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Figure 3
Targeted insertion next to a single IR in vivo: influence of the IS911 proteins. (A) Experimental scheme. Top panel: circle formation from the donor plasmid. The general structure of the donor plasmids is indicated. They are derivatives of p15A and contain an artificial transposon composed of 52 bp of the left and 52 bp of the right IS911 end (dark grey boxes) flanking a gene for resistance to chloramphenicol (Cm; light grey box). The individual plasmids are described in more detail in (B). The cartoon shows the formation of transposon circle intermediates, which are subsequently inserted into the target plasmid (bottom panel). Bottom panel: structure of the target plasmid and the product of targeted insertion. Plasmid pBST1 carries a mutated IRL sequence (grey box) with the indigenous promoter pIRL and a gene fusion between the 5' part of orfA and lacZ from the eighth codon (orfA−lacZ) (cross hatched box), a pBR322 origin of replication (unfilled oval) and the  -lactamase (bla) gene. The IR-targeted product that would result from the insertion of the CmR transposon 3 bp from the resident IRL* reconstitutes the strong pjunc promoter and activates the orfA−lacZ gene. The position and orientation of the two promoters are indicated by an arrow. This type of targeted IRL* insertion is observed, after transformation, on MacConkey lactose plates. (B) Structure of the donor plasmids, transposition frequency and percentage of lac+ colonies. The left column presents the different plasmids used to supply IS911 proteins and the artificial IS911 transposon. pCL19 carries the wild-type configuration of IS911 orfs, which drives expression of both OrfA and OrfAB proteins under the control of the lacUV5 promoter (plac). pCL21 and pCL22 carry the orfAB gene alone, where the A6G frameshift signal was exchanged for CA5G (Polard et al., 1992) to artificially fuse the orfA and orfB reading frames under the control of placUV5. It also carries the E.coli paraBAD and the araC gene, which encodes the arabinose-inducible repressor. In addition, pCL22 carries the orfA gene, which drives expression of OrfA alone (orfA), under the control of paraBAD. The direction of gene transcription is indicated by curved arrows, and the position and orientation of the promoter are indicated by an arrow. The small open circles correspond to transcription terminators of the E.coli rrnB operon, and the small white boxes represent phage T4 transcription and translation termination signals flanking the streptomycin/spectinomycin resistance cassette (SpSm). lacIq, the lacIq allele. The two right-hand columns indicate the transposition frequency (Transptn;  106) and the percentage of lac+ (% lac+) colonies obtained on MacConkey lactose 1% Cm in the absence (-) or presence (+) of 1 mM IPTG, and in the absence (-) or presence (+) of 1% arabinose. The transposition frequency is expressed as the ratio of CmR transformants compared with ApR transformants and the percentage of lac+ is expressed as the number of CmR lac+ transformants as a percentage of total transformants (CmR).
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