CRISPR-Cas systems preferentially target the leading regions of MOB_Fconjugative 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.

The CRISPR-Cas Immune System and Genetic Transfers: Reaching an Equilibrium

“…Plasmids with the pHV1 ori were degraded by the CRISPR-Cas system (few transformants), while plasmids with the pHV2 ori were successfully transformed into cells (64). This observation is analogous to the results obtained with the nonrandom MOB-family conjugative plasmid spacer acquisition in CRISPRs (12). Thus, spacer acquisition from plasmids is potentially dependent on their mode of entry into the cells or their mode of replication.…”

“…Indeed, MOB P family plasmids are usually targeted within the lagging regions while the protospacers of the MOB F family are mostly located in the leading region (the first plasmid section entering the cell). Using synthetic constructions, the authors demonstrated that the E. coli K12 type I-E CRISPR-Cas system interferes with the conjugation of the plasmid F (MOB F family) (12). Surprisingly, the level of protection achieved by the CRISPRCas system is independent of the protospacer position (leading or lagging) and independent of the DNA strand (parental or newly synthesized strand).…”

“…Westra and coworkers screened public databases to analyze the spacer content of CRISPR loci related to conjugative plasmids (12). They found that targeted protospacers are not randomly distributed on conjugative plasmids: there is a MOB family-dependent bias.…”

“…These authors showed that the lactic acid bacterium Streptococcus thermophilus could acquire resistance against a bacteriophage by integrating a genome fragment (the protospacer) from this infectious bacterial virus into its CRISPR locus (spacer). Later studies discovered the expanded role of CRISPRs in preventing horizontal gene transfer (HGT) through conjugation and transformation (12)(13)(14). The CRISPR spacer sequences of archaeal species frequently match those of their own resident viruses or plasmids, suggesting a regulatory rather than inhibitory role for CRISPR-Cas.…”

The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

“…In 2007, Barrangou et al provided the first experimental evidence that the CRISPR-Cas system is an adaptive immune system that protects its host against invading viruses (11). A role for CRISPR-Cas in defense was further established in a range of subsequent studies on viral transfections and plasmid transformations (5,(12)(13)(14)(15)(16)(17)(18)(19)(20). In 2011, Makarova et al (21) suggested the classification of the different CRISPR-Cas systems into the following three main types: type I CRISPR-Cas systems, based on the presence of the cas3 gene; type II CRISPR-Cas systems, based on the presence of the cas9 gene; and type III CRISPR-Cas systems, based on the presence of the cas10 gene.…”