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

Abstract

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_3758, 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_3758 using PacBio sequencing, which produces some of the longest average read lengths available in the industry at this moment. According to our sequencing data, YrIlm is composed of 2603 amino acids (7812bp) and has a molecular mass of 256.4kDa. 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.

Metabolic and genetic basis for auxotrophies in Gram-negative species

“…We proceeded to validate our predictions experimentally by evaluating growth requirements of the predicted auxotrophs. We were able to obtain six E. coli (61)(62)(63)(64), three Yersinia (65), three Salmonella (66,67), three Shigella (68,69), and one Klebsiella (70) strain (Fig. 6).…”

Application of molecular tools to elucidate the microbiota of seafood

“…Mehinto et al (2012) suggested the use of this platform use for de novo sequencing of nonmodel fish species, due to the long reading length. PacBio was also used to improve the sequencing of the genome of an African fish species (Conte and Kocher 2015), to perform the complete sequencing of Piscirickettsia salmonis genome, the major pathogen of farmed salmonid fish (Pulgar et al 2015), the complete sequencing of the fish pathogen Flavobacterium columnare strain C#2 genome (Bartelme et al 2016) and in the evaluation of the adhesive capacities of a fish pathogen (Wrobel et al 2017).…”

The inverse autotransporters of Yersinia ruckeri, YrInv and YrIlm, contribute to biofilm formation and virulence

“…We recently identified two genes coding for two IATs in Y. ruckeri , namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin‐like molecule) (Leo et al ., 2015; Wrobel et al ., 2017). Their corresponding proteins are composed of an N‐terminal signal peptide, a short periplasmic region containing a peptidoglycan‐binding LysM minidomain, a translocator domain as well as a large, repetitive passenger domain made of immunoglobulin (Ig)‐like domains, capped by a C‐type lectin domain at the C‐terminus in each case.…”

“…YrIlm is composed of a variable number of Ig‐like domains depending on the strain, usually 20, whereas YrInv always contains three Ig‐like domains. The expression of YrInv and YrIlm is regulated by environmental factors such as temperature, osmolarity, iron availability as well as the presence of nutrients (Wrobel et al ., 2017). Many IATs are involved in biofilm formation, such as FdeC, IatC and YeeJ from E. coli , and EtInvA from Edwardsiella tarda (Nesta et al ., 2012; Dong et al ., 2013; Martinez‐Gil et al ., 2017; Goh et al ., 2019).…”