Figure 1.
HFIR may account for the occurrence of insertions coupled with deletions, observed upon multiple genomes alignments.
A. During Homology-Assisted Illegitimate Recombination (HFIR), a linear fragment sharing homology with the chromosome (black part) enters into the recipient chromosome by an atypical double crossing-over event, with homologous recombination (HR) at the left-end of the molecule, and illegitimate recombination (IR) at the other end. B. Alignment of the resulting recombinant with the ancestral genome will produce two small “variable regions” flanked by the backbone sequences, common to both strains. C. Diversification of strains produced by HFIR, leads to the detection of insertions (variable segment VS2 in genomes G3-G5) coupled with deletions (variable segment VS1 in genomes G1-G2) upon multiple genome alignments of various E. coli strains (here 5 strains).
Table 1.
Strains and plasmids used in this study.
Figure 2.
Maps of plasmids pJA1, pJA2 and pJA3 after linearization in the cut cassette.
The 1 kb 5′ part of lacZ is shown with a grey box, and the 1 kb 3′ part of lacZ (lacZend) by a white arrow. The cat gene encoding resistance to chloramphenicol (CmR) is shown as a stripped arrow. The cut cassette is indicated by a small black box, linearization of pJA plasmids in vivo might happen either at the IsceI recognition sequence, or between the two EcoK sites. The cut cassette map is shown in details, with two grey boxes indicating the Chi sites. Below the three plasmids, the E. coli chromosome with which plasmids might recombine is shown (wavy line) in the lacZ region (grey to white arrow).
Table 2.
Sequence of the oligonucleotides used in this study.
Figure 3.
Inferred chromosomal recombination products obtained with plasmid pJA2.
Left part: Single crossing-over (SCO) between circular pJA2 and the chromosome, as produced if pJA2 remains uncut (or if pJA2 is cut and resealed). The resulting exconjugants are Lac+ CmR. Central part: HFIR between linear pJA2 and the chromosome. Exconjugants are Lac− CmR, and therefore distinguishable from the previous SCO events. Right part: If pJA2 is cut, degraded up into the lacZ sequence, and recircularized, a SCO between a circular, deleted (wavy bold line) pJA2 molecule and the chromosome will give rise to Lac− CmR exconjugants. White arrow, 3′ part of the lacZ gene. Grey box, remaining part of the lacZ gene. Wavy lines, other sequences of the E. coli chromosome. Straight line, pJA2 plasmid sequences. Black box, “cut cassette”. Primers j18, j19, j17, j5, j16 complementary to the plasmid pJA2 and the chromosome, are shown above each chromosomal recombination product.
Figure 4.
Efficiency of post-conjugative recombination with plasmids pJA3, pJA2 and pJA1.
The Lac+ (blue bars) and Lac− (white bars, when present) CmR ex-conjugants reported to the viable recipient cells, are scored separately.
Table 3.
Properties of the HFIR clones obtained.
Table 4.
Recombination frequencies with circular pJA2 in E. coli mutant derivatives.
Table 5.
Recombination frequencies with linear pJA2 in E. coli mutant derivatives.