title: CRISPR-Cas systems preferentially target the leading regions of MOBF conjugative plasmids.
abstract: Most prokaryotes contain CRISPR-Cas immune systems that provide protection against mobile genetic elements. We have focused on the ability of CRISPR-Cas to block plasmid conjugation, and analyzed the position of target sequences (protospacers) on conjugative plasmids. The analysis reveals that protospacers are non-uniformly distributed over plasmid regions in a pattern that is determined by the plasmid's mobilization type (MOB). While MOBP plasmids are most frequently targeted in the region entering the recipient cell last (lagging region), MOBF plasmids are mostly targeted in the region entering the recipient cell first (leading region). To explain this protospacer distribution bias, we propose two mutually non-exclusive hypotheses: (1) spacers are acquired more frequently from either the leading or lagging region depending on the MOB type (2) CRISPR-interference is more efficient when spacers target these preferred regions. To test the latter hypothesis, we analyzed Type I-E CRISPR-interference against MOBF prototype plasmid F in Escherichia coli. Our results show that plasmid conjugation is effectively inhibited, but the level of immunity is not affected by targeting the plasmid in the leading or lagging region. Moreover, CRISPR-immunity levels do not depend on whether the incoming single-stranded plasmid DNA, or the DNA strand synthesized in the recipient is targeted. Our findings indicate that single-stranded DNA may not be a target for Type I-E CRISPR-Cas systems, and suggest that the protospacer distribution bias might be due to spacer acquisition preferences.
authors.affiliation: Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, Wageningen, The Netherlands.
authors.forename: Edze R
authors.initials: ER
authors.lastname: Westra
authors.forename: Raymond H J
authors.initials: RH
authors.lastname: Staals
authors.forename: Gerrit
authors.initials: G
authors.lastname: Gort
authors.forename: Søren
authors.initials: S
authors.lastname: Høgh
authors.forename: Sarah
authors.initials: S
authors.lastname: Neumann
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cruz
authors.forename: Peter C
authors.initials: PC
authors.lastname: Fineran
authors.forename: Stan J J
authors.initials: SJ
authors.lastname: Brouns
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000276:immunology; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000276:immunology; D003227:Conjugation, Genetic; D004277:DNA, Single-Stranded; D048168:Escherichia coli K12 / Q000235:genetics* / Q000276:immunology; D005144:F Factor / Q000235:genetics* / Q000276:immunology
chemical_list: D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; C083180:Mob protein, Bacteria
keywords: CRISPR; adaptive immunity; conjugation; mobile genetic element; plasmid mobilization; protospacer distribution
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