Genome diversification within a clonal population of pandemic Vibrio parahaemolyticus seems to depend on the life circumstances of each individual bacteria

Background

New strains of Vibrio parahaemolyticus that cause diarrhea in humans by seafood ingestion periodically emerge through continuous evolution in the ocean. Influx and expansion in the Southern Chilean ocean of a highly clonal V. parahaemolyticus (serotype O3:K6) population from South East Asia caused one of the largest seafood-related diarrhea outbreaks in the world. Here, genomics analyses of isolates from this rapidly expanding clonal population offered an opportunity to observe the molecular evolutionary changes often obscured in more diverse populations.

Results

Whole genome sequence comparison of eight independent isolates of this population from mussels or clinical cases (from different years) was performed. Differences of 1366 to 217,729 bp genome length and 13 to 164 bp single nucleotide variants (SNVs) were found. Most genomic differences corresponded to the presence of regions unique to only one or two isolates, and were probably acquired by horizontal gene transfer (HGT). Some DNA gain was chromosomal but most was in plasmids. One isolate had a large region (8,644 bp) missing, which was probably caused by excision of a prophage. Genome innovation by the presence of unique DNA, attributable to HGT from related bacteria, varied greatly among the isolates, with values of 1,366 (ten times the number of highest number of SNVs) to 217,729 (a thousand times more than the number of highest number of SNVs).

Conclusions

The evolutionary forces (SNVs, HGT) acting on each isolate of the same population were found to differ to an extent that probably depended on the ecological scenario and life circumstances of each bacterium.

Electronic supplementary material

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ITIS,a bioinformatics tool for accurate identification of transposon insertion sites using next-generation sequencing data

Background

Transposable elements constitute an important part of the genome and are essential in adaptive mechanisms. Transposition events associated with phenotypic changes occur naturally or are induced in insertional mutant populations. Transposon mutagenesis results in multiple random insertions and recovery of most/all the insertions is critical for forward genetics study. Using genome next-generation sequencing data and appropriate bioinformatics tool, it is plausible to accurately identify transposon insertion sites, which could provide candidate causal mutations for desired phenotypes for further functional validation.

Results

We developed a novel bioinformatics tool, ITIS (Identification of Transposon Insertion Sites), for localizing transposon insertion sites within a genome. It takes next-generation genome re-sequencing data (NGS data), transposon sequence, and reference genome sequence as input, and generates a list of highly reliable candidate insertion sites as well as zygosity information of each insertion. Using a simulated dataset and a case study based on an insertional mutant line from Medicago truncatula, we showed that ITIS performed better in terms of sensitivity and specificity than other similar algorithms such as RelocaTE, RetroSeq, TEMP and TIF. With the case study data, we demonstrated the efficiency of ITIS by validating the presence and zygosity of predicted insertion sites of the Tnt1 transposon within a complex plant system, M. truncatula.

Conclusion

This study showed that ITIS is a robust and powerful tool for forward genetic studies in identifying transposable element insertions causing phenotypes. ITIS is suitable in various systems such as cell culture, bacteria, yeast, insect, mammal and plant.

Electronic supplementary material

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De novo mutations in ARID1B associated with both syndromic and non-syndromic short stature

Background

Human height is a complex trait with a strong genetic basis. Recently, a significant association between rare copy number variations (CNVs) and short stature has been identified, and candidate genes in these rare CNVs are being explored. This study aims to evaluate the association between mutations in ARID1B gene and short stature, both the syndromic and non-syndromic form.

Results

Based on a case-control study of whole genome chromosome microarray analysis (CMA), three overlapping CNVs were identified in patients with developmental disorders who exhibited short stature. ARID1B, a causal gene for Coffin Siris syndrome, is the only gene encompassed by all three CNVs. A following retrospective genotype-phenotype analysis based on a literature review confirmed that short stature is a frequent feature in those Coffin-Siris syndrome patients with ARID1B mutations. Mutation screening of ARID1B coding regions was further conducted in a cohort of 48 non-syndromic short stature patients,andfour novel missense variants including two de novo mutations were found.

Conclusion

These results suggest that haploinsufficient mutations of ARID1B are associated with syndromic short stature including Coffin-Siris syndrome and intellectual disability, while rare missense variants in ARID1B are associated with non-syndromic short stature. This study supports the notion that mutations in genes related to syndromic short stature may exert milder effect and contribute to short stature in the general population.

Electronic supplementary material

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Genome-wide next-generation DNA and RNA sequencing reveals a mutation that perturbs splicing of the phosphatidylinositol glycan anchor biosynthesis class H gene (PIGH) and causes arthrogryposis in Belgian Blue cattle

Background

Cattle populations are characterized by regular outburst of genetic defects as a result of the extensive use of elite sires. The causative genes and mutations can nowadays be rapidly identified by means of genome-wide association studies combined with next generation DNA sequencing, provided that the causative mutations are conventional loss-of-function variants. We show in this work how the combined use of next generation DNA and RNA sequencing allows for the rapid identification of otherwise difficult to identify splice-site variants.

Results

We report the use of haplotype-based association mapping to identify a locus on bovine chromosome 10 that underlies autosomal recessive arthrogryposis in Belgian Blue Cattle. We identify 31 candidate mutations by resequencing the genome of four cases and 15 controls at ~10-fold depth. By analyzing RNA-Seq data from a carrier fetus, we observe skipping of the second exon of the PIGH gene, which we confirm by RT-PCR to be fully penetrant in tissues from affected calves. We identify - amongst the 31 candidate variants - a C-to-G transversion in the first intron of the PIGH gene (c211-10C > G) that is predicted to affect its acceptor splice-site. The resulting PIGH protein is likely to be non-functional as it lacks essential domains, and hence to cause arthrogryposis.

Conclusions

This work illustrates how the growing arsenal of genome exploration tools continues to accelerate the identification of an even broader range of disease causing mutations, therefore improving the management and control of genetic defects in livestock.

Electronic supplementary material

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Genome-Wide ENU Mutagenesis in Combination with High Density SNP Analysis and Exome Sequencing Provides Rapid Identification of Novel Mouse Models of Developmental Disease

Background

Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU).

Methodology/Principal Findings

ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1.

Conclusions/Significance

In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.     

Mono-allelic retrotransposon insertion addresses epigenetic transcriptional repression in human genome

Background

Retrotransposons have been extensively studied in plants and animals and have been shown to have an impact on human genome dynamics and evolution. Their ability to move within genomes gives retrotransposons to affect genome instability.

Methods

we examined the polymorphic inserted AluYa5, evolutionary young Alu, in the progesterone receptor gene to determine the effects of Alu insertion on molecular environment. We used mono-allelic inserted cell lines which carry both Alu-present and Alu-absent alleles. To determine the epigenetic change and gene expression, we performed restriction enzyme digestion, Pyrosequencing, and Chromatin Immunoprecipitation.

Results

We observed that the polymorphic insertion of evolutionally young Alu causes increasing levels of DNA methylation in the surrounding genomic area and generates inactive histone tail modifications. Consequently the Alu insertion deleteriously inactivates the neighboring gene expression.

Conclusion

The mono-allelic Alu insertion cell line clearly showed that polymorphic inserted repetitive elements cause the inactivation of neighboring gene expression, bringing aberrant epigenetic changes.     

Single-molecule sequencing reveals the molecular basis of multidrug-resistance in ST772 methicillin-resistant Staphylococcus aureus

Background

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of hospital-associated infection, but there is growing awareness of the emergence of multidrug-resistant lineages in community settings around the world. One such lineage is ST772-MRSA-V, which has disseminated globally and is increasingly prevalent in India. Here, we present the complete genome sequence of DAR4145, a strain of the ST772-MRSA-V lineage from India, and investigate its genomic characteristics in regards to antibiotic resistance and virulence factors.

Results

Sequencing using single-molecule real-time technology resulted in the assembly of a single continuous chromosomal sequence, which was error-corrected, annotated and compared to nine draft genome assemblies of ST772-MRSA-V from Australia, Malaysia and India. We discovered numerous and redundant resistance genes associated with mobile genetic elements (MGEs) and known core genome mutations that explain the highly antibiotic resistant phenotype of DAR4145. Staphylococcal toxins and superantigens, including the leukotoxin Panton-Valentinin Leukocidin, were predominantly associated with genomic islands and the phage φ-IND772PVL. Some of these mobile resistance and virulence factors were variably present in other strains of the ST772-MRSA-V lineage.

Conclusions

The genomic characteristics presented here emphasize the contribution of MGEs to the emergence of multidrug-resistant and highly virulent strains of community-associated MRSA. Antibiotic resistance was further augmented by chromosomal mutations and redundancy of resistance genes. The complete genome of DAR4145 provides a valuable resource for future investigations into the global dissemination and phylogeography of ST772-MRSA-V.

Electronic supplementary material

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Mutations upstream of fabI in triclosan resistant Staphylococcus aureus strains are associated with elevated fabI gene expression

Background

The enoyl-acyl carrier protein (ACP) reductase enzyme (FabI) is the target for a series of antimicrobial agents including novel compounds in clinical trial and the biocide triclosan. Mutations in fabI and heterodiploidy for fabI have been shown to confer resistance in S. aureus strains in a previous study. Here we further determined the fabI upstream sequence of a selection of these strains and the gene expression levels in strains with promoter region mutations.

Results

Mutations in the fabI promoter were found in 18% of triclosan resistant clinical isolates, regardless the previously identified molecular mechanism conferring resistance. Although not significant, a higher rate of promoter mutations were found in strains without previously described mechanisms of resistance. Some of the mutations identified in the clinical isolates were also detected in a series of laboratory mutants. Microarray analysis of selected laboratory mutants with fabI promoter region mutations, grown in the absence of triclosan, revealed increased fabI expression in three out of four tested strains. In two of these strains, only few genes other than fabI were upregulated. Consistently with these data, whole genome sequencing of in vitro selected mutants identified only few mutations except the upstream and coding regions of fabI, with the promoter mutation as the most probable cause of fabI overexpression. Importantly the gene expression profiling of clinical isolates containing similar mutations in the fabI promoter also showed, when compared to unrelated non-mutated isolates, a significant up-regulation of fabI.

Conclusions

In conclusion, we have demonstrated the presence of C34T, T109G, and A101C mutations in the fabI promoter region of strains with fabI up-regulation, both in clinical isolates and/or laboratory mutants. These data provide further observations linking mutations upstream fabI with up-regulated expression of the fabI gene.

Electronic supplementary material

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Cytogeography and genome size variation in the Claytonia perfoliata (Portulacaceae) polyploid complex

Background and Aims

Genome duplication is a central process in plant evolution and contributes to patterns of variation in genome size within and among lineages. Studies that combine cytogeography with genome size measurements contribute to our basic knowledge of cytotype distributions and their associations with variation in genome size.

Methods

Ploidy and genome size were assessed with direct chromosome counts and flow cytometry for 78 populations within the Claytonia perfoliata complex, comprised of three diploid taxa with numerous polyploids that range to the decaploid level. The relationship between genome size and temperature and precipitation was investigated within and across cytotypes to test for associations between environmental factors and nuclear DNA content.

Key Results

A euploid series (n = 6) of diploids to octoploids was documented through chromosome counts, and decaploids were suggested by flow cytometry. Increased variation in genome size among populations was found at higher ploidy levels, potentially associated with differential contributions of diploid parental genomes, variation in rates of genomic loss or gain, or undetected hybridization. Several accessions were detected with atypical genome sizes, including a diploid population of C. parviflora ssp. grandiflora with an 18 % smaller genome than typical, and hexaploids of C. perfoliata and C. parviflora with genomes 30 % larger than typical. There was a slight but significant association of larger genome sizes with colder winter temperature across the C. perfoliata complex as a whole, and a strong association between lower winter temperatures and large genome size for tetraploid C. parviflora.

Conclusions

The C. perfoliata complex is characterized by polyploids ranging from tetraploid to decaploid, with large magnitude variation in genome size at higher ploidy levels, associated in part with environmental variation in temperature.     

Polyploidy influences sexual system and mating patterns in the moss Atrichum undulatum sensu lato

Background and Aims

Evolutionary transitions between separate and combined sexes have frequently occurred across various plant lineages. In mosses, which are haploid-dominant, evolutionary transitions from separate to combined sexes are often associated with genome doubling. Polyploidy and hermaphroditism have strong effects on the inbreeding depression of a population, and are subsequently predicted to affect the mating system.

Methods

We tested the association between ploidy (haploid, diploid or triploid gametophytes) and mating system in 21 populations of Atrichum undulatum sensu lato, where sex ratios vary widely. For each population, we measured the sex ratio, estimated selfing rates using allozyme markers and determined the level of ploidy through flow cytometry.

Key Results

Hermaphrodites in A. undulatum were either diploid or triploid. However, many diploid populations were strictly separate-sexed, suggesting that hermaphroditism is not a necessary result of genome doubling. Levels of selfing were strongly supported as being greater than zero in one population with strictly separate-sexed individuals, and one-third of populations with hermaphrodites.

Conclusions

Although hermaphrodites are associated with triploidy, hermaphroditism is not a necessary outcome of genome duplication. Hermaphroditism, but not genome duplication alone, increased estimated selfing rates, probably due to the occurrence of selfing within a gametophyte. Thus, genome duplication can influence the mating system and the associated evolution and maintenance of reproductive traits.     

Extensive duplication of the Wolbachia DNA in chromosome four of Drosophila ananassae

Background

Lateral gene transfer (LGT) from bacterial Wolbachia endosymbionts has been detected in ~20% of arthropod and nematode genome sequencing projects. Many of these transfers are large and contain a substantial part of the Wolbachia genome.

Results

Here, we re-sequenced three D. ananassae genomes from Asia and the Pacific that contain large LGTs from Wolbachia. We find that multiple copies of the Wolbachia genome are transferred to the Drosophila nuclear genome in all three lines. In the D. ananassae line from Indonesia, the copies of Wolbachia DNA in the nuclear genome are nearly identical in size and sequence yielding an even coverage of mapped reads over the Wolbachia genome. In contrast, the D. ananassae lines from Hawaii and India show an uneven coverage of mapped reads over the Wolbachia genome suggesting that different parts of these LGTs are present in different copy numbers. In the Hawaii line, we find that this LGT is underrepresented in third instar larvae indicative of being heterochromatic. Fluorescence in situ hybridization of mitotic chromosomes confirms that the LGT in the Hawaii line is heterochromatic and represents ~20% of the sequence on chromosome 4 (dot chromosome, Muller element F).

Conclusions

This collection of related lines contain large lateral gene transfers composed of multiple Wolbachia genomes that constitute >2% of the D. ananassae genome (~5 Mbp) and partially explain the abnormally large size of chromosome 4 in D. ananassae.

Electronic supplementary material

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Population genetic evidence for speciation pattern and gene flow between Picea wilsonii,P. morrisonicola and P. neoveitchii

Background and Aims

Genetic drift due to geographical isolation, gene flow and mutation rates together make it difficult to determine the evolutionary relationships of present-day species. In this study, population genetic data were used to model and decipher interspecific relationships, speciation patterns and gene flow between three species of spruce with similar morphology, Picea wilsonii, P. neoveitchii and P. morrisonicola. Picea wilsonii and P. neoveitchii occur from central to north-west China, where they have overlapping distributions. Picea morrisonicola, however, is restricted solely to the island of Taiwan and is isolated from the other two species by a long distance.

Methods

Sequence variations were examined in 18 DNA fragments for 22 populations, including three fragments from the chloroplast (cp) genome, two from the mitochondrial (mt) genome and 13 from the nuclear genome.

Key Results

In both the cpDNA and the mtDNA, P. morrisonicola accumulated more species-specific mutations than the other two species. However, most nuclear haplotypes of P. morrisonicola were shared by P. wilsonii, or derived from the dominant haplotypes found in that species. Modelling of population genetic data supported the hypothesis that P. morrisonicola derived from P. wilsonii within the more recent past, most probably indicating progenitor–derivative speciation with a distinct bottleneck, although further gene flow from the progenitor to the derivative continued. In addition, the occurrence was detected of an obvious mtDNA introgression from P. neoveitchii to P. wilsonii despite their early divergence.

Conclusions

The extent of mutation, introgression and lineage sorting taking place during interspecific divergence and demographic changes in the three species had varied greatly between the three genomes. The findings highlight the complex evolutionary histories of these three Asian spruce species.     

Complete sequencing of Novosphingobium sp. PP1Y reveals a biotechnologically meaningful metabolic pattern

Background

Novosphingobium sp. strain PP1Y is a marine α-proteobacterium adapted to grow at the water/fuel oil interface. It exploits the aromatic fraction of fuel oils as a carbon and energy source. PP1Y is able to grow on a wide range of mono-, poly- and heterocyclic aromatic hydrocarbons. Here, we report the complete functional annotation of the whole Novosphingobium genome.

Results

PP1Y genome analysis and its comparison with other Sphingomonadal genomes has yielded novel insights into the molecular basis of PP1Y’s phenotypic traits, such as its peculiar ability to encapsulate and degrade the aromatic fraction of fuel oils. In particular, we have identified and dissected several highly specialized metabolic pathways involved in: (i) aromatic hydrocarbon degradation; (ii) resistance to toxic compounds; and (iii) the quorum sensing mechanism.

Conclusions

In summary, the unraveling of the entire PP1Y genome sequence has provided important insight into PP1Y metabolism and, most importantly, has opened new perspectives about the possibility of its manipulation for bioremediation purposes.

Electronic supplementary material

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