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July 7, 2019  |  

Tigecycline resistance in clinical isolates of Enterococcus faecium is mediated by an upregulation of plasmid-encoded tetracycline determinants tet(L) and tet(M).

Tigecycline represents one of the last-line therapeutics to combat multidrug-resistant bacterial pathogens, including VRE and MRSA. The German National Reference Centre for Staphylococci and Enterococci has received 73 tigecycline-resistant Enterococcus faecium and Enterococcus faecalis isolates in recent years. The precise mechanism of how enterococci become resistant to tigecycline remains undetermined. This study documents an analysis of the role of efflux pumps in tigecycline resistance in clinical isolates of Enterococcus spp.Various tigecycline MICs were found for the different isolates analysed. Tigecycline-resistant strains were analysed with respect to genome and transcriptome differences by means of WGS and RT-qPCR. Genes of interest were cloned and expressed in Listeria monocytogenes for verification of their functionality.Detailed comparative whole-genome analyses of three isogenic strains, showing different levels of tigecycline resistance, revealed the major facilitator superfamily (MFS) efflux pump TetL and the ribosomal protection protein TetM as possible drug resistance proteins. Subsequent RT-qPCR confirmed up-regulation of the respective genes. A correlation of gene copy number and level of MIC was inferred from further qPCR analyses. Expression of both tet(L) and tet(M) in L. monocytogenes unequivocally demonstrated the potential to increase tigecycline MICs upon acquisition of either locus.Our results indicate that increased expression of two tetracycline resistance determinants, a tet(L)-encoded MFS pump and a tet(M)-encoded ribosomal protection protein, is capable of conferring tigecycline resistance in enterococcal clinical isolates.© The Author 2015. 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  |  

Streptomyces thermoautotrophicus does not fix nitrogen.

Streptomyces thermoautotrophicus UBT1 has been described as a moderately thermophilic chemolithoautotroph with a novel nitrogenase enzyme that is oxygen-insensitive. We have cultured the UBT1 strain, and have isolated two new strains (H1 and P1-2) of very similar phenotypic and genetic characters. These strains show minimal growth on ammonium-free media, and fail to incorporate isotopically labeled N2 gas into biomass in multiple independent assays. The sdn genes previously published as the putative nitrogenase of S. thermoautotrophicus have little similarity to anything found in draft genome sequences, published here, for strains H1 and UBT1, but share >99% nucleotide identity with genes from Hydrogenibacillus schlegelii, a draft genome for which is also presented here. H. schlegelii similarly lacks nitrogenase genes and is a non-diazotroph. We propose reclassification of the species containing strains UBT1, H1, and P1-2 as a non-Streptomycete, non-diazotrophic, facultative chemolithoautotroph and conclude that the existence of the previously proposed oxygen-tolerant nitrogenase is extremely unlikely.

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July 7, 2019  |  

Identifying potential mechanisms enabling acidophily in the ammonia-oxidising archaeon ‘Candidatus Nitrosotalea devanaterra’.

Ammonia oxidation is the first and rate-limiting step in nitrification and is dominated by two distinct groups of microorganisms in soil: ammonia-oxidising archaea (AOA) and bacteria (AOB). AOA are often more abundant than AOB, and dominate activity in acid soils. The mechanism of ammonia oxidation in acidic conditions has been a long-standing paradox. While high rates of ammonia oxidation are frequently measured in acid soils, cultivated ammonia oxidisers only grew at near-neutral pH when grown in standard laboratory culture. Although a number of mechanisms have been demonstrated to enable neutrophilic AOB growth at low pH in the laboratory, these have not been demonstrated in soil, and the recent cultivation of the obligately acidophilic ammonia oxidiser ‘Candidatus Nitrosotalea devanaterra’ provides a more parsimonious explanation for the observed high rates of activity. Analysis of the sequenced genome, transcriptional activity and lipid content of ‘Ca. N. devanaterra’ reveals that previously proposed mechanisms used by AOB for growth at low pH are not essential for archaeal ammonia oxidation in acidic environments. Instead, the genome indicates that ‘Ca. N. devanaterra’ contains genes encoding both a predicted high-affinity substrate acquisition system and potential pH homeostasis mechanisms absent in neutrophilic AOA. Analysis of mRNA revealed that candidate genes encoding for the proposed homeostasis mechanisms were all expressed during acidophilic growth, and lipid profiling by HPLC-MS demonstrated that the membrane lipids of ‘Ca. N. devanaterra’ were not dominated by crenarchaeol, as found in neutrophilic AOA. This study describes the first genome of an obligately acidophilic ammonia oxidiser and identifies potential mechanisms enabling this unique phenotype for future biochemical characterisation. Copyright © 2016 Lehtovirta-Morley et al.

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July 7, 2019  |  

Global phylogeography and evolutionary history of Shigella dysenteriae type 1

Together with plague, smallpox and typhus, epidemics of dysentery have been a major scourge of human populations for centuries1. A previous genomic study concluded that Shigella dysenteriae type 1 (Sd1), the epidemic dysentery bacillus, emerged and spread worldwide after the First World War, with no clear pattern of transmission2. This is not consistent with the massive cyclic dysentery epidemics reported in Europe during the eighteenth and nineteenth centuries1,3,4 and the first isolation of Sd1 in Japan in 18975. Here, we report a whole-genome analysis of 331 Sd1 isolates from around the world, collected between 1915 and 2011, providing us with unprecedented insight into the historical spread of this pathogen. We show here that Sd1 has existed since at least the eighteenth century and that it swept the globe at the end of the nineteenth century, diversifying into distinct lineages associated with the First World War, Second World War and various conflicts or natural disasters across Africa, Asia and Central America. We also provide a unique historical perspective on the evolution of antibiotic resistance over a 100-year period, beginning decades before the antibiotic era, and identify a prevalent multiple antibiotic-resistant lineage in South Asia that was transmitted in several waves to Africa, where it caused severe outbreaks of disease.

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July 7, 2019  |  

The identification of novel diagnostic marker genes for the detection of beer spoiling Pediococcus damnosus strains using the BlAst Diagnostic Gene findEr.

As the number of bacterial genomes increases dramatically, the demand for easy to use tools with transparent functionality and comprehensible output for applied comparative genomics grows as well. We present BlAst Diagnostic Gene findEr (BADGE), a tool for the rapid prediction of diagnostic marker genes (DMGs) for the differentiation of bacterial groups (e.g. pathogenic / nonpathogenic). DMG identification settings can be modified easily and installing and running BADGE does not require specific bioinformatics skills. During the BADGE run the user is informed step by step about the DMG finding process, thus making it easy to evaluate the impact of chosen settings and options. On the basis of an example with relevance for beer brewing, being one of the oldest biotechnological processes known, we show a straightforward procedure, from phenotyping, genome sequencing, assembly and annotation, up to a discriminant marker gene PCR assay, making comparative genomics a means to an end. The value and the functionality of BADGE were thoroughly examined, resulting in the successful identification and validation of an outstanding novel DMG (fabZ) for the discrimination of harmless and harmful contaminations of Pediococcus damnosus, which can be applied for spoilage risk determination in breweries. Concomitantly, we present and compare five complete P. damnosus genomes sequenced in this study, finding that the ability to produce the unwanted, spoilage associated off-flavor diacetyl is a plasmid encoded trait in this important beer spoiling species.

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July 7, 2019  |  

Draft genome sequences of three European laboratory derivatives from enterohemorrhagic Escherichia coli O157:H7 strain EDL933, including two plasmids.

Escherichia coliO157:H7 EDL933, isolated in 1982 in the United States, was the first enterohemorrhagicE. coli(EHEC) strain sequenced. Unfortunately, European labs can no longer receive the original strain. We checked three European EDL933 derivatives and found major genetic deviations (deletions, inversions) in two strains. All EDL933 strains contain the cryptic EHEC-plasmid, not reported before. Copyright © 2016 Fellner et al.

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July 7, 2019  |  

Finished genome sequence and methylome of the cyanide-degrading Pseudomonas pseudoalcaligenes strain CECT5344 as resolved by single-molecule real-time sequencing.

Pseudomonas pseudoalcaligenes CECT5344 tolerates cyanide and is also able to utilize cyanide and cyano-derivatives as a nitrogen source under alkaline conditions. The strain is considered as candidate for bioremediation of habitats contaminated with cyanide-containing liquid wastes. Information on the genome sequence of the strain CECT5344 became available previously. The P. pseudoalcaligenes CECT5344 genome was now resequenced by applying the single molecule, real-time (SMRT(®)) sequencing technique developed by Pacific Biosciences. The complete and finished genome sequence of the strain consists of a 4,696,984 bp chromosome featuring a GC-content of 62.34%. Comparative analyses between the new and previous versions of the P. pseudoalcaligenes CECT5344 genome sequence revealed additional regions in the new sequence that were missed in the older version. These additional regions mostly represent mobile genetic elements. Moreover, five additional genes predicted to play a role in sulfoxide reduction are present in the newly established genome sequence. The P. pseudoalcaligenes CECT5344 genome sequence is highly related to the genome sequences of different Pseudomonas mendocina strains. Approximately, 70% of all genes are shared between P. pseudoalcaligenes and P. mendocina. In contrast to P. mendocina, putative pathogenicity genes were not identified in the P. pseudoalcaligenes CECT5344 genome. P. pseudoalcaligenes CECT5344 possesses unique genes for nitrilases and mercury resistance proteins that are of importance for survival in habitats contaminated with cyano- and mercury compounds. As an additional feature of the SMRT sequencing technology, the methylome of P. pseudoalcaligenes was established. Six sequence motifs featuring methylated adenine residues (m6A) were identified in the genome. The genome encodes several methyltransferases, some of which may be considered for methylation of the m6A motifs identified. The complete genome sequence of the strain CECT5344 now provides the basis for exploitation of genetic features for biotechnological purposes. Copyright © 2016 Elsevier B.V. All rights reserved.

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July 7, 2019  |  

Refined Pichia pastoris reference genome sequence.

Strains of the species Komagataella phaffii are the most frequently used “Pichia pastoris” strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were manually curated and confirmed by RNA-seq and published LC-MS/MS data, including the addition of new open reading frames (ORFs) and a reduction in the number of spliced genes from 797 to 571. One chromosomal fragment of 76kbp between two previous gaps on chromosome 1 and another 134kbp fragment at the end of chromosome 4, as well as several shorter fragments needed re-orientation. In total more than 500 positions in the genome have been corrected. This reference genome is presented with new chromosomal numbering, positioning ribosomal repeats at the distal ends of the four chromosomes, and includes predicted chromosomal centromeres as well as the sequence of two linear cytoplasmic plasmids of 13.1 and 9.5kbp found in some strains of P. pastoris. Copyright © 2016. Published by Elsevier B.V.

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July 7, 2019  |  

Diverse CRISPR-Cas responses and dramatic cellular DNA changes and cell death in pKEF9-conjugated Sulfolobus species.

The Sulfolobales host a unique family of crenarchaeal conjugative plasmids some of which undergo complex rearrangements intracellularly. Here we examined the conjugation cycle of pKEF9 in the recipient strain Sulfolobus islandicus REY15A. The plasmid conjugated and replicated rapidly generating high average copy numbers which led to strong growth retardation that was coincident with activation of CRISPR-Cas adaptation. Simultaneously, intracellular DNA was extensively degraded and this also occurred in a conjugated ?cas6 mutant lacking a CRISPR-Cas immune response. Furthermore, the integrated forms of pKEF9 in the donor Sulfolobus solfataricus P1 and recipient host were specifically corrupted by transposable orfB elements, indicative of a dual mechanism for inactivating free and integrated forms of the plasmid. In addition, the CRISPR locus of pKEF9 was progressively deleted when conjugated into the recipient strain. Factors influencing activation of CRISPR-Cas adaptation in the recipient strain are considered, including the first evidence for a possible priming effect in Sulfolobus. The 3-Mbp genome sequence of the donor P1 strain is presented..© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019  |  

Suppressed recombination and unique candidate genes in the divergent haplotype encoding Fhb1, a major Fusarium head blight resistance locus in wheat.

Fine mapping and sequencing revealed 28 genes in the non-recombining haplotype containing Fhb1 . Of these, only a GDSL lipase gene shows a pathogen-dependent expression pattern. Fhb1 is a prominent Fusarium head blight resistance locus of wheat, which has been successfully introgressed in adapted breeding material, where it confers a significant increase in overall resistance to the causal pathogen Fusarium graminearum and the fungal virulence factor and mycotoxin deoxynivalenol. The Fhb1 region has been resolved for the susceptible wheat reference genotype Chinese Spring, yet the causal gene itself has not been identified in resistant cultivars. Here, we report the establishment of a 1 Mb contig embracing Fhb1 in the donor line CM-82036. Sequencing revealed that the region of Fhb1 deviates from the Chinese Spring reference in DNA size and gene content, which explains the repressed recombination at the locus in the performed fine mapping. Differences in genes expression between near-isogenic lines segregating for Fhb1 challenged with F. graminearum or treated with mock were investigated in a time-course experiment by RNA sequencing. Several candidate genes were identified, including a pathogen-responsive GDSL lipase absent in susceptible lines. The sequence of the Fhb1 region, the resulting list of candidate genes, and near-diagnostic KASP markers for Fhb1 constitute a valuable resource for breeding and further studies aiming to identify the gene(s) responsible for F. graminearum and deoxynivalenol resistance.

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July 7, 2019  |  

The Solanum demissum R8 late blight resistance gene is an Sw-5 homologue that has been deployed worldwide in late blight resistant varieties.

The potato late blight resistance gene R8 has been cloned. R8 is found in five late blight resistant varieties deployed in three different continents. R8 recognises Avr8 and is homologous to the NB-LRR protein Sw-5 from tomato. The broad spectrum late blight resistance gene R8 from Solanum demissum was cloned based on a previously published coarse map position on the lower arm of chromosome IX. Fine mapping in a recombinant population and bacterial artificial chromosome (BAC) library screening resulted in a BAC contig spanning 170 kb of the R8 haplotype. Sequencing revealed a cluster of at least ten R gene analogues (RGAs). The seven RGAs in the genetic window were subcloned for complementation analysis. Only one RGA provided late blight resistance and caused recognition of Avr8. From these results, it was concluded that the newly cloned resistance gene was indeed R8. R8 encodes a typical intracellular immune receptor with an N-terminal coiled coil, a central nucleotide binding site and 13 C-terminal leucine rich repeats. Phylogenetic analysis of a set of representative Solanaceae R proteins shows that R8 resides in a clearly distinct clade together with the Sw-5 tospovirus R protein from tomato. It was found that the R8 gene is present in late blight resistant potato varieties from Europe (Sarpo Mira), USA (Jacqueline Lee, Missaukee) and China (PB-06, S-60). Indeed, when tested under field conditions, R8 transgenic potato plants showed broad spectrum resistance to the current late blight population in the Netherlands, similar to Sarpo Mira.

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July 7, 2019  |  

Complete genome sequences of two methicillin-sensitive Staphylococcus aureus isolates representing a population subset highly prevalent in human colonization.

Here, we report the high-quality draft genome sequences of two methicillin-susceptible Staphylococcus aureus isolates, 08-02119 and 08-02300. Belonging to sequence type 582 (ST582) and ST7, both isolates are representatives of clonal lineages often associated with asymptomatic colonization of humans. Copyright © 2016 Weber et al.

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July 7, 2019  |  

Microsatellite length scoring by Single Molecule Real Time Sequencing – Effects of sequence structure and PCR regime.

Microsatellites are DNA sequences consisting of repeated, short (1-6 bp) sequence motifs that are highly mutable by enzymatic slippage during replication. Due to their high intrinsic variability, microsatellites have important applications in population genetics, forensics, genome mapping, as well as cancer diagnostics and prognosis. The current analytical standard for microsatellites is based on length scoring by high precision electrophoresis, but due to increasing efficiency next-generation sequencing techniques may provide a viable alternative. Here, we evaluated single molecule real time (SMRT) sequencing, implemented in the PacBio series of sequencing apparatuses, as a means of microsatellite length scoring. To this end we carried out multiplexed SMRT sequencing of plasmid-carried artificial microsatellites of varying structure under different pre-sequencing PCR regimes. For each repeat structure, reads corresponding to the target length dominated. We found that pre-sequencing amplification had large effects on scoring accuracy and error distribution relative to controls, but that the effects of the number of amplification cycles were generally weak. In line with expectations enzymatic slippage decreased proportionally with microsatellite repeat unit length and increased with repetition number. Finally, we determined directional mutation trends, showing that PCR and SMRT sequencing introduced consistent but opposing error patterns in contraction and expansion of the microsatellites on the repeat motif and single nucleotide level.

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July 7, 2019  |  

Variation of 45S rDNA intergenic spacers in Arabidopsis thaliana.

Approximately seven hundred 45S rRNA genes (rDNA) in the Arabidopsis thaliana genome are organised in two 4 Mbp-long arrays of tandem repeats arranged in head-to-tail fashion separated by an intergenic spacer (IGS). These arrays make up 5?% of the A. thaliana genome. IGS are rapidly evolving sequences and frequent rearrangements inside the rDNA loci have generated considerable interspecific and even intra-individual variability which allows to distinguish among otherwise highly conserved rRNA genes. The IGS has not been comprehensively described despite its potential importance in regulation of rDNA transcription and replication. Here we describe the detailed sequence variation in the complete IGS of A. thaliana WT plants and provide the reference/consensus IGS sequence, as well as genomic DNA analysis. We further investigate mutants dysfunctional in chromatin assembly factor-1 (CAF-1) (fas1 and fas2 mutants), which are known to have a reduced number of rDNA copies, and plant lines with restored CAF-1 function (segregated from a fas1xfas2 genetic background) showing major rDNA rearrangements. The systematic rDNA loss in CAF-1 mutants leads to the decreased variability of the IGS and to the occurrence of distinct IGS variants. We present for the first time a comprehensive and representative set of complete IGS sequences, obtained by conventional cloning and by Pacific Biosciences sequencing. Our data expands the knowledge of the A. thaliana IGS sequence arrangement and variability, which has not been available in full and in detail until now. This is also the first study combining IGS sequencing data with RFLP analysis of genomic DNA.

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July 7, 2019  |  

A viral immunity chromosome in the marine picoeukaryote, Ostreococcus tauri.

Micro-algae of the genus Ostreococcus and related species of the order Mamiellales are globally distributed in the photic zone of world’s oceans where they contribute to fixation of atmospheric carbon and production of oxygen, besides providing a primary source of nutrition in the food web. Their tiny size, simple cells, ease of culture, compact genomes and susceptibility to the most abundant large DNA viruses in the sea render them attractive as models for integrative marine biology. In culture, spontaneous resistance to viruses occurs frequently. Here, we show that virus-producing resistant cell lines arise in many independent cell lines during lytic infections, but over two years, more and more of these lines stop producing viruses. We observed sweeping over-expression of all genes in more than half of chromosome 19 in resistant lines, and karyotypic analyses showed physical rearrangements of this chromosome. Chromosome 19 has an unusual genetic structure whose equivalent is found in all of the sequenced genomes in this ecologically important group of green algae.

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