloacae harboring blaKPC-3 in the upper midwestern United States.

Carbapenemase-producing, carbapenem-resistant Enterobacteriaceae, or CP-CRE, are an emerging threat to human and animal health, because they are resistant to many of the last-line antimicrobials available for disease treatment. Carbapenemase-producing Enterobacter cloacae harboring blaKPC-3 recently was reported in the upper midwestern United States and implicated in a hospital outbreak in Fargo, North Dakota (L. M. Kiedrowski, D. M. Guerrero, F. Perez, R. A. Viau, L. J. Rojas, M. F. Mojica, S. D. Rudin, A. M. Hujer, S. H. Marshall, and R. A. Bonomo, Emerg Infect Dis 20:1583-1585, 2014, http://dx.doi.org/10.3201/eid2009.140344). In early 2009, the Minnesota Department of Health began collecting and screening CP-CRE from patients throughout Minnesota. Here, we analyzed a retrospective group of CP-E. cloacae isolates (n = 34) collected between 2009 and 2013. Whole-genome sequencing and analysis revealed that 32 of the strains were clonal, belonging to the ST171 clonal complex and differing collectively by 211 single-nucleotide polymorphisms, and it revealed a dynamic clone under positive selection. The phylogeography of these strains suggests that this clone existed in eastern North Dakota and western Minnesota prior to 2009 and subsequently was identified in the Minneapolis and St. Paul metropolitan area. All strains harbored identical IncFIA-like plasmids conferring a CP-CRE phenotype and an additional IncX3 plasmid. In a single patient with multiple isolates submitted over several months, we found evidence that these plasmids had transferred from the E. cloacae clone to an Escherichia coli ST131 bacterium, rendering it as a CP-CRE. The spread of this clone throughout the upper midwestern United States is unprecedented for E. cloacae and highlights the importance of continued surveillance to identify such threats to human health. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

The complete genome sequence of the emerging pathogen Mycobacterium haemophilum explains its unique culture requirements.

Mycobacterium haemophilum is an emerging pathogen associated with a variety of clinical syndromes, most commonly skin infections in immunocompromised individuals. M. haemophilum exhibits a unique requirement for iron supplementation to support its growth in culture, but the basis for this property and how it may shape pathogenesis is unclear. Using a combination of Illumina, PacBio, and Sanger sequencing, the complete genome sequence of M. haemophilum was determined. Guided by this sequence, experiments were performed to define the basis for the unique growth requirements of M. haemophilum. We found that M. haemophilum, unlike many other mycobacteria, is unable to synthesize iron-binding siderophores known as mycobactins or to utilize ferri-mycobactins to support growth. These differences correlate with the absence of genes associated with mycobactin synthesis, secretion, and uptake. In agreement with the ability of heme to promote growth, we identified genes encoding heme uptake machinery. Consistent with its propensity to infect the skin, we show at the whole-genome level the genetic closeness of M. haemophilum with Mycobacterium leprae, an organism which cannot be cultivated in vitro, and we identify genes uniquely shared by these organisms. Finally, we identify means to express foreign genes in M. haemophilum. These data explain the unique culture requirements for this important pathogen, provide a foundation upon which the genome sequence can be exploited to improve diagnostics and therapeutics, and suggest use of M. haemophilum as a tool to elucidate functions of genes shared with M. leprae.Mycobacterium haemophilum is an emerging pathogen with an unknown natural reservoir that exhibits unique requirements for iron supplementation to grow in vitro. Understanding the basis for this iron requirement is important because it is fundamental to isolation of the organism from clinical samples and environmental sources. Defining the molecular basis for M. haemophilium’s growth requirements will also shed new light on mycobacterial strategies to acquire iron and can be exploited to define how differences in such strategies influence pathogenesis. Here, through a combination of sequencing and experimental approaches, we explain the basis for the iron requirement. We further demonstrate the genetic closeness of M. haemophilum and Mycobacterium leprae, the causative agent of leprosy which cannot be cultured in vitro, and we demonstrate methods to genetically manipulate M. haemophilum. These findings pave the way for the use of M. haemophilum as a model to elucidate functions of genes shared with M. leprae. Copyright © 2015 Tufariello et al.

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July 7, 2019

Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade.

Horizontal gene transfer (HGT), or the transfer of genes between species, has been recognized recently as more pervasive than previously suspected. Here, we report evidence for an unprecedented degree of HGT into an animal genome, based on a draft genome of a tardigrade, Hypsibius dujardini. Tardigrades are microscopic eight-legged animals that are famous for their ability to survive extreme conditions. Genome sequencing, direct confirmation of physical linkage, and phylogenetic analysis revealed that a large fraction of the H. dujardini genome is derived from diverse bacteria as well as plants, fungi, and Archaea. We estimate that approximately one-sixth of tardigrade genes entered by HGT, nearly double the fraction found in the most extreme cases of HGT into animals known to date. Foreign genes have supplemented, expanded, and even replaced some metazoan gene families within the tardigrade genome. Our results demonstrate that an unexpectedly large fraction of an animal genome can be derived from foreign sources. We speculate that animals that can survive extremes may be particularly prone to acquiring foreign genes.

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July 7, 2019

Comparative genomics and metabolic profiling of the genus Lysobacter.

Lysobacter species are Gram-negative bacteria widely distributed in soil, plant and freshwater habitats. Lysobacter owes its name to the lytic effects on other microorganisms. To better understand their ecology and interactions with other (micro)organisms, five Lysobacter strains representing the four species L. enzymogenes, L. capsici, L. gummosus and L. antibioticus were subjected to genomics and metabolomics analyses.Comparative genomics revealed a diverse genome content among the Lysobacter species with a core genome of 2,891 and a pangenome of 10,028 coding sequences. Genes encoding type I, II, III, IV, V secretion systems and type IV pili were highly conserved in all five genomes, whereas type VI secretion systems were only found in L. enzymogenes and L. gummosus. Genes encoding components of the flagellar apparatus were absent in the two sequenced L. antibioticus strains. The genomes contained a large number of genes encoding extracellular enzymes including chitinases, glucanases and peptidases. Various nonribosomal peptide synthase (NRPS) and polyketide synthase (PKS) gene clusters encoding putative bioactive metabolites were identified but only few of these clusters were shared between the different species. Metabolic profiling by imaging mass spectrometry complemented, in part, the in silico genome analyses and allowed visualisation of the spatial distribution patterns of several secondary metabolites produced by or induced in Lysobacter species during interactions with the soil-borne fungus Rhizoctonia solani.Our work shows that mining the genomes of Lysobacter species in combination with metabolic profiling provides novel insights into the genomic and metabolic potential of this widely distributed but understudied and versatile bacterial genus.

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July 7, 2019

Complete genome sequence of Novosphingobium pentaromativorans US6-1(T).

Novosphingobium pentaromativorans US6-1(T) is a species in the family Sphingomonadaceae. According to the phylogenetic analysis based on 16S rRNA gene sequence of the N. pentaromativorans US6-1(T) and nine genome-sequenced strains in the genus Novosphingobium, the similarity ranged from 93.9 to 99.9 % and the highest similarity was found with Novosphingobium sp. PP1Y (99.9 %), whereas the ANI value based on genomes ranged from 70.9 to 93 % and the highest value was 93 %. This microorganism was isolated from muddy coastal bay sediments where the environment is heavily polluted by polycyclic aromatic hydrocarbons (PAHs). It was previously shown to be capable of degrading multiple PAHs, including benzo[a]pyrene. To further understand the PAH biodegradation pathways the previous draft genome of this microorganism was revised to obtain a complete genome using Illumina MiSeq and PacBio platform. The genome of strain US6-1(T) consists of 5,457,578 bp, which includes the 3,979,506 bp chromosome and five megaplasmids. It comprises 5110 protein-coding genes and 82 RNA genes. Here, we provide an analysis of the complete genome sequence which enables the identification of new characteristics of this strain.

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July 7, 2019

Genome sequence and description of the anaerobic lignin-degrading bacterium Tolumonas lignolytica sp. nov.

Tolumonas lignolytica BRL6-1(T) sp. nov. is the type strain of T. lignolytica sp. nov., a proposed novel species of the Tolumonas genus. This strain was isolated from tropical rainforest soils based on its ability to utilize lignin as a sole carbon source. Cells of Tolumonas lignolytica BRL6-1(T) are mesophilic, non-spore forming, Gram-negative rods that are oxidase and catalase negative. The genome for this isolate was sequenced and returned in seven unique contigs totaling 3.6Mbp, enabling the characterization of several putative pathways for lignin breakdown. Particularly, we found an extracellular peroxidase involved in lignin depolymerization, as well as several enzymes involved in ß-aryl ether bond cleavage, which is the most abundant linkage between lignin monomers. We also found genes for enzymes involved in ferulic acid metabolism, which is a common product of lignin breakdown. By characterizing pathways and enzymes employed in the bacterial breakdown of lignin in anaerobic environments, this work should assist in the efficient engineering of biofuel production from lignocellulosic material.

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July 7, 2019

Complete genome sequence of Thioalkalivibrio paradoxus type strain ARh 1(T), an obligately chemolithoautotrophic haloalkaliphilic sulfur-oxidizing bacterium isolated from a Kenyan soda lake.

Thioalkalivibrio paradoxus strain ARh 1(T) is a chemolithoautotrophic, non-motile, Gram-negative bacterium belonging to the Gammaproteobacteria that was isolated from samples of haloalkaline soda lakes. It derives energy from the oxidation of reduced sulfur compounds and is notable for its ability to grow on thiocyanate as its sole source of electrons, sulfur and nitrogen. The full genome consists of 3,756,729 bp and comprises 3,500 protein-coding and 57 RNA-coding genes. This organism was sequenced as part of the community science program at the DOE Joint Genome Institute.

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July 7, 2019

The genomic sequence of lymphocryptovirus from cynomolgus macaque.

Lymphocryptoviruses such as Epstein-Barr virus (EBV) cause persistent infections in human and non-human primates, and suppression of the immune system can increase the risk of lymphocryptovirus (LCV)-associated tumor development in both human and non-human primates. To enable LCV infection as a non-clinical model to study effects of therapeutics on EBV immunity, we determined the genomic DNA sequence of the LCV from cynomolgus macaque, a species commonly used for non-clinical testing. Comparison to rhesus macaque LCV and human EBV sequences indicates that LCV from the cynomolgus macaque has the same genomic arrangement and a high degree of similarity in most genes, especially with rhesus macaque LCV. Genes showing lower similarity were those encoding proteins involved in latency and/or tumor promotion or immune evasion. The genomic sequence of LCV from cynomolgus macaque should aid the development of non-clinical tools for identifying therapeutics that impact LCV immunity and carry potential lymphoma risk. Copyright © 2015 Elsevier Inc. All rights reserved.

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July 7, 2019

Genome analysis of Staphylococcus agnetis, an agent of lameness in broiler chickens.

Lameness in broiler chickens is a significant animal welfare and financial issue. Lameness can be enhanced by rearing young broilers on wire flooring. We have identified Staphylococcus agnetis as significantly involved in bacterial chondronecrosis with osteomyelitis (BCO) in proximal tibia and femorae, leading to lameness in broiler chickens in the wire floor system. Administration of S. agnetis in water induces lameness. Previously reported in some cases of cattle mastitis, this is the first report of this poorly described pathogen in chickens. We used long and short read next generation sequencing to assemble single finished contigs for the genome and a large plasmid from the chicken pathogen. Comparison of the S. agnetis genome to those of other pathogenic Staphylococci shows that S.agnetis contains a distinct repertoire of virulence determinants. Additionally, the S. agnetis genome has several regions that differ substantially from the genomes of other pathogenic Staphylococci. Comparison of our finished genome to a recent draft genome for a cattle mastitis isolate suggests that future investigations focus on the evolutionary epidemiology of this emerging pathogen of domestic animals.

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July 7, 2019

De novo assembly of Dekkera bruxellensis: a multi technology approach using short and long-read sequencing and optical mapping.

It remains a challenge to perform de novo assembly using next-generation sequencing (NGS). Despite the availability of multiple sequencing technologies and tools (e.g., assemblers) it is still difficult to assemble new genomes at chromosome resolution (i.e., one sequence per chromosome). Obtaining high quality draft assemblies is extremely important in the case of yeast genomes to better characterise major events in their evolutionary history. The aim of this work is two-fold: on the one hand we want to show how combining different and somewhat complementary technologies is key to improving assembly quality and correctness, and on the other hand we present a de novo assembly pipeline we believe to be beneficial to core facility bioinformaticians. To demonstrate both the effectiveness of combining technologies and the simplicity of the pipeline, here we present the results obtained using the Dekkera bruxellensis genome.In this work we used short-read Illumina data and long-read PacBio data combined with the extreme long-range information from OpGen optical maps in the task of de novo genome assembly and finishing. Moreover, we developed NouGAT, a semi-automated pipeline for read-preprocessing, de novo assembly and assembly evaluation, which was instrumental for this work.We obtained a high quality draft assembly of a yeast genome, resolved on a chromosomal level. Furthermore, this assembly was corrected for mis-assembly errors as demonstrated by resolving a large collapsed repeat and by receiving higher scores by assembly evaluation tools. With the inclusion of PacBio data we were able to fill about 5 % of the optical mapped genome not covered by the Illumina data.

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