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September 22, 2019  |  

Functional metagenomics identifies an exosialidase with an inverting catalytic mechanism that defines a new glycoside hydrolase family (GH156).

Exosialidases are glycoside hydrolases that remove a single terminal sialic acid residue from oligosaccharides. They are widely distributed in biology, having been found in prokaryotes, eukaryotes, and certain viruses. Most characterized prokaryotic sialidases are from organisms that are pathogenic or commensal with mammals. However, in this study, we used functional metagenomic screening to seek microbial sialidases encoded by environmental DNA isolated from an extreme ecological niche, a thermal spring. Using recombinant expression of potential exosialidase candidates and a fluorogenic sialidase substrate, we discovered an exosialidase having no homology to known sialidases. Phylogenetic analysis indicated that this protein is a member of a small family of bacterial proteins of previously unknown function. Proton NMR revealed that this enzyme functions via an inverting catalytic mechanism, a biochemical property that is distinct from those of known exosialidases. This unique inverting exosialidase defines a new CAZy glycoside hydrolase family we have designated GH156.© 2018 Chuzel et al.

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September 22, 2019  |  

Genomic evidence for asymmetric introgression by sexual selection in the common wall lizard.

Strongly selected characters can be transferred from one lineage to another with limited genetic exchange, resulting in asymmetric introgression and a mosaic genome in the receiving population. However, systems are rarely sufficiently well studied to link the pattern of introgression to its underlying process. Male common wall lizards in western Italy exhibit exaggeration of a suite of sexually selected characters that make them outcompete males from a distantly related lineage that lack these characters. This results in asymmetric hybridization and adaptive introgression of the suite of characters following secondary contact. We developed genomewide markers to infer the demographic history of gene flow between different genetic lineages, identify the spread of the sexually selected syndrome, and test the prediction that introgression should be asymmetric and heterogeneous across the genome. Our results show that secondary contact was accompanied by gene flow in both directions across most of the genome, but with approximately 3% of the genome showing highly asymmetric introgression in the predicted direction. Demographic simulations reveal that this asymmetric gene flow is more recent than the initial secondary contact, and the data suggest that the exaggerated male sexual characters originated within the Italian lineage and subsequently spread throughout this lineage before eventually reaching the contact zone. These results demonstrate that sexual selection can cause a suite of characters to spread throughout both closely and distantly related lineages with limited gene flow across the genome at large.© 2018 John Wiley & Sons Ltd.

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September 22, 2019  |  

Genomic discovery of the hypsin gene and biosynthetic pathways for terpenoids in Hypsizygus marmoreus.

Hypsizygus marmoreus (Beech mushroom) is a popular ingredient in Asian cuisine. The medicinal effects of its bioactive compounds such as hypsin and hypsiziprenol have been reported, but the genetic basis or biosynthesis of these components is unknown.In this study, we sequenced a reference strain of H. marmoreus (Haemi 51,987-8). We evaluated various assembly strategies, and as a result the Allpaths and PBJelly produced the best assembly. The resulting genome was 42.7 Mbp in length and annotated with 16,627 gene models. A putative gene (Hypma_04324) encoding the antifungal and antiproliferative hypsin protein with 75% sequence identity with the previously known N-terminal sequence was identified. Carbohydrate active enzyme analysis displayed the typical feature of white-rot fungi where auxiliary activity and carbohydrate-binding modules were enriched. The genome annotation revealed four terpene synthase genes responsible for terpenoid biosynthesis. From the gene tree analysis, we identified that terpene synthase genes can be classified into six clades. Four terpene synthase genes of H. marmoreus belonged to four different groups that implies they may be involved in the synthesis of different structures of terpenes. A terpene synthase gene cluster was well-conserved in Agaricomycetes genomes, which contained known biosynthesis and regulatory genes.Genome sequence analysis of this mushroom led to the discovery of the hypsin gene. Comparative genome analysis revealed the conserved gene cluster for terpenoid biosynthesis in the genome. These discoveries will further our understanding of the biosynthesis of medicinal bioactive molecules in this edible mushroom.

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September 22, 2019  |  

Comprehensive profiling of four base overhang ligation fidelity by T4 DNA Ligase and application to DNA assembly.

Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson-Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, high-accuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.

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September 22, 2019  |  

Streptococcus suis contains multiple phase-variable methyltransferases that show a discrete lineage distribution.

Streptococcus suis is a major pathogen of swine, responsible for a number of chronic and acute infections, and is also emerging as a major zoonotic pathogen, particularly in South-East Asia. Our study of a diverse population of S. suis shows that this organism contains both Type I and Type III phase-variable methyltransferases. In all previous examples, phase-variation of methyltransferases results in genome wide methylation differences, and results in differential regulation of multiple genes, a system known as the phasevarion (phase-variable regulon). We hypothesized that each variant in the Type I and Type III systems encoded a methyltransferase with a unique specificity, and could therefore control a distinct phasevarion, either by recombination-driven shuffling between different specificities (Type I) or by biphasic on-off switching via simple sequence repeats (Type III). Here, we present the identification of the target specificities for each Type III allelic variant from S. suis using single-molecule, real-time methylome analysis. We demonstrate phase-variation is occurring in both Type I and Type III methyltransferases, and show a distinct association between methyltransferase type and presence, and population clades. In addition, we show that the phase-variable Type I methyltransferase was likely acquired at the origin of a highly virulent zoonotic sub-population.

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September 22, 2019  |  

Unraveling microbial communities associated with methylmercury production in paddy soils.

Rice consumption is now recognized as an important pathway of human exposure to the neurotoxin methylmercury (MeHg), particularly in countries where rice is a staple food. Although the discovery of a two-gene cluster hgcAB has linked Hg methylation to several phylogenetically diverse groups of anaerobic microorganisms converting inorganic mercury (Hg) to MeHg, the prevalence and diversity of Hg methylators in microbial communities of rice paddy soils remain unclear. We characterized the abundance and distribution of hgcAB genes using third-generation PacBio long-read sequencing and Illumina short-read metagenomic sequencing, in combination with quantitative PCR analyses in several mine-impacted paddy soils from southwest China. Both Illumina and PacBio sequencing analyses revealed that Hg methylating communities were dominated by iron-reducing bacteria (i.e., Geobacter) and methanogens, with a relatively low abundance of hgcA + sulfate-reducing bacteria in the soil. A positive correlation was observed between the MeHg content in soil and the relative abundance of Geobacter carrying the hgcA gene. Phylogenetic analysis also uncovered some hgcAB sequences closely related to three novel Hg methylators, Geobacter anodireducens, Desulfuromonas sp. DDH964, and Desulfovibrio sp. J2, among which G. anodireducens was validated for its ability to methylate Hg. These findings shed new light on microbial community composition and major clades likely driving Hg methylation in rice paddy soils.

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September 22, 2019  |  

Excision-reintegration at a pneumococcal phase-variable restriction-modification locus drives within- and between-strain epigenetic differentiation and inhibits gene acquisition.

Phase-variation of Type I restriction-modification systems can rapidly alter the sequence motifs they target, diversifying both the epigenetic patterns and endonuclease activity within clonally descended populations. Here, we characterize the Streptococcus pneumoniae SpnIV phase-variable Type I RMS, encoded by the translocating variable restriction (tvr) locus, to identify its target motifs, mechanism and regulation of phase variation, and effects on exchange of sequence through transformation. The specificity-determining hsdS genes were shuffled through a recombinase-mediated excision-reintegration mechanism involving circular intermediate molecules, guided by two types of direct repeat. The rate of rearrangements was limited by an attenuator and toxin-antitoxin system homologs that inhibited recombinase gene transcription. Target motifs for both the SpnIV, and multiple Type II, MTases were identified through methylation-sensitive sequencing of a panel of recombinase-null mutants. This demonstrated the species-wide diversity observed at the tvr locus can likely specify nine different methylation patterns. This will reduce sequence exchange in this diverse species, as the native form of the SpnIV RMS was demonstrated to inhibit the acquisition of genomic islands by transformation. Hence the tvr locus can drive variation in genome methylation both within and between strains, and limits the genomic plasticity of S. pneumoniae.

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September 22, 2019  |  

Phenotypic and genomic comparison of Photorhabdus luminescens subsp. laumondii TT01 and a widely used rifampicin-resistant Photorhabdus luminescens laboratory strain.

Photorhabdus luminescens is an enteric bacterium, which lives in mutualistic association with soil nematodes and is highly pathogenic for a broad spectrum of insects. A complete genome sequence for the type strain P. luminescens subsp. laumondii TT01, which was originally isolated in Trinidad and Tobago, has been described earlier. Subsequently, a rifampicin resistant P. luminescens strain has been generated with superior possibilities for experimental characterization. This strain, which is widely used in research, was described as a spontaneous rifampicin resistant mutant of TT01 and is known as TT01-RifR.Unexpectedly, upon phenotypic comparison between the rifampicin resistant strain and its presumed parent TT01, major differences were found with respect to bioluminescence, pigmentation, biofilm formation, haemolysis as well as growth. Therefore, we renamed the strain TT01-RifR to DJC. To unravel the genomic basis of the observed differences, we generated a complete genome sequence for strain DJC using the PacBio long read technology. As strain DJC was supposed to be a spontaneous mutant, only few sequence differences were expected. In order to distinguish these from potential sequencing errors in the published TT01 genome, we re-sequenced a derivative of strain TT01 in parallel, also using the PacBio technology. The two TT01 genomes differed at only 30 positions. In contrast, the genome of strain DJC varied extensively from TT01, showing 13,000 point mutations, 330 frameshifts, and 220 strain-specific regions with a total length of more than 300 kb in each of the compared genomes.According to the major phenotypic and genotypic differences, the rifampicin resistant P. luminescens strain, now named strain DJC, has to be considered as an independent isolate rather than a derivative of strain TT01. Strains TT01 and DJC both belong to P. luminescens subsp. laumondii.

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September 22, 2019  |  

Genotype to phenotype: Diet-by-mitochondrial DNA haplotype interactions drive metabolic flexibility and organismal fitness.

Diet may be modified seasonally or by biogeographic, demographic or cultural shifts. It can differentially influence mitochondrial bioenergetics, retrograde signalling to the nuclear genome, and anterograde signalling to mitochondria. All these interactions have the potential to alter the frequencies of mtDNA haplotypes (mitotypes) in nature and may impact human health. In a model laboratory system, we fed four diets varying in Protein: Carbohydrate (P:C) ratio (1:2, 1:4, 1:8 and 1:16 P:C) to four homoplasmic Drosophila melanogaster mitotypes (nuclear genome standardised) and assayed their frequency in population cages. When fed a high protein 1:2 P:C diet, the frequency of flies harbouring Alstonville mtDNA increased. In contrast, when fed the high carbohydrate 1:16 P:C food the incidence of flies harbouring Dahomey mtDNA increased. This result, driven by differences in larval development, was generalisable to the replacement of the laboratory diet with fruits having high and low P:C ratios, perturbation of the nuclear genome and changes to the microbiome. Structural modelling and cellular assays suggested a V161L mutation in the ND4 subunit of complex I of Dahomey mtDNA was mildly deleterious, reduced mitochondrial functions, increased oxidative stress and resulted in an increase in larval development time on the 1:2 P:C diet. The 1:16 P:C diet triggered a cascade of changes in both mitotypes. In Dahomey larvae, increased feeding fuelled increased ß-oxidation and the partial bypass of the complex I mutation. Conversely, Alstonville larvae upregulated genes involved with oxidative phosphorylation, increased glycogen metabolism and they were more physically active. We hypothesise that the increased physical activity diverted energy from growth and cell division and thereby slowed development. These data further question the use of mtDNA as an assumed neutral marker in evolutionary and population genetic studies. Moreover, if humans respond similarly, we posit that individuals with specific mtDNA variations may differentially metabolise carbohydrates, which has implications for a variety of diseases including cardiovascular disease, obesity, and perhaps Parkinson’s Disease.

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September 22, 2019  |  

The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals

The DNA sequence of the two human mucin genes MUC2 and MUC6 have not been completely resolved due to the repetitive nature of their central exon coding for Proline, Threonine and Serine rich sequences. The exact nucleotide sequence of these exons has remained unknown for a long time due to limitations in traditional sequencing techniques. These are still very poorly covered in new whole genome sequencing projects with the corresponding protein sequences partly missing. We used a BAC clone containing both these genes and third generation sequencing technology, SMRT sequencing, to obtain the full-length contiguous MUC2 and MUC6 tandem repeat sequences. The new sequences span the entire repeat regions with good coverage revealing their length, variation in repeat sequences and their internal organization. The sequences obtained were used to compare with available sequences from whole genome sequencing projects indicating variation in number of repeats and their internal organization between individuals. The lack of these sequences has limited the association of genetic alterations with disease. The full sequences of these mucins will now allow such studies, which could be of importance for inflammatory bowel diseases for MUC2 and gastric ulcer diseases for MUC6 where deficient mucus protection is assumed to play an important role.

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September 22, 2019  |  

Cryptocurrencies and Zero Mode Wave guides: An unclouded path to a more contiguous Cannabis sativa L. genome assembly

We describe the use ofa Decentralized Autonomous Organization (DAO) to crypto- fund the single molecule sequencing and publication ofa Type ll Cannabis plant. This resulted in the construction of the most contiguous Cannabis genome assembly to date. The combined use of the Dash cryptocurrency, DAOs, and Pacific Biosciences sequencing delivered a 1.03 Gb genome with a N50 of 665Kb in 77 days from funding to public upload. This represents a 230 fold improvement in the contiguity of the first cannabis assemblies in 2011 and a 4 fold improvement over all cannabis assemblies to date. 34Gb ofadditional sequencing pushed the assembly to a N50 of 3.8Mb. Hi-C data from Phase Genomics further scaffolded the assembly to 35 contigs at an N50 of 74Mb but requires additional curation. The genome is partially phased and larger than previously reported (2N : 1.33Gb). The CBCA, THCA and CBDA synthase gene clusters have been phased onto respective contigs demonstrating tandem repeat expansions.

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September 22, 2019  |  

Microevolution of Neisseria lactamica during nasopharyngeal colonisation induced by controlled human infection.

Neisseria lactamica is a harmless coloniser of the infant respiratory tract, and has a mutually-excluding relationship with the pathogen Neisseria meningitidis. Here we report controlled human infection with genomically-defined N. lactamica and subsequent bacterial microevolution during 26 weeks of colonisation. We find that most mutations that occur during nasopharyngeal carriage are transient indels within repetitive tracts of putative phase-variable loci associated with host-microbe interactions (pgl and lgt) and iron acquisition (fetA promotor and hpuA). Recurrent polymorphisms occurred in genes associated with energy metabolism (nuoN, rssA) and the CRISPR-associated cas1. A gene encoding a large hypothetical protein was often mutated in 27% of the subjects. In volunteers who were naturally co-colonised with meningococci, recombination altered allelic identity in N. lactamica to resemble meningococcal alleles, including loci associated with metabolism, outer membrane proteins and immune response activators. Our results suggest that phase variable genes are often mutated during carriage-associated microevolution.

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September 22, 2019  |  

Genomic surveillance of Enterococcus faecium reveals limited sharing of strains and resistance genes between livestock and humans in the United Kingdom.

Vancomycin-resistant Enterococcus faecium (VREfm) is a major cause of nosocomial infection and is categorized as high priority by the World Health Organization global priority list of antibiotic-resistant bacteria. In the past, livestock have been proposed as a putative reservoir for drug-resistant E. faecium strains that infect humans, and isolates of the same lineage have been found in both reservoirs. We undertook cross-sectional surveys to isolate E. faecium (including VREfm) from livestock farms, retail meat, and wastewater treatment plants in the United Kingdom. More than 600 isolates from these sources were sequenced, and their relatedness and antibiotic resistance genes were compared with genomes of almost 800 E. faecium isolates from patients with bloodstream infection in the United Kingdom and Ireland. E. faecium was isolated from 28/29 farms; none of these isolates were VREfm, suggesting a decrease in VREfm prevalence since the last UK livestock survey in 2003. However, VREfm was isolated from 1% to 2% of retail meat products and was ubiquitous in wastewater treatment plants. Phylogenetic comparison demonstrated that the majority of human and livestock-related isolates were genetically distinct, although pig isolates from three farms were more genetically related to human isolates from 2001 to 2004 (minimum of 50?single-nucleotide polymorphisms [SNPs]). Analysis of accessory (variable) genes added further evidence for distinct niche adaptation. An analysis of acquired antibiotic resistance genes and their variants revealed limited sharing between humans and livestock. Our findings indicate that the majority of E. faecium strains infecting patients are largely distinct from those from livestock in this setting, with limited sharing of strains and resistance genes.IMPORTANCE The rise in rates of human infection caused by vancomycin-resistant Enterococcus faecium (VREfm) strains between 1988 to the 2000s in Europe was suggested to be associated with acquisition from livestock. As a result, the European Union banned the use of the glycopeptide drug avoparcin as a growth promoter in livestock feed. While some studies reported a decrease in VREfm in livestock, others reported no reduction. Here, we report the first livestock VREfm prevalence survey in the UK since 2003 and the first large-scale study using whole-genome sequencing to investigate the relationship between E. faecium strains in livestock and humans. We found a low prevalence of VREfm in retail meat and limited evidence for recent sharing of strains between livestock and humans with bloodstream infection. There was evidence for limited sharing of genes encoding antibiotic resistance between these reservoirs, a finding which requires further research. Copyright © 2018 Gouliouris et al.

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September 22, 2019  |  

Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

DNA N6-adenine methylation (6mA) has recently been reported in diverse eukaryotes, spanning unicellular organisms to metazoans. Yet the functional significance of 6mA remains elusive due to its low abundance, difficulty of manipulation within native DNA, and lack of understanding of eukaryotic 6mA writers. Here, we report a novel DNA 6mA methyltransferase in ciliates, termed MTA1. The enzyme contains an MT-A70 domain but is phylogenetically distinct from all known RNA and DNA methyltransferases. Disruption of MTA1 in vivo leads to the genome-wide loss of 6mA in asexually growing cells and abolishment of the consensus ApT dimethylated motif. Genes exhibit subtle changes in chromatin organization or RNA expression upon loss of 6mA, depending on their starting methylation level. Mutants fail to complete the sexual cycle, which normally coincides with a peak of MTA1 expression. Thus, MTA1 functions in a developmental stage-specific manner. We determine the impact of 6mA on chromatin organization in vitro by reconstructing complete, full-length ciliate chromosomes harboring 6mA in native or ectopic positions. Using these synthetic chromosomes, we show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a novel MT-A70 protein necessary for eukaryotic 6mA methylation and defines the impact of 6mA on chromatin organization using epigenetically defined synthetic chromosomes.

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September 22, 2019  |  

Loss of Rap1 supports recombination-based telomere maintenance independent of RNA-DNA hybrids in fission yeast

To investigate the molecular changes needed for cells to maintain their telomeres by recombination, we monitored telomere appearance during serial culture of fission yeast cells lacking the telomerase recruitment factor Ccq1. Rad52 is loaded onto critically short telomeres shortly after germination despite continued telomere erosion, suggesting that recruitment of recombination factors is not sufficient to maintain telomeres in the absence of telomerase function. Instead, survivor formation coincides with the derepression of telomeric repeat-containing RNA (TERRA). Degradation of telomere-associated TERRA in this context drives a severe growth crisis, ultimately leading to a distinct type of linear survivor with altered cytological telomere characteristics and the eviction of the shelterin component Rap1 (but not the TRF1/TRF2 orthologue, Taz1) from the telomere. We demonstrate that deletion of Rap1 is protective, preventing the growth crisis that is otherwise triggered by degradation of telomere-engaged TERRA in survivors with linear chromosomes. Thus, modulating the stoichiometry of shelterin components appears to support recombination-dependent survivors to persist in the absence of telomere-engaged TERRA.

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