Studies On Nosema Bombycis Genomics

Horizontal gene transfer (HGT), which is defined as the movement of genetic material between different organisms, cells, organelles and even DNA within the genome, plays a highly significant role in the evolution of both the species and their genome. To date, a great number of the studies on HGT in the prokaryotic genomes are finished, but the ones in the eukaryotic genomes fall behind because the methods of detecting HGT in them are hard, and just several unicellular eukaryotes identified the horizontally transferred genes. There repersents in a report claimed that fungi will be a compatible module to inquiry the eukaryotic HGT.Related to the fungi kingdom, microsporidia are a group of obligate intracellular eukaryotic parasites that infect a wide variety of species, including humans. The microsporidiosis caused by them are severe and hard to cure radically, thus microsporidia draws a great interest to the scientists around the world. Nosema bombycis, as the first discovered microsporidia, have been studied since the middle of the nineteenth century. In 2003, the genome project of Nosema bombycis was started in our laboratory. So far, a whole genome shotgun database with 7.8-fold coverage of the haploid genome, a Solexa database with 25-fold coverage, a miniBAC library with 0.3-fold coverage and a cDNA library with 11,155 expressed sequence tags were constructed successfully. Based on these databases, we carried out the analyses of horizontal gene transfer within the genome of N. bombycis.1. Identification of horizontally transferred genes in the N. bombycis genomeBased on the whole genome database of N. bombycis, we identified 55 horizontally transferred genes using the phylogenetic analysis and Darkhorse method. Forty-eight of them were confirmed by both methods, indicative of a reliable result. We randomly chose four of them to carry out the Southern blot, and the result shows that these genes exist in the genome of N. bombycis rather than the contamination.2. Characterization of horizontally transferred genes in the N. bombycis genomeThere are lots of differences between the features of horizontally transferred genes and the ones of whole genomic genes, such as nucleotide composition, codon usage bias, transcriptional activity and selection pressure. For example, the horizontally transferred genes show higher GC content, weaker codon usage bias, different optimal codon, usage and their higher GC content, lower transcription and bigger Ka/Ks value than the whole genomic genes. These results reconfirm that the N. bombycis horizontally transferred genes are exogenous.According to the status of the homologues of horizontally transferred genes in the other microsporidian genomes, we divided 55 these genes into two categories:ones before the divergence of microsporidia phylum (bHGT); while the other is after that divergence (aHGT). Moreover, this classification was supported by the above-mentioned features, the syntenic maps and the self-organizing clustering method. Compared with aHGT genes, the features of bHGT are more similar to the ones of whole genomic genes, suggesting that there is more time for bHGT genes to change themselves to adapt to their host genome, and this is the so-called amelioration process of horizontally transferred genes.3. Source species, transferred direction and transferred time of horizontally transferred genes in the N. bombycis genomeTaxonomic distribution of 55 horizontally transferred genes was identified. All of them are prokaryote/virus-to-eukaryote, but no genes origined from its host Bombyx mori were found. The majority of such entries have one or more members of the proteobacteria as the most possible origin in aHGT while firmicutes in bHGT. This distribution of N. bombycis horizontally transferred genes is widespread, where transfer events from many phylums of both Bacteria and Archaea domains of life plus Viruses are uncovered, in a highly resolved phylogenetic tree of life.All of the detected transferred directions are the HGT from other organism to microsporidia or N. bombycis. The calculated transferred time of bHGT show 357-760 MYr (million years) while the one of aHGT show 11-73 MYr. Further qualitative analysis show that the transferred time of bHGT is 1.55-fold than the one of aHGT, fully affirming the classification of bHGT and aHGT.4. Function of horizontally transferred genes in the N. bombycis genomeMost horizontally transferred genes of N. bombycis are functionally unknown, while the definitive one play role in many functions, specifically in metabolism. This phenomenon is similar with the known HGT from the genomes of Entamoeba histolytica and Trichomonas vaginalis, which can be interpreted by the hypothesis that HGT favors the acquisition of operational genes but not informational genes.Generally speaking, bHGT genes play the supplementary role in the genome of N. bombycis, while aHGT take the newly-increased function. In addition, among the KEGG metabolic maps of 55 horizontally transferred genes, we discovered that the phosphomevalonate kinase (EC 2.7.4.2) encoded by a horizontally transferred gene play an important role in the mevalonate pathway.5. Two cases of horizontally transferred genes in the N. bombycis genomeA comprehensive analysis of manganese superoxide dismutase (MnSOD) sequences from eukaryotes, Archaea and bacteria shows the microsporidial MnSODs to be grouped within bacteria MnSODs rather than eukarotic MnSODs, in terms of sequence similarity and predicted oligomeric structure and subcellular localization, indicative of a possible horizontal gene transfer.Tandem duplicated NbMnSOD2 gene seems to have changed more rapidly and is under less evolutionary constraint than the original NbMnSODl. NbMnSOD2 shows some sequence properties of tetramers and other characteristics quite different from NbMnSODl. This suggests that NbMnSOD2 may function under different conditions or in different tissues of its host rather than simply resulting in an increase in expression.The serine proteinase inhibitors (serpin) genes were found in a few organisms of many wide phylums, inferring that only the HGT can be used to explain this circumstance. Thus, we suggested that the serpin of N. bombycis had possibly come from a poxviridae according to its phylogenetic analysis.6. Transposable elements in the N. bombycis genomeAs the horizontal gene transfer within the genome, transposable elements took account of 36.12%of whole genome sequences in the N. bombycis genome, suggesting this is one of the reasons for the bigger genome size of N. bombycis than other microsporidia. We also found four transposable elements were acquired from its host silkworm by horizontal transfer. The considerable transposable elements are an important factor for the reshuffling and evolution of the N. bombycis genome. Moreover, a positive correlation between the transposable elements and horizontally transferred genes was observed by our statistical analysis.The bulk of transposable elements are unclassed, and the Ty3/gypsy LTR retrotransposons (Nbr), which constitute the largest part of known classes, have been studied already. Following previously reported eight Nbr elements, we detected six novel transcribed ones (Nbr9-Nbr14). Retrotransposon diversity and incomplete domains with insertions, deletions and in-frame stop codons in coding regions were detected in them, suggesting that both defective and loss events of LTR retrotransposon have happened in N. bombycis genome. Analysis of selection showed that strong purifying selection acts on all elements except Nbrl 1. This implies that selective pressure keeps both these Nbrs and their functions in genome. Interestingly, Nbrl 1 is under positive selection and some positively selected codons were identified, indicating that new functionality might have evolved in the Nbrl 1 retrotransposon. Unlike other transposable elements, Nbrl 1 has integrated into a conserved syntenic block and probably resulted in the inversion of both flanking regions. This demonstrates that transposable elements are an important factor for the reshuffling and evolution of their host genomes, and may be maintained under natural selection.