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Asset Tag: Microbiology

July 7, 2019

Sequence assembly of Yarrowia lipolytica strain W29/CLIB89 shows transposable element diversity.

Yarrowia lipolytica, an oleaginous yeast, is capable of accumulating significant cellular mass in lipid making it an important source of biosustainable hydrocarbon-based chemicals. In spite of a similar number of protein-coding genes to that in other Hemiascomycetes, the Y. lipolytica genome is almost double that of model yeasts. Despite its economic importance and several distinct strains in common use, an independent genome assembly exists for only one strain. We report here a de novo annotated assembly of the chromosomal genome of an industrially-relevant strain, W29/CLIB89, determined by hybrid next-generation sequencing. For the first time, each Y. lipolytica chromosome is represented by a single contig. The telomeric rDNA repeats were localized by Irys long-range genome mapping and one complete copy of the rDNA sequence is reported. Two large structural variants and retroelement differences with reference strain CLIB122 including a full-length, novel Ty3/Gypsy long terminal repeat (LTR) retrotransposon and multiple LTR-like sequences are described. Strikingly, several of these are adjacent to RNA polymerase III-transcribed genes, which are almost double in number in Y. lipolytica compared to other Hemiascomycetes. In addition to previously-reported dimeric RNA polymerase III-transcribed genes, tRNA pseudogenes were identified. Multiple full-length and truncated LINE elements are also present. Therefore, although identified transposons do not constitute a significant fraction of the Y. lipolytica genome, they could have played an active role in its evolution. Differences between the sequence of this strain and of the existing reference strain underscore the utility of an additional independent genome assembly for this economically important organism.

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July 7, 2019

Genomic analysis reveals novel diversity among the 1976 Philadelphia Legionnaires’ disease outbreak isolates and additional ST36 strains.

Legionella pneumophila was first recognized as a cause of severe and potentially fatal pneumonia during a large-scale outbreak of Legionnaires’ disease (LD) at a Pennsylvania veterans’ convention in Philadelphia, 1976. The ensuing investigation and recovery of four clinical isolates launched the fields of Legionella epidemiology and scientific research. Only one of the original isolates, “Philadelphia-1”, has been widely distributed or extensively studied. Here we describe the whole-genome sequencing (WGS), complete assembly, and comparative analysis of all Philadelphia LD strains recovered from that investigation, along with L. pneumophila isolates sharing the Philadelphia sequence type (ST36). Analyses revealed that the 1976 outbreak was due to multiple serogroup 1 strains within the same genetic lineage, differentiated by an actively mobilized, self-replicating episome that is shared with L. pneumophila str. Paris, and two large, horizontally-transferred genomic loci, among other polymorphisms. We also found a completely unassociated ST36 strain that displayed remarkable genetic similarity to the historical Philadelphia isolates. This similar strain implies the presence of a potential clonal population, and suggests important implications may exist for considering epidemiological context when interpreting phylogenetic relationships among outbreak-associated isolates. Additional extensive archival research identified the Philadelphia isolate associated with a non-Legionnaire case of “Broad Street pneumonia”, and provided new historical and genetic insights into the 1976 epidemic. This retrospective analysis has underscored the utility of fully-assembled WGS data for Legionella outbreak investigations, highlighting the increased resolution that comes from long-read sequencing and a sequence type-matched genomic data set.

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July 7, 2019

Complete circular genome sequence of successful ST8/SCCmecIV community-associated methicillin-resistant Staphylococcus aureus (OC8) in Russia: one-megabase genomic inversion, IS256’s spread, and evolution of Russia ST8-IV.

ST8/SCCmecIV community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) has been a common threat, with large USA300 epidemics in the United States. The global geographical structure of ST8/SCCmecIV has not yet been fully elucidated. We herein determined the complete circular genome sequence of ST8/SCCmecIVc strain OC8 from Siberian Russia. We found that 36.0% of the genome was inverted relative to USA300. Two IS256, oppositely oriented, at IS256-enriched hot spots were implicated with the one-megabase genomic inversion (MbIN) and vSaß split. The behavior of IS256 was flexible: its insertion site (att) sequences on the genome and junction sequences of extrachromosomal circular DNA were all divergent, albeit with fixed sizes. A similar multi-IS256 system was detected, even in prevalent ST239 healthcare-associated MRSA in Russia, suggesting IS256’s strong transmission potential and advantage in evolution. Regarding epidemiology, all ST8/SCCmecIVc strains from European, Siberian, and Far Eastern Russia, examined had MbIN, and geographical expansion accompanied divergent spa types and resistance to fluoroquinolones, chloramphenicol, and often rifampicin. Russia ST8/SCCmecIVc has been associated with life-threatening infections such as pneumonia and sepsis in both community and hospital settings. Regarding virulence, the OC8 genome carried a series of toxin and immune evasion genes, a truncated giant surface protein gene, and IS256 insertion adjacent to a pan-regulatory gene. These results suggest that unique single ST8/spa1(t008)/SCCmecIVc CA-MRSA (clade, Russia ST8-IVc) emerged in Russia, and this was followed by large geographical expansion, with MbIN as an epidemiological marker, and fluoroquinolone resistance, multiple virulence factors, and possibly a multi-IS256 system as selective advantages.

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July 7, 2019

Novel m4C modification in type I restriction-modification systems.

We identify a new subgroup of Type I Restriction-Modification enzymes that modify cytosine in one DNA strand and adenine in the opposite strand for host protection. Recognition specificity has been determined for ten systems using SMRT sequencing and each recognizes a novel DNA sequence motif. Previously characterized Type I systems use two identical copies of a single methyltransferase (MTase) subunit, with one bound at each half site of the specificity (S) subunit to form the MTase. The new m4C-producing Type I systems we describe have two separate yet highly similar MTase subunits that form a heterodimeric M1M2S MTase. The MTase subunits from these systems group into two families, one of which has NPPF in the highly conserved catalytic motif IV and modifies adenine to m6A, and one having an NPPY catalytic motif IV and modifying cytosine to m4C. The high degree of similarity among their cytosine-recognizing components (MTase and S) suggest they have recently evolved, most likely from the far more common m6A Type I systems. Type I enzymes that modify cytosine exclusively were formed by replacing the adenine target recognition domain (TRD) with a cytosine-recognizing TRD. These are the first examples of m4C modification in Type I RM systems.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019

Emergence of endemic MLST non-typeable vancomycin-resistant Enterococcus faecium.

Enterococcus faecium is a major nosocomial pathogen causing significant morbidity and mortality worldwide. Assessment of E. faecium using MLST to understand the spread of this organism is an important component of hospital infection control measures. Recent studies, however, suggest that MLST might be inadequate for E. faecium surveillance.To use WGS to characterize recently identified vancomycin-resistant E. faecium (VREfm) isolates non-typeable by MLST that appear to be causing a multi-jurisdictional outbreak in Australia.Illumina NextSeq and Pacific Biosciences SMRT sequencing platforms were used to determine the genome sequences of 66 non-typeable E. faecium (NTEfm) isolates. Phylogenetic and bioinformatics analyses were subsequently performed using a number of in silico tools.Sixty-six E. faecium isolates were identified by WGS from multiple health jurisdictions in Australia that could not be typed by MLST due to a missing pstS allele. SMRT sequencing and complete genome assembly revealed a large chromosomal rearrangement in representative strain DMG1500801, which likely facilitated the deletion of the pstS region. Phylogenomic analysis of this population suggests that deletion of pstS within E. faecium has arisen independently on at least three occasions. Importantly, the majority of these isolates displayed a vancomycin-resistant genotype.We have identified NTEfm isolates that appear to be causing a multi-jurisdictional outbreak in Australia. Identification of these isolates has important implications for MLST-based typing activities designed to monitor the spread of VREfm and provides further evidence supporting the use of WGS for hospital surveillance of E. faecium.© 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.

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July 7, 2019

Complete genome sequence of Brevibacterium linens SMQ-1335.

Brevibacterium linens is one of the main bacteria found in the smear of surface-ripened cheeses. The genome of the industrial strain SMQ-1335 was sequenced using PacBio. It has 4,209,935 bp, a 62.6% G+C content, 3,848 open reading frames, and 61 structural RNAs. A new type I restriction-modification system was identified. Copyright © 2016 de Melo et al.

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July 7, 2019

Production of the bioactive compounds violacein and indolmycin is conditional in a maeA mutant of Pseudoalteromonas luteoviolacea S4054 lacking the malic enzyme.

It has previously been reported that some strains of the marine bacterium Pseudoalteromonas luteoviolacea produce the purple bioactive pigment violacein as well as the antibiotic compound indolmycin, hitherto only found in Streptomyces. The purpose of the present study was to determine the relative role of each of these two compounds as antibacterial compounds in P. luteoviolacea S4054. Using Tn10 transposon mutagenesis, a mutant strain that was significantly reduced in violacein production in mannose-containing substrates was created. Full genome analyses revealed that the vio-biosynthetic gene cluster was not interrupted by the transposon; instead the insertion was located to the maeA gene encoding the malic enzyme. Supernatant of the mutant strain inhibited Vibrio anguillarum and Staphylococcus aureus in well diffusion assays and in MIC assays at the same level as the wild type strain. The mutant strain killed V. anguillarum in co-culture experiments as efficiently as the wild type. Using UHPLC-UV/Vis analyses, we quantified violacein and indolmycin, and the mutant strain only produced 7-10% the amount of violacein compared to the wild type strain. In contrast, the amount of indolmycin produced by the mutant strain was about 300% that of the wild type. Since inhibition of V. anguillarum and S. aureus by the mutant strain was similar to that of the wild type, it is concluded that violacein is not the major antibacterial compound in P. luteoviolacea. We furthermore propose that production of violacein and indolmycin may be metabolically linked and that yet unidentified antibacterial compound(s) may be play a role in the antibacterial activity of P. luteoviolacea.

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July 7, 2019

Complete genome sequence of Pseudomonas citronellolis P3B5, a candidate for microbial phyllo-remediation of hydrocarbon-contaminated sites

Pseudomonas citronellolis is a Gram negative, motile gammaproteobacterium belonging to the order Pseudomonadales and the family Pseudomonadaceae. We isolated strain P3B5 from the phyllosphere of basil plants (Ocimum basilicum L.). Here we describe the physiology of this microorganism, its full genome sequence, and detailed annotation. The 6.95 Mbp genome contains 6071 predicted protein coding sequences and 96 RNA coding sequences. P. citronellolis has been the subject of many studies including the investigation of long-chain aliphatic compounds and terpene degradation. Plant leaves are covered by long-chain aliphates making up a waxy layer that is associated with the leaf cuticle. In addition, basil leaves are known to contain high amounts of terpenoid substances, hinting to a potential nutrient niche that might be exploited by P. citronellolis. Furthermore, the isolated strain exhibited resistance to several antibiotics. To evaluate the potential of this strain as source of transferable antibiotic resistance genes on raw consumed herbs we therefore investigated if those resistances are encoded on mobile genetic elements. The availability of the genome will be helpful for comparative genomics of the phylogenetically broad pseudomonads, in particular with the sequence of the P. citronellolis type strain PRJDB205 not yet publicly available. The genome is discussed with respect to a phyllosphere related lifestyle, aliphate and terpenoid degradation, and antibiotic resistance.

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July 7, 2019

Divergent isoprenoid biosynthesis pathways in Staphylococcus species constitute a drug target for treating infections in companion animals.

Staphylococcus species are a leading cause of skin and soft tissue infections in humans and animals, and the antibiotics used to treat these infections are often the same. Methicillin- and multidrug-resistant staphylococcal infections are becoming more common in human and veterinary medicine. From a “One Health” perspective, this overlap in antibiotic use and resistance raises concerns over the potential spread of antibiotic resistance genes. Whole-genome sequencing and comparative genomics analysis revealed that Staphylococcus species use divergent pathways to synthesize isoprenoids. Species frequently associated with skin and soft tissue infections in companion animals, including S. schleiferi and S. pseudintermedius, use the nonmevalonate pathway. In contrast, S. aureus, S. epidermidis, and S. lugdunensis use the mevalonate pathway. The antibiotic fosmidomycin, an inhibitor of the nonmevalonate pathway, was effective in killing canine clinical staphylococcal isolates but had no effect on the growth or survival of S. aureus and S. epidermidis. These data identify an essential metabolic pathway in Staphylococcus that differs among members of this genus and suggest that drugs such as fosmidomycin, which targets enzymes in the nonmevalonate pathway, may be an effective treatment for certain staphylococcal infections. IMPORTANCE Drug-resistant Staphylococcus species are a major concern in human and veterinary medicine. There is a need for new antibiotics that exhibit a selective effect in treating infections in companion and livestock animals and that would not be used to treat human bacterial infections. We have identified fosmidomycin as an antibiotic that selectively targets certain Staphylococcus species that are often encountered in skin infections in cats and dogs. These findings expand our understanding of Staphylococcus evolution and may have direct implications for treating staphylococcal infections in veterinary medicine.

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July 7, 2019

Complete genome sequence of pigmentation-negative Yersinia pestis strain Cadman.

Here, we report the genome sequence of Yersinia pestis strain Cadman, an attenuated strain lacking the pgm locus. Y. pestis is the causative agent of plague and generally must be worked with under biosafety level 3 (BSL-3) conditions. However, strains lacking the pgm locus are considered safe to work with under BSL-2 conditions. Copyright © 2016 Lovett et al.

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July 7, 2019

MethSMRT: an integrative database for DNA N6-methyladenine and N4-methylcytosine generated by single-molecular real-time sequencing

DNA methylation is an important type of epigenetic modifications, where 5- methylcytosine (5mC), 6-methyadenine (6mA) and 4-methylcytosine (4mC) are the most common types. Previous efforts have been largely focused on 5mC, providing invaluable insights into epigenetic regulation through DNA methylation. Recently developed single-molecule real-time (SMRT) sequencing technology provides a unique opportunity to detect the less studied DNA 6mA and 4mC modifications at single-nucleotide resolution. With a rapidly increased amount of SMRT sequencing data generated, there is an emerging demand to systematically explore DNA 6mA and 4mC modifications from these data sets. MethSMRT is the first resource hosting DNA 6mA and 4mC methylomes. All the data sets were processed using the same analysis pipeline with the same quality control. The current version of the database provides a platform to store, browse, search and download epigenome-wide methylation profiles of 156 species, including seven eukaryotes such as Arabidopsis, C. elegans, Drosophila, mouse and yeast, as well as 149 prokaryotes. It also offers a genome browser to visualize the methylation sites and related information such as single nucleotide polymorphisms (SNP) and genomic annotation. Furthermore, the database provides a quick summary of statistics of methylome of 6mA and 4mC and predicted methylation motifs for each species. MethSMRT is publicly available at http://sysbio.sysu.edu.cn/methsmrt/ without use restriction.

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