ABSTRACT: The repeat structure of two paralogous genes, Yersinia ruckeri Invasin (yrInv) and a “Y. ruckeri Invasin-like molecule”, (yrIlm) sheds light on the evolution of adhesive capacities of a fish pathogen
Agnieszka Wrobel
Inverse autotransporters comprise the recently identified type Ve secretion system and are exemplified by intimin from enterohaemorrhagic Escherichia coli and invasin from enteropathogenic Yersiniae. These proteins share a common domain architecture and promote bacterial adhesion to host cells. Here, we identified and characterized two putative inverse autotransporter genes in the fish pathogen Yersinia ruckeri NVH_1, namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin-like molecule). When trying to clone the highly repetitive genes for structural and functional studies, we experienced problems in obtaining PCR products. PCR failures and the highly repetitive nature of inverse autotransporters prompted us to sequence the genome of Y. ruckeri NVH_1 using PacBio sequencing. Based on the new genome information, we performed PCR analysis on four non-sequenced Y. ruckeri strains as well as the sequenced Y.ruckeri type strain. We found that the genes are variably present in the strains, and that the length of yrIlm, when present, also varies. In addition, the length of the gene product for all strains, including the type strain, was much longer than expected based on deposited sequences. The internal repeats of the yrInv gene product are highly diverged, but represent the same bacterial immunoglobulin-like domains as in yrIlm. Using qRT-PCR, we found that yrIlm and yrInv are differentially expressed under conditions relevant for pathogenesis. In addition, we compared the genomic context of both genes in the newly sequenced Y.ruckeri strain to all available PacBio-sequenced Y. ruckeri genomes, and found indications of recent events of horizontal gene transfer. Taken together, this study demonstrates and highlights the power of Single Molecule Real-Time technology for sequencing highly repetitive proteins, and sheds light on the genetic events that gave rise to these highly repetitive genes in a commercially important fish pathogen.
ABSTRACT: Unicellular origin of the Microprocessor and microRNAs
Arthur A. B. Haraldsen
In the past decade we have learned that the unicellular sister lineages of animals have surprisingly complex genetic machineries, and share many of the gene pathways and regulatory networks that are key to animal development. One group of genes that is believed to be key to the acquisition of metazoan complexity is the miRNAs. However, the protein machinery essential for biogenesis of miRNAs in animals, the so-called Microprocessor complex, has not been identified outside animals and could therefore be a genetic innovation specific to animals. We have therefore investigated the presence of genes involved in miRNA biogenesis, as well as the miRNA genes themselves, in unicellular relatives of animals by transcriptome and small RNA sequencing. We find homologues of the entire animal miRNA biogenesis machinery, including Drosha and Pasha (the components of the Microprocessor), as well as miRNA genes, in a wide range of ichthyosporeans. This implies that miRNAs and the Microprocessor complex evolved before the metazoan last common ancestor. This is consistent with the view that most of the animal developmental toolkit evolved prior to animals. Furthermore, it implies that absence of the miRNA biogenesis pathway in other lineages of Choanozoa, Ctenophores and Placozoa are result of secondary loss, which also seems to correspond to the absence of miRNA genes.