Comparative genomics analysis of metallothioneins in twelve Drosophila species

Metallothioneins (MTs) are a superfamily of Cys-rich polypeptides that bind heavy metal ions, both for physiological and detoxification purposes. They are present in all organisms, but their origin is probably polyphyletic, so that MT evolutionary studies are rather scarce. We present a thorough search and analysis of the MT coding sequences in the 12 Drosophila genomes completely sequenced, taking as reference the features reported for D. melanogaster, where four isogenes (MtnA to MtnD) are known and deeply characterized. We include a fifth isoform in this study, named MtnE, and recently annotated. The MTs polymorphism pattern is essentially the same for the 12 Drosophila species. Invariably, a MtnA form and an MtnB-cluster, comprising the MtnB-to-MtnE forms in tandem array, are observed. The whole set of genes are kept in the same synteny element (Muller E), but implicated in rearrangement events (mainly inversions), encompassing all or some of the isogenes. Gene exon/intron architecture, and cDNA and protein sequences appear highly conserved through Drosophila speciation, concordantly with an essential function for MT isoforms in flies, even for those previously considered as minor products. Data presented here will be comprehensively analyzed to provide a valuable guide for future MT evolutionary, structure and function studies.     

Comparative genomics of Riemerella anatipestifer reveals genetic diversity

Background

Riemerella anatipestifer is one of the most important pathogens of ducks. However, the molecular mechanisms of R. anatipestifer infection are poorly understood. In particular, the lack of genomic information from a variety of R. anatipestifer strains has proved severely limiting.

Results

In this study, we present the complete genomes of two R. anatipestifer strains, RA-CH-1 (2,309,519 bp, Genbank accession {"type":"entrez-nucleotide","attrs":{"text":"CP003787","term_id":"403311769","term_text":"CP003787"}}CP003787) and RA-CH-2 (2,166,321 bp, Genbank accession {"type":"entrez-nucleotide","attrs":{"text":"CP004020","term_id":"441482619","term_text":"CP004020"}}CP004020). Both strains are from isolates taken from two different sick ducks in the SiChuang province of China. A comparative genomics approach was used to identify similarities and key differences between RA-CH-1 and RA-CH-2 and the previously sequenced strain RA-GD, a clinical isolate from GuangDong, China, and ATCC11845.

Conclusion

The genomes of RA-CH-2 and RA-GD were extremely similar, while RA-CH-1 was significantly different than ATCC11845. RA-CH-1 is 140,000 bp larger than the three other strains and has 16 unique gene families. Evolutionary analysis shows that RA-CH-1 and RA-CH-2 are closed and in a branch with ATCC11845, while RA-GD is located in another branch. Additionally, the detection of several iron/heme-transport related proteins and motility mechanisms will be useful in elucidating factors important in pathogenicity. This information will allow a better understanding of the phenotype of different R. anatipestifer strains and molecular mechanisms of infection.     

Comparative genomics of yeast species: new insights into their biology

The genomes of two hemiascomycetous yeasts (Saccharomyces cerevisiae and Candida albicans) and one archiascomycete (Schizosaccharomyces pombe) have been completely sequenced and the genes have been annotated. In addition, the genomes of 13 more Hemiascomycetes have been partially sequenced. The amount of data thus obtained provides information on the evolutionary relationships between yeast species. In addition, the differential genetic characteristics of the microorganisms explain a number of distinctive biological traits. Gene order conservation is observed between phylogenetically close species and is lost in distantly related species, probably due to rearrangements of short regions of DNA. However, gene function is much more conserved along evolution. Compared to S. cerevisiae and S. pombe, C. albicans has a larger number of specific genes, i.e., genes not found in other organisms, a fact that can account for the biological characteristics of this pathogenic dimorphic yeast which is able to colonize a large variety of environments.     

From phenotype to genotype: a Bayesian solution

The study of biological systems commonly depends on inferring the state of a 'hidden' variable, such as an underlying genotype, from that of an 'observed' variable, such as an expressed phenotype. However, this cannot be achieved using traditional quantitative methods when more than one genetic mechanism exists for a single observable phenotype. Using a novel latent class Bayesian model, it is possible to infer the prevalence of different genetic elements in a population given a sample of phenotypes. As an exemplar, data comprising phenotypic resistance to six antimicrobials obtained from passive surveillance of Salmonella Typhimurium DT104 are analysed to infer the prevalence of individual resistance genes, as well as the prevalence of a genomic island known as SGI1 and its variants. Three competing models are fitted to the data and distinguished between using posterior predictive p-values to assess their ability to predict the observed number of unique phenotypes. The results suggest that several SGI1 variants circulate in a few fixed forms through the population from which our data were derived. The methods presented could be applied to other types of phenotypic data, and represent a useful and generic mechanism of inferring the genetic population structure of organisms.     

Comparative genomics of the Bifidobacterium breve taxon

Background

Bifidobacteria are commonly found as part of the microbiota of the gastrointestinal tract (GIT) of a broad range of hosts, where their presence is positively correlated with the host’s health status. In this study, we assessed the genomes of thirteen representatives of Bifidobacterium breve, which is not only a frequently encountered component of the (adult and infant) human gut microbiota, but can also be isolated from human milk and vagina.

Results

In silico analysis of genome sequences from thirteen B. breve strains isolated from different environments (infant and adult faeces, human milk, human vagina) shows that the genetic variability of this species principally consists of hypothetical genes and mobile elements, but, interestingly, also genes correlated with the adaptation to host environment and gut colonization. These latter genes specify the biosynthetic machinery for sortase-dependent pili and exopolysaccharide production, as well as genes that provide protection against invasion of foreign DNA (i.e. CRISPR loci and restriction/modification systems), and genes that encode enzymes responsible for carbohydrate fermentation. Gene-trait matching analysis showed clear correlations between known metabolic capabilities and characterized genes, and it also allowed the identification of a gene cluster involved in the utilization of the alcohol-sugar sorbitol.

Conclusions

Genome analysis of thirteen representatives of the B. breve species revealed that the deduced pan-genome exhibits an essentially close trend. For this reason our analyses suggest that this number of B. breve representatives is sufficient to fully describe the pan-genome of this species. Comparative genomics also facilitated the genetic explanation for differential carbon source utilization phenotypes previously observed in different strains of B. breve.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-170) contains supplementary material, which is available to authorized users.     

Functional genomics of hsp-90 in parasitic and free-living nematodes

Heat shock protein 90 (Hsp-90) is a highly conserved essential protein in eukaryotes. Here we describe the molecular characterisation of hsp-90 from three nematodes, the free-living Caenorhabditis elegans (Ce) and the parasitic worms Brugia pahangi (Bp) and Haemonchus contortus (Hc). These molecules were functionally characterised by rescue of a Ce-daf-21 (hsp-90) null mutant. Our results show a gradient of rescue: the C. elegans endogenous gene provided full rescue of the daf-21 mutant, while Hc-hsp-90 provided partial rescue. In contrast, no rescue could be obtained using a variety of Bp-hsp-90 constructs, despite the fact that Bp-hsp-90 was transcribed and translated in the mutant worms. daf-21 RNA interference (RNAi) experiments were carried out to determine whether knock-down of the endogenous daf-21 mRNA in N2 worms could be complemented by expression of either parasite gene. However neither parasite gene could rescue the daf-21 (RNAi) phenotypes. These results indicate that factors other than the level of sequence identity are important for determining whether parasite genes can functionally complement in C. elegans.     

Comparative transcriptional profiling and evolutionary analysis of the GRAM domain family in eukaryotes

The GRAM domain was found in glucosyltransferases, myotubularins and other membrane-associated proteins. So far, functions for majority of these proteins are yet to be uncovered. In order to address the evolutionary and functional significance of this family members, we have performed a comprehensive investigation on their genome-wide identification, phylogenetic relationship and expression divergence in five different organisms representing monocot/dicot plants, vertebrate/invertebrate animals and yeast, namely, Oryza sativa, Arabidopsis thaliana, Mus musculus, Drosophila melanogaster and Saccharomyces cerevisiae, respectively. We have identified 65 members of GRAM domain family from these organisms. Our data revealed that this family was an ancient group and various organisms had evolved into different family sizes. Large-scale genome duplication and divergence in both expression patterns and functions were significantly contributed to the expansion and retention of this family. Mouse and Drosophila members showed higher divergences in their proteins as indicated by higher Ka/Ks ratios and possessed multiple domains in various combinations. However, in plants, their protein functions were possibly retained with a relatively low divergence as signified by lower Ka/Ks ratios and only one additional domain was combined during evolution. On the other hand, this family in all five organisms exhibited high divergence in their expression patterns both at tissue level and under various biotic and abiotic stresses. These highly divergent expression patterns unraveled the complexity of functions of GRAM domain family. Each member may play specialized roles in a specific tissue or stress condition and may function as regulators of environmental and hormonal signaling.     

Bursts of transposition from non-long terminal repeat retrotransposon families of the RTE clade in Schistosoma mansoni

The genus Schistosoma is composed of blood flukes that infect vertebrates, from which three species are major causative agents of human schistosomiasis, a tropical disease that affects more than 200 million people. Current models of the recent evolution of Schistosoma indicate multiple events of migration and speciation from an Asian ancestral species. Transposable elements are important drivers of genome evolution and have been hypothesised to have an important role in speciation. In this work, we describe a comprehensive inventory of Schistosoma mansoni and Schistosoma japonicum retrotransposons, based on their recently published genomic data. We find a considerable difference in retrotransposon representation between the two species (22% and 13%, respectively). A large part of this difference can be attributed to higher representation of two previously described families of S. mansoni retrotransposons (SR2 and Perere-3/SR3), compared with the representation of their closest relative families in S. japonicum. A more detailed analysis suggests that these two S. mansoni families were the subject of recent bursts of transposition that were not paralleled by their S. japonicum counterparts. We hypothesise that these bursts could be a consequence of the evolutionary pressure resulting from migration of Schistosoma from Asia to Africa and their establishment in this new environment, helping both speciation and adaptation.     

Comparative proteomics profiling of a gentamicin-attenuated Leishmania infantum cell line identifies key changes in parasite thiol-redox metabolism

We have previously described an attenuated line of Leishmania infantum (H-line), selected by culturing promastigotes in vitro in the presence of gentamicin. To elucidate the molecular basis for this attenuation, we undertook a comparative proteomic analysis using multiplex 2-dimensional (2D) difference gel electrophoresis. Eighteen proteins that showed significant and reproducible changes in expression were identified. Many of these were components of the thiol-redox control system in Leishmania and this observation, validated by Western blot, prompted us to investigate the sensitivity of the attenuated line to oxidative stress. The attenuated line was found to be significantly more susceptible to hydrogen peroxide, a change which may explain the loss of virulence. In a direct assay of trypanothione-dependent peroxidase activity, hydrogen peroxide metabolism in the H-line was significantly lower than in wild type. Furthermore, trypanothione reductase activity was significantly lower in the H-line, suggesting that gentamicin selection may result in pleiotropic affects on thiol metabolism in Leishmania. A putative RNA-binding protein was very strongly up-regulated in the attenuated line, suggesting a possible target for gentamicin in Leishmania.     

Comparative genomics of first available bovine Anaplasma phagocytophilum genome obtained with targeted sequence capture

Background

Anaplasma phagocytophilum is a zoonotic and obligate intracellular bacterium transmitted by ticks. In domestic ruminants, it is the causative agent of tick-borne fever, which causes significant economic losses in Europe. As A. phagocytophilum is difficult to isolate and cultivate, only nine genome sequences have been published to date, none of which originate from a bovine strain.Our goals were to; 1/ develop a sequencing methodology which efficiently circumvents the difficulties associated with A. phagocytophilum isolation and culture; 2/ describe the first genome of a bovine strain; and 3/ compare it with available genomes, in order to both explore key genomic features at the species level, and to identify candidate genes that could be specific to bovine strains.

Results

DNA was extracted from a bovine blood sample infected by A. phagocytophilum. Following a whole genome capture approach, A. phagocytophilum DNA was enriched 197-fold in the sample and then sequenced using Illumina technology. In total, 58.9% of obtained reads corresponded to the A. phagocytophilum genome, covering 85.3% of the HZ genome. Then by performing comparisons with nine previously-sequenced A. phagocytophilum genomes, we determined the core genome of these ten strains. Following analysis, 1281 coding DNA sequences, including 1001 complete sequences, were detected in the A. phagocytophilum bovine genome, of which four appeared to be unique to the bovine isolate. These four coding DNA sequences coded for "hypothetical proteins of unknown function” and require further analysis. We also identified nine proteins common to both European domestic ruminants tested.

Conclusion

Using a whole genome capture approach, we have sequenced the first A. phagocytophilum genome isolated from a cow. To the best of our knowledge, this is the first time that this method has been used to selectively enrich pathogenic bacterial DNA from samples also containing host DNA. The four proteins unique to the A. phagocytophilum bovine genome could be involved in host tropism, therefore their functions need to be explored.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-973) contains supplementary material, which is available to authorized users.     

Parasites, emerging disease and wildlife conservation

In this review some emerging issues of parasite infections in wildlife, particularly in Australia, are considered. We discuss the importance of understanding parasite biodiversity in wildlife in terms of conservation, the role of wildlife as reservoirs of parasite infection, and the role of parasites within the broader context of the ecosystem. Using a number of parasite species, the value of undertaking longitudinal surveillance in natural systems using non-invasive sampling and molecular tools to characterise infectious agents is illustrated in terms of wildlife health, parasite biodiversity and ecology.     

Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms

This is the first report of cutaneous leishmaniasis in kangaroos where infection was acquired within Australia. The diagnosis is based on the clinical criteria used for humans, the lesion histopathology, the detection and isolation of parasites from the lesions, and the analysis of the small subunit ribosomal RNA genes using the polymerase chain reaction. Despite a clear indication that the parasites belong to the genus Leishmania, no assignation to a known Leishmania species could be made using these or other less conserved genetic loci such as the non-transcribed spacer of the mini-exon repeat. As is the case in humans, some but not all animals harbouring lesions had antibodies to the isolated parasites or to several other Leishmania species. The isolated parasites displayed two well characterised Leishmania glycoconjugates, the lipophosphoglycan and proteophosphoglycan. They were infectious for mouse macrophages in vitro and established long-term infection at 33 degrees C but not at 37 degrees C. Our findings raise the possibility of transmission to humans, which may be unrecognised and suggest the possibility that imported species of Leishmania could become endemic in Australia.     

Imaging of the host/parasite interplay in cutaneous leishmaniasis

An understanding of host-parasite interplay is essential for the development of therapeutics and vaccines. Immunoparasitologists have learned a great deal from ‘conventional’ in vitro and in vivo approaches, but recent developments in imaging technologies have provided us (immunologists and parasitologists) with the ability to ask new and exciting questions about the dynamic nature of the parasite-immune system interface. These studies are providing us with new insights into the mechanisms involved in the initiation of a Leishmania infection and the consequent induction and regulation of the immune response. Here, we review some of the recent developments and discuss how these observations can be further developed to understand the immunology of cutaneous Leishmania infection in vivo.     

Comparative genomics reveals expansion of the FLC region in the genus Arabidopsis

Mechanisms of genome evolution are poorly understood although recent genome sequencing is providing the tools to begin to illuminate such mechanisms. Using high-resolution molecular cytogenetic tools, we examined the structural evolution of 790 kb surrounding the evolutionarily important FLC locus of Arabidopsis thaliana in three of its relatives, Arabidopsis halleri, Arabidopsis neglecta and Arabidopsis arenosa. Sequenced BACs from A. thaliana were used as heterologous probes across these species and genome expansion was found in all three species relative to A. thaliana, ranging from 16 to 27%. Expansion was seen along the length of the entire region but molecular analyses revealed no characteristic pattern of either intra- or intergenic expansion among these species. Mapping of BACs on DNA fibers from A. thaliana revealed one possible error, ~14 kb missing from the reported sequence, indicating that for comparative studies it is important to confirm the reference sequence to which comparison will be made.     

Virulence related sequences; insights provided by comparative genomics of Streptococcus uberis of differing virulence

Background

Streptococcus uberis, a Gram-positive, catalase-negative member of the family Streptococcaceae is an important environmental pathogen responsible for a significant proportion of subclinical and clinical bovine intramammary infections. Currently, the genome of only a single reference strain (0140J) has been described. Here we present a comparative analysis of complete draft genome sequences of an additional twelve S. uberis strains.

Results

Pan and core genome analysis revealed the core genome common to all strains to be 1,550 genes in 1,509 orthologous clusters, complemented by 115-246 accessory genes present in one or more S. uberis strains but absent in the reference strain 0140J. Most of the previously predicted virulent genes were present in the core genome of all 13 strains but gene gain/loss was observed between the isolates in CDS associated with clustered regularly interspaced short palindromic repeats (CRISPRs), prophage and bacteriocin production. Experimental challenge experiments confirmed strain EF20 as non-virulent; only able to infect in a transient manner that did not result in clinical mastitis. Comparison of the genome sequence of EF20 with the validated virulent strain 0140J identified genes associated with virulence, however these did not relate clearly with clinical/non-clinical status of infection.

Conclusion

The gain/loss of mobile genetic elements such as CRISPRs and prophage are a potential driving force for evolutionary change. This first “whole-genome” comparison of strains isolated from clinical vs non-clinical intramammary infections including the type virulent vs non-virulent strains did not identify simple gene gain/loss rules that readily explain, or be confidently associated with, differences in virulence. This suggests that a more complex dynamic determines infection potential and clinical outcome not simply gene content.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1512-6) contains supplementary material, which is available to authorized users.     

Comparative genomic analysis of Ralstonia solanacearum reveals candidate genes for host specificity

Background

Ralstonia solanacearum is a vascular soil-borne plant pathogen with an unusually broad host range. This economically destructive and globally distributed bacterium has thousands of distinct lineages within a heterogeneous and taxonomically disputed species complex. Some lineages include highly host-adapted strains (ecotypes), such as the banana Moko disease-causing strains, the cold-tolerant potato brown rot strains (also known as R3bv2) and the recently emerged Not Pathogenic to Banana (NPB) strains.

Results

These distinct ecotypes offer a robust model to study host adaptation and the emergence of ecotypes because the polyphyletic Moko strains include lineages that are phylogenetically close to the monophyletic brown rot and NPB strains. Draft genomes of eight new strains belonging to these three model ecotypes were produced to complement the eleven publicly available R. solanacearum genomes. Using a suite of bioinformatics methods, we searched for genetic and evolutionary features that distinguish ecotypes and propose specific hypotheses concerning mechanisms of host adaptation in the R. solanacearum species complex. Genome-wide, few differences were identified, but gene loss events, non-synonymous polymorphisms, and horizontal gene transfer were identified among type III effectors and were associated with host range differences.

Conclusions

This extensive comparative genomics analysis uncovered relatively few divergent features among closely related strains with contrasting biological characteristics; however, several virulence factors were associated with the emergence of Moko, NPB and brown rot and could explain host adaptation.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1474-8) contains supplementary material, which is available to authorized users.