Enrichment of sequencing targets from the human genome by solution hybridization

Background

Genome sequences, now available for most pathogens, hold promise for the rational design of new therapies. However, biological resources for genome-scale identification of gene function (notably genes involved in pathogenesis) and/or genes essential for cell viability, which are necessary to achieve this goal, are often sorely lacking. This holds true for Neisseria meningitidis, one of the most feared human bacterial pathogens that causes meningitis and septicemia.

Results

By determining and manually annotating the complete genome sequence of a serogroup C clinical isolate of N. meningitidis (strain 8013) and assembling a library of defined mutants in up to 60% of its non-essential genes, we have created NeMeSys, a biological resource for Neisseria meningitidis systematic functional analysis. To further enhance the versatility of this toolbox, we have manually (re)annotated eight publicly available Neisseria genome sequences and stored all these data in a publicly accessible online database. The potential of NeMeSys for narrowing the gap between sequence and function is illustrated in several ways, notably by performing a functional genomics analysis of the biogenesis of type IV pili, one of the most widespread virulence factors in bacteria, and by identifying through comparative genomics a complete biochemical pathway (for sulfur metabolism) that may potentially be important for nasopharyngeal colonization.

Conclusions

By improving our capacity to understand gene function in an important human pathogen, NeMeSys is expected to contribute to the ongoing efforts aimed at understanding a prokaryotic cell comprehensively and eventually to the design of new therapies.     

A Computational Approach towards a Gene Regulatory Network for the Developing Nematostella vectensis Gut

Background

The starlet sea anemone Nematostella vectensis is a diploblastic cnidarian that expresses a set of conserved genes for gut formation during its early development. During the last decade, the spatial distribution of many of these genes has been visualized with RNA hybridization or protein immunolocalization techniques. However, due to N. vectensis'' curved and changing morphology, quantification of these spatial data is problematic. A method is developed for two-dimensional gene expression quantification, which enables a numerical analysis and dynamic modeling of these spatial patterns.

Methods/Result

In this work, first standardized gene expression profiles are generated from publicly available N. vectensis embryo images that display mRNA and/or protein distributions. Then, genes expressed during gut formation are clustered based on their expression profiles, and further grouped based on temporal appearance of their gene products in embryonic development. Representative expression profiles are manually selected from these clusters, and used as input for a simulation-based optimization scheme. This scheme iteratively fits simulated profiles to the selected profiles, leading to an optimized estimation of the model parameters. Finally, a preliminary gene regulatory network is derived from the optimized model parameters.

Outlook

While the focus of this study is N. vectensis, the approach outlined here is suitable for inferring gene regulatory networks in the embryonic development of any animal, thus allowing to comparatively study gene regulation of gut formation in silico across various species.     

The genome of the truffle-parasite Tolypocladium ophioglossoides and the evolution of antifungal peptaibiotics

Background

Two major mycoparasitic lineages, the family Hypocreaceae and the genus Tolypocladium, exist within the fungal order, Hypocreales. Peptaibiotics are a group of secondary metabolites almost exclusively described from Trichoderma species of Hypocreaceae. Peptaibiotics are produced by nonribosomal peptide synthetases (NRPSs) and have antibiotic and antifungal activities. Tolypocladium species are mainly truffle parasites, but a few species are insect pathogens.

Results

The draft genome sequence of the truffle parasite Tolypocladium ophioglossoides was generated and numerous secondary metabolite clusters were discovered, many of which have no known putative product. However, three large peptaibiotic gene clusters were identified using phylogenetic analyses. Peptaibiotic genes are absent from the predominantly plant and insect pathogenic lineages of Hypocreales, and are therefore exclusive to the largely mycoparasitic lineages. Using NRPS adenylation domain phylogenies and reconciliation of the domain tree with the organismal phylogeny, it is demonstrated that the distribution of these domains is likely not the product of horizontal gene transfer between mycoparasitic lineages, but represents independent losses in insect pathogenic lineages. Peptaibiotic genes are less conserved between species of Tolypocladium and are the product of complex patterns of lineage sorting and module duplication. In contrast, these genes are more conserved within the genus Trichoderma and consistent with diversification through speciation.

Conclusions

Peptaibiotic NRPS genes are restricted to mycoparasitic lineages of Hypocreales, based on current sampling. Phylogenomics and comparative genomics can provide insights into the evolution of secondary metabolite genes, their distribution across a broader range of taxa, and their possible function related to host specificity.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1777-9) contains supplementary material, which is available to authorized users.     

A genomic survey of proteases in Aspergilli

Background

Proteases can hydrolyze peptides in aqueous environments. This property has made proteases the most important industrial enzymes by taking up about 60% of the total enzyme market. Microorganisms are the main sources for industrial protease production due to their high yield and a wide range of biochemical properties. Several Aspergilli have the ability to produce a variety of proteases, but no comprehensive comparative study has been carried out on protease productivity in this genus so far.

Results

We have performed a combined analysis of comparative genomics, proteomics and enzymology tests on seven Aspergillus species grown on wheat bran and sugar beet pulp. Putative proteases were identified by homology search and Pfam domains. These genes were then clusters based on orthology and extracellular proteases were identified by protein subcellular localization prediction. Proteomics was used to identify the secreted enzymes in the cultures, while protease essays with and without inhibitors were performed to determine the overall protease activity per protease class. All this data was then integrated to compare the protease productivities in Aspergilli.

Conclusions

Genomes of Aspergillus species contain a similar proportion of protease encoding genes. According to comparative genomics, proteomics and enzymatic experiments serine proteases make up the largest group in the protease spectrum across the species. In general wheat bran gives higher induction of proteases than sugar beet pulp. Interesting differences of protease activity, extracellular enzyme spectrum composition, protein occurrence and abundance were identified for species. By combining in silico and wet-lab experiments, we present the intriguing variety of protease productivity in Aspergilli.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-523) contains supplementary material, which is available to authorized users.     

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.     

Comparative population genomics reveals the domestication history of the peach,Prunus persica,and human influences on perennial fruit crops

Background

Recently, many studies utilizing next generation sequencing have investigated plant evolution and domestication in annual crops. Peach, Prunus persica, is a typical perennial fruit crop that has ornamental and edible varieties. Unlike other fruit crops, cultivated peach includes a large number of phenotypes but few polymorphisms. In this study, we explore the genetic basis of domestication in peach and the influence of humans on its evolution.

Results

We perform large-scale resequencing of 10 wild and 74 cultivated peach varieties, including 9 ornamental, 23 breeding, and 42 landrace lines. We identify 4.6 million SNPs, a large number of which could explain the phenotypic variation in cultivated peach. Population analysis shows a single domestication event, the speciation of P. persica from wild peach. Ornamental and edible peach both belong to P. persica, along with another geographically separated subgroup, Prunus ferganensis.We identify 147 and 262 genes under edible and ornamental selection, respectively. Some of these genes are associated with important biological features. We perform a population heterozygosity analysis in different plants that indicates that free recombination effects could affect domestication history. By applying artificial selection during the domestication of the peach and facilitating its asexual propagation, humans have caused a sharp decline of the heterozygote ratio of SNPs.

Conclusions

Our analyses enhance our knowledge of the domestication history of perennial fruit crops, and the dataset we generated could be useful for future research on comparative population genomics.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0415-1) contains supplementary material, which is available to authorized users.     

An efficient method for transient gene expression in monocots applied to modify the Brachypodium distachyon cell wall

Background

Agrobacterium-mediated transformation is widely used to produce insertions into plant genomes. There are a number of well-developed Agrobacterium-mediated transformation methods for dicotyledonous plants, but there are few for monocotyledonous plants.

Methods

Three hydrolase genes were transiently expressed in Brachypodium distachyon plants using specially designed vectors that express the gene product of interest and target it to the plant cell wall. Expression of functional hydrolases in genotyped plants was confirmed using western blotting, activity assays, cell wall compositional analysis and digestibility tests.

Key Results

An efficient, new, Agrobacterium-mediated approach was developed for transient gene expression in the grass B. distachyon, using co-cultivation of mature seeds with bacterial cells. This method allows transformed tissues to be obtained rapidly, within 3–4 weeks after co-cultivation. Also, the plants carried transgenic tissue and maintained transgenic protein expression throughout plant maturation. The efficiency of transformation was estimated at around 5 % of initially co-cultivated seeds. Application of this approach to express three Aspergillus nidulans hydrolases in the Brachypodium cell wall successfully confirmed its utility and resulted in the expected expression of active microbial proteins and alterations of cell wall composition. Cell wall modifications caused by expression of A. nidulans α-arabinofuranosidase and α-galactosidase increased the biodegradability of plant biomass.

Conclusions

This newly developed approach is a quick and efficient technique for expressing genes of interest in Brachypodium plants, which express the gene product throughout development. In the future, this could be used for broad functional genomics studies of monocots and for biotechnological applications, such as plant biomass modification for biofuel production.     

Enrichment of Triticum aestivum gene annotations using ortholog cliques and gene ontologies in other plants

Background

While the gargantuan multi-nation effort of sequencing T. aestivum gets close to completion, the annotation process for the vast number of wheat genes and proteins is in its infancy. Previous experimental studies carried out on model plant organisms such as A. thaliana and O. sativa provide a plethora of gene annotations that can be used as potential starting points for wheat gene annotations, proven that solid cross-species gene-to-gene and protein-to-protein correspondences are provided.

Results

DNA and protein sequences and corresponding annotations for T. aestivum and 9 other plant species were collected from Ensembl Plants release 22 and curated. Cliques of predicted 1-to-1 orthologs were identified and an annotation enrichment model was defined based on existing gene-GO term associations and phylogenetic relationships among wheat and 9 other plant species. A total of 13 cliques of size 10 were identified, which represent putative functionally equivalent genes and proteins in the 10 plant species. Eighty-five new and more specific GO terms were associated with wheat genes in the 13 cliques of size 10, which represent a 65% increase compared with the previously 130 known GO terms. Similar expression patterns for 4 genes from Arabidopsis, barley, maize and rice in cliques of size 10 provide experimental evidence to support our model. Overall, based on clique size equal or larger than 3, our model enriched the existing gene-GO term associations for 7,838 (8%) wheat genes, of which 2,139 had no previous annotation.

Conclusions

Our novel comparative genomics approach enriches existing T. aestivum gene annotations based on cliques of predicted 1-to-1 orthologs, phylogenetic relationships and existing gene ontologies from 9 other plant species.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1496-2) contains supplementary material, which is available to authorized users.     

The bovine lactation genome: insights into the evolution of mammalian milk

Background

The newly assembled Bos taurus genome sequence enables the linkage of bovine milk and lactation data with other mammalian genomes.

Results

Using publicly available milk proteome data and mammary expressed sequence tags, 197 milk protein genes and over 6,000 mammary genes were identified in the bovine genome. Intersection of these genes with 238 milk production quantitative trait loci curated from the literature decreased the search space for milk trait effectors by more than an order of magnitude. Genome location analysis revealed a tendency for milk protein genes to be clustered with other mammary genes. Using the genomes of a monotreme (platypus), a marsupial (opossum), and five placental mammals (bovine, human, dog, mice, rat), gene loss and duplication, phylogeny, sequence conservation, and evolution were examined. Compared with other genes in the bovine genome, milk and mammary genes are: more likely to be present in all mammals; more likely to be duplicated in therians; more highly conserved across Mammalia; and evolving more slowly along the bovine lineage. The most divergent proteins in milk were associated with nutritional and immunological components of milk, whereas highly conserved proteins were associated with secretory processes.

Conclusions

Although both copy number and sequence variation contribute to the diversity of milk protein composition across species, our results suggest that this diversity is primarily due to other mechanisms. Our findings support the essentiality of milk to the survival of mammalian neonates and the establishment of milk secretory mechanisms more than 160 million years ago.