title: CRISPR-Cas and Contact-Dependent Secretion Systems Present on Excisable Pathogenicity Islands with Conserved Recombination Modules.
abstract: Pathogenicity islands (PAIs) are mobile integrated genetic elements that contain a diverse range of virulence factors. PAIs integrate into the host chromosome at a tRNA locus that contains their specific bacterial attachment site,attB, via integrase-mediated site-specific recombination generatingattLandattRsites. We identified conserved recombination modules (integrases andattsites) previously described in choleragenicVibrio choleraePAIs but with novel cargo genes. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins) and a type VI secretion system (T6SS) gene cluster were identified at theVibriopathogenicity island 1 (VPI-1) insertion site in 19V. choleraestrains and contained the same recombination module. Two divergent type I-F CRISPR-Cas systems were identified, which differed in Cas protein homology and content. The CRISPR repeat sequence was identical among allV. choleraestrains, but the CRISPR spacer sequences and the number of spacers varied.In silicoanalysis suggests that the CRISPR-Cas systems were active against phages and plasmids. A type III secretion system (T3SS) was present in 12V. choleraestrains on a 68-kb island inserted at the same tRNA-serine insertion site as VPI-2 and contained the same recombination module. Bioinformatics analysis showed that two divergent T3SSs exist among the strains examined. Both the CRISPR and T3SS islands excised site specifically from the bacterial chromosome as complete units, and the cognate integrases were essential for this excision. These data demonstrated that identical recombination modules that catalyze integration and excision from the chromosome can acquire diverse cargo genes, signifying a novel method of acquisition for both CRISPR-Cas systems and T3SSs.IMPORTANCEThis work demonstrated the presence of CRISPR-Cas systems and T3SSs on PAIs. Our work showed that similar recombination modules can associate with different cargo genes and catalyze their incorporation into bacterial chromosomes, which could convert a strain into a pathogen with very different disease pathologies. Each island had the ability to excise from the chromosome as distinct, complete units for possible transfer. Evolutionary analysis of these regions indicates that they were acquired by horizontal transfer and that PAIs are the units of transfer. Similar to the case for phage evolution, PAIs have a modular structure where different functional regions are acquired by identical recombination modules.
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, USA.
authors.forename: Megan R
authors.initials: MR
authors.lastname: Carpenter
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, USA.
authors.forename: Sai S
authors.initials: SS
authors.lastname: Kalburge
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, USA.
authors.forename: Joseph D
authors.initials: JD
authors.lastname: Borowski
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, USA.
authors.forename: Molly C
authors.initials: MC
authors.lastname: Peters
authors.affiliation: Center of Bioinformatics and Computational Biology, University of Maryland, College Park, Maryland, USA.
authors.forename: Rita R
authors.initials: RR
authors.lastname: Colwell
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, USA fboyd@udel.edu.
authors.forename: E Fidelma
authors.initials: EF
authors.lastname: Boyd
mesh_terms: D058947:Bacterial Secretion Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D019295:Computational Biology; D005798:Genes, Bacterial; D014644:Genetic Variation; D044404:Genomic Islands; D020071:Interspersed Repetitive Sequences; D011995:Recombination, Genetic; D017385:Sequence Homology; D014734:Vibrio cholerae / Q000145:classification / Q000235:genetics*
chemical_list: D058947:Bacterial Secretion Systems
keywords: CRISPR-Cas; Vibrio cholerae; pathogenicity islands; type III secretion