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July 7, 2019  |  

Neisseria lactamica Y92-1009 complete genome sequence.

We present the high quality, complete genome assembly of Neisseria lactamica Y92-1009 used to manufacture an outer membrane vesicle (OMV)-based vaccine, and a member of the Neisseria genus. The strain is available on request from the Public Health England Meningococcal Reference Unit. This Gram negative, dipplococcoid bacterium is an organism of worldwide clinical interest because human nasopharyngeal carriage is related inversely to the incidence of meningococcal disease, caused by Neisseria meningitidis. The organism sequenced was isolated during a school carriage survey in Northern Ireland in 1992 and has been the subject of a variety of laboratory and clinical studies. Four SMRT cells on a RSII machine by Pacific Biosystems were used to produce a complete, closed genome assembly. Sequence data were obtained for a total of 30,180,391 bases from 2621 reads and assembled using the HGAP algorithm. The assembly was corrected using short reads obtained from an Illumina HiSeq 2000instrument. This resulted in a 2,146,723 bp assembly with approximately 460 fold mean coverage depth and a GC ratio of 52.3%.

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July 7, 2019  |  

Genome and plasmid sequences of Escherichia coli KV7, an extended-spectrum ß-lactamase isolate derived from feces of a healthy pig.

We present single-contig assemblies for Escherichia coli strain KV7 (serotype O27, phylogenetic group D) and its six plasmids, isolated from a healthy pig, as determined by PacBio RS II and Illumina MiSeq sequencing. The chromosome of 4,997,475 bp and G+C content of 50.75% harbored 4,540 protein-encoding genes. Copyright © 2017 Bateman et al.

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July 7, 2019  |  

Genetic plasticity of the Shigella virulence plasmid is mediated by intra- and inter-molecular events between insertion sequences.

Acquisition of a single copy, large virulence plasmid, pINV, led to the emergence of Shigella spp. from Escherichia coli. The plasmid encodes a Type III secretion system (T3SS) on a 30 kb pathogenicity island (PAI), and is maintained in a bacterial population through a series of toxin:antitoxin (TA) systems which mediate post-segregational killing (PSK). The T3SS imposes a significant cost on the bacterium, and strains which have lost the plasmid and/or genes encoding the T3SS grow faster than wild-type strains in the laboratory, and fail to bind the indicator dye Congo Red (CR). Our aim was to define the molecular events in Shigella flexneri that cause loss of Type III secretion (T3S), and to examine whether TA systems exert positional effects on pINV. During growth at 37°C, we found that deletions of regions of the plasmid including the PAI lead to the emergence of CR-negative colonies; deletions occur through intra-molecular recombination events between insertion sequences (ISs) flanking the PAI. Furthermore, by repositioning MvpAT (which belongs to the VapBC family of TA systems) near the PAI, we demonstrate that the location of this TA system alters the rearrangements that lead to loss of T3S, indicating that MvpAT acts both globally (by reducing loss of pINV through PSK) as well as locally (by preventing loss of adjacent sequences). During growth at environmental temperatures, we show for the first time that pINV spontaneously integrates into different sites in the chromosome, and this is mediated by inter-molecular events involving IS1294. Integration leads to reduced PAI gene expression and impaired secretion through the T3SS, while excision of pINV from the chromosome restores T3SS function. Therefore, pINV integration provides a reversible mechanism for Shigella to circumvent the metabolic burden imposed by pINV. Intra- and inter-molecular events between ISs, which are abundant in Shigella spp., mediate plasticity of S. flexneri pINV.

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July 7, 2019  |  

First detailed genetic characterization of the structural organization of type III arginine catabolic mobile elements harbored by Staphylococcus epidermidis by using whole-genome sequencing.

The type III arginine catabolic mobile element (ACME) was detected in three Staphylococcus epidermidis oral isolates recovered from separate patients (one healthy, one healthy with dental implants, and one with periodontal disease) based on ACME-arc-operon- and ACME-opp3-operon-directed PCR. These isolates were subjected to whole-genome sequencing to characterize the precise structural organization of ACME III for the first time, which also revealed that all three isolates were the same sequence type, ST329. Copyright © 2017 McManus et al.

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July 7, 2019  |  

Building a locally diploid genome and transcriptome of the diatom Fragilariopsis cylindrus.

The genome of the cold-adapted diatom Fragilariopsis cylindrus is characterized by highly diverged haplotypes that intersperse its homozygous genome. Here, we describe how a combination of PacBio DNA and Illumina RNA sequencing can be used to resolve this complex genomic landscape locally into the highly diverged haplotypes, and how to map various environmentally controlled transcripts onto individual haplotypes. We assembled PacBio sequence data with the FALCON assembler and created a haplotype resolved annotation of the assembly using annotations of a Sanger sequenced F. cylindrus genome. RNA-seq datasets from six different growth conditions were used to resolve allele-specifc gene expression in F. cylindrus. This approach enables to study differential expression of alleles in a complex genomic landscape and provides a useful tool to study how diverged haplotypes in diploid organisms are used for adaptation and evolution to highly variable environments.

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July 7, 2019  |  

Novel multiresistance cfr plasmids in linezolid-resistant methicillin-resistant Staphylococcus epidermidis and vancomycin-resistant Enterococcus faecium (VRE) from a hospital outbreak: co-location of cfr and optrA in VRE.

Linezolid is often the drug of last resort to treat infections caused by Gram-positive cocci. Linezolid resistance can be mutational (23S rRNA or L-protein) or, less commonly, acquired [predominantly cfr, conferring resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins and streptogramin A compounds (PhLOPSA) or optrA, encoding oxazolidinone and phenicol resistance].To investigate the clonality and genetic basis of linezolid resistance in 13 linezolid-resistant (LZDR) methicillin-resistant Staphylococcus epidermidis (MRSE) isolates recovered during a 2013/14 outbreak in an ICU in an Irish hospital and an LZDR vancomycin-resistant Enterococcus faecium (VRE) isolate from an LZDR-MRSE-positive patient.All isolates underwent PhLOPSA susceptibility testing, 23S rRNA sequencing, DNA microarray profiling and WGS.All isolates exhibited the PhLOPSA phenotype. The VRE harboured cfr and optrA on a novel 73?kb plasmid (pEF12-0805) also encoding erm(A), erm(B), lnu(B), lnu(E), aphA3 and aadE. One MRSE (M13/0451, from the same patient as the VRE) harboured cfr on a novel 8.5?kb plasmid (pSEM13-0451). The remaining 12 MRSE lacked cfr but exhibited linezolid resistance-associated mutations and were closely related to (1-52 SNPs) but distinct from M13/0451 (202-223 SNPs).Using WGS, novel and distinct cfr and cfr/optrA plasmids were identified in an MRSE and VRE isolate, respectively, as well as a cfr-negative LZDR-MRSE ICU outbreak and a distinct cfr-positive LZDR-MRSE from the same ICU. To our knowledge, this is the first report of cfr and optrA on a single VRE plasmid. Ongoing surveillance of linezolid resistance is essential to maintain its therapeutic efficacy.© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

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July 7, 2019  |  

Diversity oriented biosynthesis via accelerated evolution of modular gene clusters.

Erythromycin, avermectin and rapamycin are clinically useful polyketide natural products produced on modular polyketide synthase multienzymes by an assembly-line process in which each module of enzymes in turn specifies attachment of a particular chemical unit. Although polyketide synthase encoding genes have been successfully engineered to produce novel analogues, the process can be relatively slow, inefficient, and frequently low-yielding. We now describe a method for rapidly recombining polyketide synthase gene clusters to replace, add or remove modules that, with high frequency, generates diverse and highly productive assembly lines. The method is exemplified in the rapamycin biosynthetic gene cluster where, in a single experiment, multiple strains were isolated producing new members of a rapamycin-related family of polyketides. The process mimics, but significantly accelerates, a plausible mechanism of natural evolution for modular polyketide synthases. Detailed sequence analysis of the recombinant genes provides unique insight into the design principles for constructing useful synthetic assembly-line multienzymes.

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July 7, 2019  |  

First report of cfr-encoding plasmids in the pandemic sequence type (ST) 22 methicillin-resistant Staphylococcus aureus Staphylococcal cassette chromosome mec type-IV clone.

Linezolid is often the drug of last resort for serious methicillin-resistant Staphylococcus aureus (MRSA) infections. Linezolid resistance is mediated by mutations in 23S rRNA and genes for ribosomal proteins, cfr encoding phenicol, lincosamide, oxazolidinone, pleuromutilin and streptogramin A (PhLOPSA) resistance, its homolgue cfr(B) or optrA conferring oxazolidinone and phenicol resistance. Linezolid resistance is rare in S. aureus, and cfr even rarer. This study investigated the clonality and linezolid resistance mechanisms of two MRSA isolates from patients in separate Irish hospitals. Isolates were subjected to cfr PCR, PhLOPSA susceptibility testing, 23S rRNA PCR and sequencing, DNA microarray profiling, spa typing, pulsed-field gel electrophoresis (PFGE), plasmid curing and conjugative transfer. Whole-genome sequencing was used for single nucleotide variant (SNV) analysis, multilocus-sequence typing, L-protein mutation identification, cfr-plasmid sequence analysis and optrA and cfr(B) detection. Isolates M12/0145 and M13/0401 exhibited linezolid MICs of 64 and 16 mg/liter, respectively, and harbored identical 23S rRNA and L22 mutations, but M12/0145 exhibited the mutation in 2/6 23S rRNA alleles compared to 1/5 in M13/0401. Both isolates were ST22-MRSA-IV/t032, harbored cfr, exhibited the PhLOPSA phenotype and lacked optrA and cfr(B). They differed by five PFGE bands and 603 SNVs. Isolate M12/0145 harbored cfr and fexA on a 41-kb conjugative pSCFS3-type plasmid, whereas M13/0401 harbored cfr and lsa(B) on a novel 27-kb plasmid. This is the first report of cfr in the pandemic ST22-MRSA-IV clone. Different cfr plasmids and mutations associated with linezolid resistance in genotypically distinct ST22-MRSA-IV isolates highlights that prudent management of linezolid use is essential. Copyright © 2016 Shore et al.

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July 7, 2019  |  

A pigeonpea gene confers resistance to Asian soybean rust in soybean.

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.

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July 7, 2019  |  

Complete genome sequence of Streptomyces formicae KY5, the formicamycin producer.

Here we report the complete genome of the new species Streptomyces formicae KY5 isolated from Tetraponera fungus growing ants. S. formicae was sequenced using the PacBio and 454 platforms to generate a single linear chromosome with terminal inverted repeats. Illumina MiSeq sequencing was used to correct base changes resulting from the high error rate associated with PacBio. The genome is 9.6 Mbps, has a GC content of 71.38% and contains 8162 protein coding sequences. Predictive analysis shows this strain encodes at least 45 gene clusters for the biosynthesis of secondary metabolites, including a type 2 polyketide synthase encoding cluster for the antibacterial formicamycins. Streptomyces formicae KY5 is a new, taxonomically distinct Streptomyces species and this complete genome sequence provides an important marker in the genus of Streptomyces. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

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July 7, 2019  |  

Short genome report of cellulose-producing commensal Escherichia coli 1094.

Bacterial surface colonization and biofilm formation often rely on the production of an extracellular polymeric matrix that mediates cell-cell and cell-surface contacts. In Escherichia coli and many Betaproteobacteria and Gammaproteobacteria cellulose is often the main component of the extracellular matrix. Here we report the complete genome sequence of the cellulose producing strain E. coli 1094 and compare it with five other closely related genomes within E. coli phylogenetic group A. We present a comparative analysis of the regions encoding genes responsible for cellulose biosynthesis and discuss the changes that could have led to the loss of this important adaptive advantage in several E. coli strains. Data deposition: The annotated genome sequence has been deposited at the European Nucleotide Archive under the accession number PRJEB21000.

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July 7, 2019  |  

First description of novel arginine catabolic mobile elements (ACMEs) types IV and V harboring a kdp operon in Staphylococcus epidermidis characterized by whole genome sequencing.

The arginine catabolic mobile element (ACME) was first described in the methicillin-resistant Staphylococcus aureus strain USA300 and is thought to facilitate survival on skin. To date three distinct ACME types have been characterized comprehensively in S. aureus and/or Staphylococcus epidermidis. Type I harbors the arc and opp3 operons encoding an arginine deaminase pathway and an oligopeptide permease ABC transporter, respectively, type II harbors the arc operon only, and type III harbors the opp3 operon only. To investigate the diversity and detailed genetic organization of ACME, whole genome sequencing (WGS) was performed on 32 ACME-harboring oro-nasal S. epidermidis isolates using MiSeq- and PacBio-based WGS platforms. In nine isolates the ACMEs lacked the opp3 operon, but harbored a complete kdp operon (kdpE/D/A/B/C) located a maximum of 2.8?kb upstream of the arc operon. The kdp operon exhibited 63% DNA sequence identity to the native S. aureus kdp operon. These findings identified a novel, previously undescribed ACME type (designated ACME IV), which could be subtyped (IVa and IVb) based on distinct 5′ flanking direct repeat sequences (DRs). Multilocus sequence typing (MLST) sequences extracted from the WGS data identified the sequence types (STs) of the isolates investigated. Four of the nine ACME IV isolates belonged to ST153, and one to ST17, a single locus variant of ST153. A tenth isolate, identified as ST5, harbored another novel ACME type (designated ACME V) containing the kdp, arc and opp3 operons and flanked by DR_F, and DR_B but lacked any internal DRs. ACME V was colocated with a staphylococcal chromosome cassette mec (SCCmec) IV element and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in a 116.9?kb composite island. The extensive genetic diversity of ACME in S. epidermidis has been further elucidated by WGS, revealing two novel ACME types IV and V for the first time. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

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