Next-generation sequencing has ushered in a new era of microbial genomics, enabling the detailed historical and geographical tracing of bacteria. This is helping to shape our understanding of bacterial evolution.
Fungi interact closely with bacteria, both on the surfaces of the hyphae and within their living tissues (i.e. endohyphal bacteria, EHB). These EHB can be obligate or facultative symbionts and can mediate diverse phenotypic traits in their hosts. Although EHB have been observed in many lineages of fungi, it remains unclear how widespread and general these associations are, and whether there are unifying ecological and genomic features can be found across EHB strains as a whole. We cultured 11 bacterial strains after they emerged from the hyphae of diverse Ascomycota that were isolated as foliar endophytes of cupressaceous trees, and generated nearly complete genome sequences for all. Unlike the genomes of largely obligate EHB, the genomes of these facultative EHB resembled those of closely related strains isolated from environmental sources. Although all analysed genomes encoded structures that could be used to interact with eukaryotic hosts, pathways previously implicated in maintenance and establishment of EHB symbiosis were not universally present across all strains. Independent isolation of two nearly identical pairs of strains from different classes of fungi, coupled with recent experimental evidence, suggests horizontal transfer of EHB across endophytic hosts. Given the potential for EHB to influence fungal phenotypes, these genomes could shed light on the mechanisms of plant growth promotion or stress mitigation by fungal endophytes during the symbiotic phase, as well as degradation of plant material during the saprotrophic phase. As such, these findings contribute to the illumination of a new dimension of functional biodiversity in fungi.
Few discoveries have been more transformative to the biological sciences than the development of DNA sequencing technologies. The rapid advancement of sequencing and bioinformatics tools has revolutionized bacterial genetics, deepening our understanding of model and clinically relevant organisms. Although application of newer sequencing technologies to studies in bacterial genetics is increasing, the implementation of DNA sequencing technologies and development of the bioinformatics tools required for analyzing the large data sets generated remains a challenge for many. In this minireview, we have chosen to summarize three sequencing approaches that are particularly useful for bacterial genetics. We provide resources for scientists new to and interested in their application. Herein, we discuss the analysis of Tn-seq data to determine gene disruptions differentially represented in a mutant population, Illumina sequencing for identification of suppressor or other mutations, and we summarize single-molecule real time (SMRT) sequencing for de novo genome assembly and the use of the output data for detection of DNA base modifications. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
Myroides odoratimimus (M. odoratimimus) has been gradually implicated as an important nosocomial pathogen that poses a serious health threat to immunocompromised patients owing to its multi-drug resistance. However, the resistance mechanism is currently unclear. To clarify the antibiotic resistance and infectivity mechanisms of M. odoratimimus, whole genome sequencing was performed on the multi-drug-resistant M. odoratimimus strain PR63039. The genome sequence was completed with single molecule real-time (SMRT) technologies. Then, annotation was performed using RAST and IMG-ER. A number of databases and software programs were used to analyze the genomic characteristics, including GC-Profile, ISfinder, CG viewer, ARDB, CARD, ResFinder, the VFDB database, PHAST and Progressive Mauve. The M. odoratimimus PR63039 genome consisted of a chromosome and a plasmid. The genome contained a large number of resistance genes and virulence factors. The distribution of the resistance genes was distinctive, and a resistance region named MY63039-RR was found. The subsystem features generated by RAST indicated that the annotated genome had 108 genes that were potentially involved in virulence, disease and defense, all of which had strong associations with resistance and pathogenicity. The prophage analysis showed two incomplete prophages in the genome. The genomic analysis of M. odoratimimus PR63039 partially clarified its antibiotic resistance mechanisms and virulence factors. Obtaining a clear understanding of its genomic characteristics will be conducive to the management of multidrug-resistant M. odoratimimus.
Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, we here report a gapless, near-finished genome sequence for B. cinerea strain B05.10. The assembly comprises 18 chromosomes and was confirmed by an optical map and a genetic map based on ~75 000 SNP markers. All chromosomes contain fully assembled centromeric regions, and 10 chromosomes have telomeres on both ends. The genetic map consisted of 4153 cM and comparison of genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that confer resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that UTRs of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress. This article is protected by copyright. All rights reserved. © 2016 BSPP AND JOHN WILEY & SONS LTD.
Plants have evolved hundreds of nucleotide-binding and leucine-rich domain proteins (NLRs) as potential intracellular immune receptors, but the evolutionary mechanism leading to the ability to recognize specific pathogen effectors is elusive. Here, we cloned Pvr4 (a Potyvirus resistance gene in Capsicum annuum) and Tsw (a Tomato spotted wilt virus resistance gene in Capsicum chinense) via a genome-based approach using independent segregating populations. The genes both encode typical NLRs and are located at the same locus on pepper chromosome 10. Despite the fact that these two genes recognize completely different viral effectors, the genomic structures and coding sequences of the two genes are strikingly similar. Phylogenetic studies revealed that these two immune receptors diverged from a progenitor gene of a common ancestor. Our results suggest that sequence variations caused by gene duplication and neofunctionalization may underlie the evolution of the ability to specifically recognize different effectors. These findings thereby provide insight into the divergent evolution of plant immune receptors.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.
From 2012 to 2014, there has been a huge increase in vancomycin-resistant (vanA) Enterococcus faecium (VREfm) in Copenhagen, Denmark, with 602 patients infected or colonized with VREfm in 2014 compared with just 22 in 2012. The objective of this study was to describe the genetic epidemiology of VREfm to assess the contribution of clonal spread and horizontal transfer of the vanA transposon (Tn1546) and plasmid in the dissemination of VREfm in hospitals.VREfm from Copenhagen, Denmark (2012-14) were whole-genome sequenced. The clonal structure was determined and the structure of Tn1546-like transposons was characterized. One VREfm isolate belonging to the largest clonal group was sequenced using long-read technology to close a 37 kb vanA plasmid.Phylogeny revealed a polyclonal structure where 495 VREfm isolates were divided into 13 main groups and 7 small groups. The majority of the isolates were located in three groups (n?=?44, 100 and 218) and clonal spread of VREfm between wards and hospitals was identified. Five Tn1546-like transposon types were identified. A dominant truncated transposon (type 4, 92%) was spread across all but one VREfm group. The closed vanA plasmid was highly covered by reads from isolates containing the type 4 transposon.This study suggests that it was the dissemination of the type 4 Tn1546-like transposon and plasmid via horizontal transfer to multiple populations of E. faecium, followed by clonal spread of new VREfm clones, that contributed to the increase in and diversity of VREfm in Danish hospitals.© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
The spike glycoprotein of the Middle East respiratory coronavirus (MERS-CoV) facilitates receptor binding and cell entry. During investigation of a multi-facility outbreak of MERS-CoV in Taif, Saudi Arabia, we identified a mixed population of wild-type and variant sequences with a large 530 nucleotide deletion in the spike gene from the serum of one patient. The out of frame deletion predicted loss of most of the S2 subunit of the spike protein leaving the S1 subunit with an intact receptor binding domain. This finding documents human infection with a novel genetic variant of MERS-CoV present as a quasispecies. J. Med. Virol. 89:542-545, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) of clonal complex (CC) 398 were first reported to cause severe infections in humans in 2005 [1]. Direct animal exposure is considered the most effective means of MRSA CC398 transmission from livestock to humans. However, about 20%–38% of MRSA CC398 cases among humans cannot be epidemiologically linked to direct livestock contact, indicating other transmission pathways [2]. As recently reported in this journal by Larsen et al [3], poultry meat may serve as a vehicle for livestock-to-human transmission. Here, we present similar findings for CC9/CC398 MRSA (displaying spa type t899 and related), which shares unique characteristics with human clinical isolates in Denmark as shown by Larsen et al [3], strongly supporting the implication of poultry, especially turkey meat, as the source of CC9/CC398.
Methylation has a profound role in the regulation of numerous biological processes in bacteria including virulence. The study of methylation in bacteria has greatly advanced thanks to next-generation sequencing technologies. These technologies have expedited the process of uncovering unique features of many bacterial methylomes such as characterizing previously uncharacterized methyltransferases, cataloging genome-wide DNA methylations in bacteria, identifying the frequency of methylation at particular genomic loci, and revealing regulatory roles of methylation in the biology of various bacterial species. For instance, methylation has been cited as a potential source for the pathogenicity differences observed in C. jejuni strains with syntenic genomes as seen in recent publications. Here, we describe the methodology for the use of Pacific Biosciences’ single molecule real-time (SMRT) sequencing for detecting methylation patterns in C. jejuni and bioinformatics tools to profile its methylome.
Malassezia species are opportunistic pathogenic fungi that are frequently associated with seborrhoeic dermatitis, including dandruff. Most Malassezia species are lipid dependent, a property that is compensated by breaking down host sebum into fatty acids by lipases. In this study, we aimed to sequence and analyse the whole genome of Malassezia restricta KCTC 27527, a clinical isolate from a Korean patient with severe dandruff, to search for lipase orthologues and identify the lipase that is the most frequently expressed on the scalp of patients with dandruff. The genome of M. restricta KCTC 27527 was sequenced using the Illumina MiSeq and PacBio platforms. Lipase orthologues were identified by comparison with known lipase genes in the genomes of Malassezia globosa and Malassezia sympodialis. The expression of the identified lipase genes was directly evaluated in swab samples from the scalps of 56 patients with dandruff. We found that, among the identified lipase-encoding genes, the gene encoding lipase homolog MRES_03670, named LIP5 in this study, was the most frequently expressed lipase in the swab samples. Our study provides an overview of the genome of a clinical isolate of M. restricta and fundamental information for elucidating the role of lipases during fungus-host interaction.© 2016 Blackwell Verlag GmbH.
From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80 kb region of recombination, also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specific conditions of hospital and healthcare environments.
During routine screening for Burkholderia pseudomallei from water wells in northern Australia in areas where it is endemic, Gram-negative bacteria (strains MSMB43(T), MSMB121, and MSMB122) with a similar morphology and biochemical pattern to B. pseudomallei and B. thailandensis were coisolated with B. pseudomallei on Ashdown’s selective agar. To determine the exact taxonomic position of these strains and to distinguish them from B. pseudomallei and B. thailandensis, they were subjected to a series of phenotypic and molecular analyses. Biochemical and fatty acid methyl ester analysis was unable to distinguish B. humptydooensis sp. nov. from closely related species. With matrix-assisted laser desorption ionization-time of flight analysis, all isolates grouped together in a cluster separate from other Burkholderia spp. 16S rRNA and recA sequence analyses demonstrated phylogenetic placement for B. humptydooensis sp. nov. in a novel clade within the B. pseudomallei group. Multilocus sequence typing (MLST) analysis of the three isolates in comparison with MLST data from 3,340 B. pseudomallei strains and related taxa revealed a new sequence type (ST318). Genome-to-genome distance calculations and the average nucleotide identity of all isolates to both B. thailandensis and B. pseudomallei, based on whole-genome sequences, also confirmed B. humptydooensis sp. nov. as a novel Burkholderia species within the B. pseudomallei complex. Molecular analyses clearly demonstrated that strains MSMB43(T), MSMB121, and MSMB122 belong to a novel Burkholderia species for which the name Burkholderia humptydooensis sp. nov. is proposed, with the type strain MSMB43(T) (American Type Culture Collection BAA-2767; Belgian Co-ordinated Collections of Microorganisms LMG 29471; DDBJ accession numbers CP013380 to CP013382).IMPORTANCEBurkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. The genus Burkholderia consists of a diverse group of species, with the closest relatives of B. pseudomallei referred to as the B. pseudomallei complex. A proposed novel species, B. humptydooensis sp. nov., was isolated from a bore water sample from the Northern Territory in Australia. B. humptydooensis sp. nov. is phylogenetically distinct from B. pseudomallei and other members of the B. pseudomallei complex, making it the fifth member of this important group of bacteria. Copyright © 2017 Tuanyok et al.
Lutibacter profundi LP1T within the family Flavobacteriaceae was isolated from a biofilm growing on the surface of a black smoker chimney at the Loki’s Castle vent field, located on the Arctic Mid-Ocean Ridge. The complete genome of L. profundi LP1T is the first genome to be published within the genus Lutibacter. L. profundi LP1T consists of a single 2,966,978 bp circular chromosome with a GC content of 29.8%. The genome comprises 2,537 protein-coding genes, 40 tRNA species and 2 rRNA operons. The microaerophilic, organotrophic isolate contains genes for all central carbohydrate metabolic pathways. However, genes for the oxidative branch of the pentose-phosphate-pathway, the glyoxylate shunt of the tricarboxylic acid cycle and the ATP citrate lyase for reverse TCA are not present. L. profundi LP1T utilizes starch, sucrose and diverse proteinous carbon sources. In accordance, the genome harbours 130 proteases and 104 carbohydrate-active enzymes, indicating a specialization in degrading organic matter. Among a small arsenal of 24 glycosyl hydrolases, which offer the possibility to hydrolyse diverse poly- and oligosaccharides, a starch utilization cluster was identified. Furthermore, a variety of enzymes may be secreted via T9SS and contribute to the hydrolytic variety of the microorganism. Genes for gliding motility are present, which may enable the bacteria to move within the biofilm. A substantial number of genes encoding for extracellular polysaccharide synthesis pathways, curli fibres and attachment to surfaces could mediate adhesion in the biofilm and may contribute to the biofilm formation. In addition to aerobic respiration, the complete denitrification pathway and genes for sulphide oxidation e.g. sulphide:quinone reductase are present in the genome. sulphide:quinone reductase and denitrification may serve as detoxification systems allowing L. profundi LP1T to thrive in a sulphide and nitrate enriched environment. The information gained from the genome gives a greater insight in the functional role of L. profundi LP1T in the biofilm and its adaption strategy in an extreme environment.
Cronobacter sakazakii is an emerging opportunistic pathogen that is associated with rare but life-threatening cases of severe diseases: meningitis, necrotizing enterocolitis, and sepsis in premature and full-term infants. However, the pathogenesis mechanism of this pathogen remains largely unknown. To determine its pathogenesis at the genomic level, the genome of C. sakazakii ATCC 29544(T) was completely sequenced and analyzed.The genomic DNA, containing a circular chromosome and three plasmids, is composed of 4,511,265 bp with a GC content of 56.71%, containing 4380 predicted open reading frames (ORFs), 22 rRNA genes, and 83 tRNA genes. The plasmids, designated pCSK29544_p1, pCSK29544_p2, and pCSK29544_p3, were 93,905-bp, 4938-bp, and 53,457-bp with GC contents of 57.02, 54.88, and 50.07%, respectively. They were also predicted to have 72, 6, and 57 ORFs without RNA genes.The strain ATCC 29544(T) genome has ompA and ibeB-homologous cusC genes, probably associated with the invasion of human brain microvascular endothelial cells (BMECs). In addition, gene clusters for siderophore production (iucABCD/iutA) and the related transport system (eitCBAD) were detected in pCSK29544_p1 plasmid, indicating better iron uptake ability for survival. Furthermore, to survive under extremely dry condition like milk powder, this genome has gene clusters for biosynthesis of capsular proteins (CSK29544_00281-00284) and cellulose (CSK29544_01124-01127) for biofilm formation and a gene cluster for utilization of sialic acid in the milk (nanKTAR). The genome information of C. sakazakii ATCC 29544(T) would provide further understanding of its pathogenesis at the molecular level for the regulation of pathogenicity and the development of a rapid detection method using biomarkers.
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