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.

Keywords: CRISPR; adaptive immunity; conjugation; mobile genetic element; plasmid mobilization; protospacer distribution.

Figure 1. Spacers from CRISPRdb targeting conjugative plasmids. (A) Conjugative plasmids, of which the oriT site and the relaxase gene could be identified, were screened for homology with spacers from the CRISPRdb. After establishing the leading and lagging regions of the plasmid, by taking into account the location of the relaxase relative to the oriT site, the distance of each spacer hit from the oriT site is expressed as a percentage of the total plasmid size. These values are depicted as open circles on the plasmid map (left). The red line indicates the protospacer density at the respective position. The protospacer distribution is also shown in a histogram (right). To this end, the plasmid is divided into 10% segments (i.e., plasmid fragments corresponding to 10% of the plasmid size). When equally distributed, each 10% segment would carry 10% of all protospacers. The actual percentages of protospacers present in each 10% segment are indicated by the blue bars. (B) A similar analysis as in (A) was performed, but using only the MOB_F family of conjugative plasmids, and using the relaxase gene start position to calculate distances of the spacer hits.

Figure 2. Spacers from CRISPRdb targeting plasmid F (A) Map of plasmid F indicating the size of the plasmid and the location of the origin of transfer (oriT), the relaxase gene, the leading region (which enters the recipient cell first) and the transfer region (which encodes the genes essential for plasmid transfer). Asterisks indicate the approximate positions of the protospacers listed in (D). (B) Similar analysis as presented in Figure 1A, where the distance of each spacer hit on plasmid F from the oriT or (C) from the relaxase gene is calculated and expressed as a percentage of the total plasmid size. (D) Alignments of spacer sequences (“Query,” top sequences) and the corresponding plasmid F sequences (“Sbjct“, bottom sequences). The species, CRISPR and spacer are indicated above each alignment, following the nomenclature used by the CRISPRdb. The numbers adjacent to the alignment indicate the position of the spacer and protospacer sequence, respectively.

Figure 3. A synthetic anti-pOX CRISPR provides resistance against conjugational plasmid transfer. (A) The Type I-E CRISPR-Cas system of E. coli contains eight cas genes and a downstream CRISPR with type 2 repeats. Promoter elements (Pcas3, Panti-cas, Pcas and P_CRISPR) are indicated by arrows. (B) A plasmid map of the F-plasmid with all HindIII sites. The origin of transfer (oriT) is indicated by a black triangle, the leading and transfer regions of the plasmid are indicated with arrows. The plasmid F derived pOX38-Tc corresponds to the largest HindIII fragment of plasmid F, and contains a tetracycline resistance marker. The positions of the protospacers targeted by the synthetic anti-pOX CRISPR indicated in (D) are indicated by colored boxes and labeled with the name of the spacer that targets the site (i.e., sp1-5). (C) Schematic representation of the non-targeting CRISPR J4, which contains four identical spacer sequences targeting the J gene of phage Lambda. (D) Schematic representation of the targeting CRISPR-F, which contains 5 different spacer sequences targeting sequences of pOX38-Tc. The positions of the target sequences are indicated in (B) by colored boxes and labeled with the name of the spacer that targets the site. (E) Conjugation efficiencies using E. coli MC4100 + pOX38-Tc as a donor strain and E. coli K12 or E. coli K12Δhns transformed with a plasmid carrying either CRISPR-J4 or CRISPR-F as recipient strains. (F) Conjugation efficiencies using donor strain as in (E) and either induced or uninduced E. coli BL21 (DE3) transformed with either CRISPR-J4 or CRISPR-F as recipient strains. Conjugation efficiencies are expressed as transconjugants/donor.

Figure 4. Interference levels of CRISPRs targeting leading regions as compared with CRISPRs targeting lagging regions. (A and B) Synthetic CRISPR constructs targeting plasmid F are shown together with the coordinates of the corresponding protospacer on plasmid F. Four CRISPRs contain a spacer at the second position in the array that targets the leading region of plasmid F (IE1, IE2, SE1, SE2) and four additional CRISPRs target the non-leading region of plasmid F (IL1, IL2, SL1, SL2). Of these CRISPRs, four target the incoming strand of plasmid F (IE1, IE2, IL1, IL2) and four CRISPRs target the synthesized strand of plasmid F (SE1, SE2, SL1, SL2). (C) Efficiency of conjugation (#transconjugants/#donor) from E. coli MC4100 donor cells carrying pOX38-Tc to E. coli K12Δhns transformed with any of the CRISPRs shown in (A). (D) Efficiency of conjugation (#transconjugants/#donor) from E. coli MC4100 donor cells carrying pOX38-Tc to E. coli K12Δhns transformed with any of the CRISPRs shown in (B).