Comparative genomic analysis of clinical and environmental strains provides insight into the pathogenicity and evolution of Vibrio parahaemolyticus

Background

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium. Infections with the bacterium could become systemic and can be life-threatening to immunocompromised individuals. Genome sequences of a few clinical isolates of V. parahaemolyticus are currently available, but the genome dynamics across the species and virulence potential of environmental strains on a genome-scale have not been described before.

Results

Here we present genome sequences of four V. parahaemolyticus clinical strains from stool samples of patients and five environmental strains in Hong Kong. Phylogenomics analysis based on single nucleotide polymorphisms revealed a clear distinction between the clinical and environmental isolates. A new gene cluster belonging to the biofilm associated proteins of V. parahaemolyticus was found in clincial strains. In addition, a novel small genomic island frequently found among clinical isolates was reported. A few environmental strains were found harboring virulence genes and prophage elements, indicating their virulence potential. A unique biphenyl degradation pathway was also reported. A database for V. parahaemolyticus (http://kwanlab.bio.cuhk.edu.hk/vp) was constructed here as a platform to access and analyze genome sequences and annotations of the bacterium.

Conclusions

We have performed a comparative genomics analysis of clinical and environmental strains of V. parahaemolyticus. Our analyses could facilitate understanding of the phylogenetic diversity and niche adaptation of this bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1135) contains supplementary material, which is available to authorized users.     

An assembly and alignment-free method of phylogeny reconstruction from next-generation sequencing data

Background

Next-generation sequencing technologies are rapidly generating whole-genome datasets for an increasing number of organisms. However, phylogenetic reconstruction of genomic data remains difficult because de novo assembly for non-model genomes and multi-genome alignment are challenging.

Results

To greatly simplify the analysis, we present an Assembly and Alignment-Free (AAF) method (https://sourceforge.net/projects/aaf-phylogeny) that constructs phylogenies directly from unassembled genome sequence data, bypassing both genome assembly and alignment. Using mathematical calculations, models of sequence evolution, and simulated sequencing of published genomes, we address both evolutionary and sampling issues caused by direct reconstruction, including homoplasy, sequencing errors, and incomplete sequencing coverage. From these results, we calculate the statistical properties of the pairwise distances between genomes, allowing us to optimize parameter selection and perform bootstrapping. As a test case with real data, we successfully reconstructed the phylogeny of 12 mammals using raw sequencing reads. We also applied AAF to 21 tropical tree genome datasets with low coverage to demonstrate its effectiveness on non-model organisms.

Conclusion

Our AAF method opens up phylogenomics for species without an appropriate reference genome or high sequence coverage, and rapidly creates a phylogenetic framework for further analysis of genome structure and diversity among non-model organisms.

Electronic supplementary material

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BEACON: automated tool for Bacterial GEnome Annotation ComparisON

Background

Genome annotation is one way of summarizing the existing knowledge about genomic characteristics of an organism. There has been an increased interest during the last several decades in computer-based structural and functional genome annotation. Many methods for this purpose have been developed for eukaryotes and prokaryotes. Our study focuses on comparison of functional annotations of prokaryotic genomes. To the best of our knowledge there is no fully automated system for detailed comparison of functional genome annotations generated by different annotation methods (AMs).

Results

The presence of many AMs and development of new ones introduce needs to: a/ compare different annotations for a single genome, and b/ generate annotation by combining individual ones. To address these issues we developed an Automated Tool for Bacterial GEnome Annotation ComparisON (BEACON) that benefits both AM developers and annotation analysers. BEACON provides detailed comparison of gene function annotations of prokaryotic genomes obtained by different AMs and generates extended annotations through combination of individual ones. For the illustration of BEACON’s utility, we provide a comparison analysis of multiple different annotations generated for four genomes and show on these examples that the extended annotation can increase the number of genes annotated by putative functions up to 27 %, while the number of genes without any function assignment is reduced.

Conclusions

We developed BEACON, a fast tool for an automated and a systematic comparison of different annotations of single genomes. The extended annotation assigns putative functions to many genes with unknown functions. BEACON is available under GNU General Public License version 3.0 and is accessible at: http://www.cbrc.kaust.edu.sa/BEACON/.

Electronic supplementary material

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Young,intact and nested retrotransposons are abundant in the onion and asparagus genomes

Background and Aims

Although monocotyledonous plants comprise one of the two major groups of angiosperms and include >65 000 species, comprehensive genome analysis has been focused mainly on the Poaceae (grass) family. Due to this bias, most of the conclusions that have been drawn for monocot genome evolution are based on grasses. It is not known whether these conclusions apply to many other monocots.

Methods

To extend our understanding of genome evolution in the monocots, Asparagales genomic sequence data were acquired and the structural properties of asparagus and onion genomes were analysed. Specifically, several available onion and asparagus bacterial artificial chromosomes (BACs) with contig sizes >35 kb were annotated and analysed, with a particular focus on the characterization of long terminal repeat (LTR) retrotransposons.

Key Results

The results reveal that LTR retrotransposons are the major components of the onion and garden asparagus genomes. These elements are mostly intact (i.e. with two LTRs), have mainly inserted within the past 6 million years and are piled up into nested structures. Analysis of shotgun genomic sequence data and the observation of two copies for some transposable elements (TEs) in annotated BACs indicates that some families have become particularly abundant, as high as 4–5 % (asparagus) or 3–4 % (onion) of the genome for the most abundant families, as also seen in large grass genomes such as wheat and maize.

Conclusions

Although previous annotations of contiguous genomic sequences have suggested that LTR retrotransposons were highly fragmented in these two Asparagales genomes, the results presented here show that this was largely due to the methodology used. In contrast, this current work indicates an ensemble of genomic features similar to those observed in the Poaceae.     

Comparative genomic analysis of Acinetobacter baumannii clinical isolates reveals extensive genomic variation and diverse antibiotic resistance determinants

Background

Acinetobacter baumannii is an important nosocomial pathogen that poses a serious health threat to immune-compromised patients. Due to its rapid ability to develop multidrug resistance (MDR), A. baumannii has increasingly become a focus of attention worldwide. To better understand the genetic variation and antibiotic resistance mechanisms of this bacterium at the genomic level, we reported high-quality draft genome sequences of 8 clinical isolates with various sequence types and drug susceptibility profiles.

Results

We sequenced 7 MDR and 1 drug-sensitive clinical A. baumannii isolates and performed comparative genomic analysis of these draft genomes with 16 A. baumannii complete genomes from GenBank. We found a high degree of variation in A. baumannii, including single nucleotide polymorphisms (SNPs) and large DNA fragment variations in the AbaR-like resistance island (RI) regions, the prophage and the type VI secretion system (T6SS). In addition, we found several new AbaR-like RI regions with highly variable structures in our MDR strains. Interestingly, we found a novel genomic island (designated as GI_BJ4) in the drug-sensitive strain BJ4 carrying metal resistance genes instead of antibiotic resistance genes inserted into the position where AbaR-like RIs commonly reside in other A. baumannii strains. Furthermore, we showed that diverse antibiotic resistance determinants are present outside the RIs in A. baumannii, including antibiotic resistance-gene bearing integrons, the bla_OXA-23-containing transposon Tn2009, and chromosomal intrinsic antibiotic resistance genes.

Conclusions

Our comparative genomic analysis revealed that extensive genomic variation exists in the A. baumannii genome. Transposons, genomic islands and point mutations are the main contributors to the plasticity of the A. baumannii genome and play critical roles in facilitating the development of antibiotic resistance in the clinical isolates.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1163) contains supplementary material, which is available to authorized users.     

Complete genome sequence and comparative genomic analyses of the vancomycin-producing Amycolatopsis orientalis

Background

Amycolatopsis orientalis is the type species of the genus and its industrial strain HCCB10007, derived from ATCC 43491, has been used for large-scale production of the vital antibiotic vancomycin. However, to date, neither the complete genomic sequence of this species nor a systemic characterization of the vancomycin biosynthesis cluster (vcm) has been reported. With only the whole genome sequence of Amycolatopsis mediterranei available, additional complete genomes of other species may facilitate intra-generic comparative analysis of the genus.

Results

The complete genome of A. orientalis HCCB10007 comprises an 8,948,591-bp circular chromosome and a 33,499-bp dissociated plasmid. In total, 8,121 protein-coding sequences were predicted, and the species-specific genomic features of A. orientalis were analyzed in comparison with that of A. mediterranei. The common characteristics of Amycolatopsis genomes were revealed via intra- and inter-generic comparative genomic analyses within the domain of actinomycetes, and led directly to the development of sequence-based Amycolatopsis molecular chemotaxonomic characteristics (MCCs). The chromosomal core/quasi-core and non-core configurations of the A. orientalis and the A. mediterranei genome were analyzed reciprocally, with respect to further understanding both the discriminable criteria and the evolutionary implementation. In addition, 26 gene clusters related to secondary metabolism, including the 64-kb vcm cluster, were identified in the genome. Employing a customized PCR-targeting-based mutagenesis system along with the biochemical identification of vancomycin variants produced by the mutants, we were able to experimentally characterize a halogenase, a methyltransferase and two glycosyltransferases encoded in the vcm cluster. The broad substrate spectra characteristics of these modification enzymes were inferred.

Conclusions

This study not only extended the genetic knowledge of the genus Amycolatopsis and the biochemical knowledge of vcm-related post-assembly tailoring enzymes, but also developed methodology useful for in vivo studies in A. orientalis, which has been widely considered as a barrier in this field.

Electronic supplementary material

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CoryneBase: Corynebacterium Genomic Resources and Analysis Tools at Your Fingertips

Corynebacteria are used for a wide variety of industrial purposes but some species are associated with human diseases. With increasing number of corynebacterial genomes having been sequenced, comparative analysis of these strains may provide better understanding of their biology, phylogeny, virulence and taxonomy that may lead to the discoveries of beneficial industrial strains or contribute to better management of diseases. To facilitate the ongoing research of corynebacteria, a specialized central repository and analysis platform for the corynebacterial research community is needed to host the fast-growing amount of genomic data and facilitate the analysis of these data. Here we present CoryneBase, a genomic database for Corynebacterium with diverse functionality for the analysis of genomes aimed to provide: (1) annotated genome sequences of Corynebacterium where 165,918 coding sequences and 4,180 RNAs can be found in 27 species; (2) access to comprehensive Corynebacterium data through the use of advanced web technologies for interactive web interfaces; and (3) advanced bioinformatic analysis tools consisting of standard BLAST for homology search, VFDB BLAST for sequence homology search against the Virulence Factor Database (VFDB), Pairwise Genome Comparison (PGC) tool for comparative genomic analysis, and a newly designed Pathogenomics Profiling Tool (PathoProT) for comparative pathogenomic analysis. CoryneBase offers the access of a range of Corynebacterium genomic resources as well as analysis tools for comparative genomics and pathogenomics. It is publicly available at http://corynebacterium.um.edu.my/.     

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae)

Background and Aims

The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification.

Methods

A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100–500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling.

Key Results

Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S–5·8S–25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species.

Conclusions

The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.     

Genome analysis and in vivo virulence of porcine extraintestinal pathogenic Escherichia coli strain PCN033

Background

Strains of extraintestinal pathogenic Escherichia coli (ExPEC) can invade and colonize extraintestinal sites and cause a wide range of infections. Genomic analysis of ExPEC has mainly focused on isolates of human and avian origins, with porcine ExPEC isolates yet to be sequenced. To better understand the genomic attributes underlying the pathogenicity of porcine ExPEC, we isolated two E. coli strains PCN033 and PCN061 from pigs, assessed their in vivo virulence, and completed and compared their genomes.

Results

Animal experiments demonstrated that strain PCN033, but not PCN061, was pathogenic in a pig model. The chromosome of PCN033 was 384 kb larger than that of PCN061. Among the PCN033-specific sequences, genes encoding adhesins, unique lipopolysaccharide, unique capsular polysaccharide, iron acquisition and transport systems, and metabolism were identified. Additionally, a large plasmid PCN033p3 harboring many typical ExPEC virulence factors was identified in PCN033. Based on the genetic variation between PCN033 and PCN061, corresponding phenotypic differences in flagellum-dependent swarming motility and metabolism were verified. Furthermore, the comparative genomic analyses showed that the PCN033 genome shared many similarities with genomic sequences of human ExPEC strains. Additionally, comparison of PCN033 genome with other nine characteristic E. coli genomes revealed 425 PCN033-special coding sequences. Genes of this subset included those encoding type I restriction-modification (R-M) system, type VI secretion system (T6SS) and membrane-associated proteins.

Conclusions

The genetic and phenotypic differences between PCN033 and PCN061 could partially explain their differences in virulence, and also provide insight towards the molecular mechanisms of porcine ExPEC infections. Additionally, the similarities between the genomes of PCN033 and human ExPEC strains suggest that some connections between porcine and human ExPEC strains exist. The first completed genomic sequence for porcine ExPEC and the genomic differences identified by comparative analyses provide a baseline understanding of porcine ExPEC genetics and lay the foundation for their further study.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1890-9) contains supplementary material, which is available to authorized users.     

New implications on genomic adaptation derived from the Helicobacter pylori genome comparison

Background

Helicobacter pylori has a reduced genome and lives in a tough environment for long-term persistence. It evolved with its particular characteristics for biological adaptation. Because several H. pylori genome sequences are available, comparative analysis could help to better understand genomic adaptation of this particular bacterium.

Principal Findings

We analyzed nine H. pylori genomes with emphasis on microevolution from a different perspective. Inversion was an important factor to shape the genome structure. Illegitimate recombination not only led to genomic inversion but also inverted fragment duplication, both of which contributed to the creation of new genes and gene family, and further, homological recombination contributed to events of inversion. Based on the information of genomic rearrangement, the first genome scaffold structure of H. pylori last common ancestor was produced. The core genome consists of 1186 genes, of which 22 genes could particularly adapt to human stomach niche. H. pylori contains high proportion of pseudogenes whose genesis was principally caused by homopolynucleotide (HPN) mutations. Such mutations are reversible and facilitate the control of gene expression through the change of DNA structure. The reversible mutations and a quasi-panmictic feature could allow such genes or gene fragments frequently transferred within or between populations. Hence, pseudogenes could be a reservoir of adaptation materials and the HPN mutations could be favorable to H. pylori adaptation, leading to HPN accumulation on the genomes, which corresponds to a special feature of Helicobacter species: extremely high HPN composition of genome.

Conclusion

Our research demonstrated that both genome content and structure of H. pylori have been highly adapted to its particular life style.     

TE-Tracker: systematic identification of transposition events through whole-genome resequencing

Background

Transposable elements (TEs) are DNA sequences that are able to move from their location in the genome by cutting or copying themselves to another locus. As such, they are increasingly recognized as impacting all aspects of genome function. With the dramatic reduction in cost of DNA sequencing, it is now possible to resequence whole genomes in order to systematically characterize novel TE mobilization in a particular individual. However, this task is made difficult by the inherently repetitive nature of TE sequences, which in some eukaryotes compose over half of the genome sequence. Currently, only a few software tools dedicated to the detection of TE mobilization using next-generation-sequencing are described in the literature. They often target specific TEs for which annotation is available, and are only able to identify families of closely related TEs, rather than individual elements.

Results

We present TE-Tracker, a general and accurate computational method for the de-novo detection of germ line TE mobilization from re-sequenced genomes, as well as the identification of both their source and destination sequences. We compare our method with the two classes of existing software: specialized TE-detection tools and generic structural variant (SV) detection tools. We show that TE-Tracker, while working independently of any prior annotation, bridges the gap between these two approaches in terms of detection power. Indeed, its positive predictive value (PPV) is comparable to that of dedicated TE software while its sensitivity is typical of a generic SV detection tool. TE-Tracker demonstrates the benefit of adopting an annotation-independent, de novo approach for the detection of TE mobilization events. We use TE-Tracker to provide a comprehensive view of transposition events induced by loss of DNA methylation in Arabidopsis. TE-Tracker is freely available at http://www.genoscope.cns.fr/TE-Tracker.

Conclusions

We show that TE-Tracker accurately detects both the source and destination of novel transposition events in re-sequenced genomes. Moreover, TE-Tracker is able to detect all potential donor sequences for a given insertion, and can identify the correct one among them. Furthermore, TE-Tracker produces significantly fewer false positives than common SV detection programs, thus greatly facilitating the detection and analysis of TE mobilization events.

Electronic supplementary material

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Comparative genomics of 274 Vibrio cholerae genomes reveals mobile functions structuring three niche dimensions

Background

Vibrio cholerae is a globally dispersed pathogen that has evolved with humans for centuries, but also includes non-pathogenic environmental strains. Here, we identify the genomic variability underlying this remarkable persistence across the three major niche dimensions space, time, and habitat.

Results

Taking an innovative approach of genome-wide association applicable to microbial genomes (GWAS-M), we classify 274 complete V. cholerae genomes by niche, including 39 newly sequenced for this study with the Ion Torrent DNA-sequencing platform. Niche metadata were collected for each strain and analyzed together with comprehensive annotations of genetic and genomic attributes, including point mutations (single-nucleotide polymorphisms, SNPs), protein families, functions and prophages.

Conclusions

Our analysis revealed that genomic variations, in particular mobile functions including phages, prophages, transposable elements, and plasmids underlie the metadata structuring in each of the three niche dimensions. This underscores the role of phages and mobile elements as the most rapidly evolving elements in bacterial genomes, creating local endemicity (space), leading to temporal divergence (time), and allowing the invasion of new habitats. Together, we take a data-driven approach for comparative functional genomics that exploits high-volume genome sequencing and annotation, in conjunction with novel statistical and machine learning analyses to identify connections between genotype and phenotype on a genome-wide scale.

Electronic supplementary material

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Characterization of the core and accessory genomes of Pseudomonas aeruginosa using bioinformatic tools Spine and AGEnt

Background

Pseudomonas aeruginosa is an important opportunistic pathogen responsible for many infections in hospitalized and immunocompromised patients. Previous reports estimated that approximately 10% of its 6.6 Mbp genome varies from strain to strain and is therefore referred to as “accessory genome”. Elements within the accessory genome of P. aeruginosa have been associated with differences in virulence and antibiotic resistance. As whole genome sequencing of bacterial strains becomes more widespread and cost-effective, methods to quickly and reliably identify accessory genomic elements in newly sequenced P. aeruginosa genomes will be needed.

Results

We developed a bioinformatic method for identifying the accessory genome of P. aeruginosa. First, the core genome was determined based on sequence conserved among the completed genomes of twelve reference strains using Spine, a software program developed for this purpose. The core genome was 5.84 Mbp in size and contained 5,316 coding sequences. We then developed an in silico genome subtraction program named AGEnt to filter out core genomic sequences from P. aeruginosa whole genomes to identify accessory genomic sequences of these reference strains. This analysis determined that the accessory genome of P. aeruginosa ranged from 6.9-18.0% of the total genome, was enriched for genes associated with mobile elements, and was comprised of a majority of genes with unknown or unclear function. Using these genomes, we showed that AGEnt performed well compared to other publically available programs designed to detect accessory genomic elements. We then demonstrated the utility of the AGEnt program by applying it to the draft genomes of two previously unsequenced P. aeruginosa strains, PA99 and PA103.

Conclusions

The P. aeruginosa genome is rich in accessory genetic material. The AGEnt program accurately identified the accessory genomes of newly sequenced P. aeruginosa strains, even when draft genomes were used. As P. aeruginosa genomes become available at an increasingly rapid pace, this program will be useful in cataloging the expanding accessory genome of this bacterium and in discerning correlations between phenotype and accessory genome makeup. The combination of Spine and AGEnt should be useful in defining the accessory genomes of other bacterial species as well.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-737) contains supplementary material, which is available to authorized users.