FIG 5.

Class V phase variants arise via an excision-reinsertion mechanism in which a variable region within mgpBC recombines with MgPar5, producing a circular intermediate that subsequently reinserts in a second MgPar. The DNA rearrangements producing the class V phase variant C11 and revertant C11-R are shown. (A) The mgpBC operon and MgPar5 located immediately downstream are shown, with the positions of the variable regions (B, EF, G, and KLM) indicated. A single recombination involving variable regions B of the expression site and MgPar5 results in the excision of mgpBC sequences and the production of a circular intermediate, shown in panel B. Subsequent insertion of the circular intermediate via recombination with region B of MgPar2, flanked by rnjA and the 16S rrnA gene, is shown in panel C. The dashed circle indicates where mgpB-specific region B sequences were found between MgPar5 and MgPar2 region B sequences, evidence for the creation of a circular intermediate to produce phase variants of this class. The AT-rich sequences between regions EF and KLM of MgPar2 and MgPar5 containing stop codons in all three reading frames are not shown (15). (D) Southern blot analysis using probes B-Nt, B-Ct, and C-Ct for digested genomic DNAs of the wild type and class V mutants. The fragment sizes resulting from BglII (Bg), EcoRI (Ec), and Acc65I (Ac) digestions are also indicated. The various band sizes for the different class V mutants depend on the variable region involved in the excision and the MgPar in which the recombination intermediate inserted. (E) Expression site of revertant C11-R, with the region confirmed by PCR and sequencing being marked by the dashed rectangle. C11-R mgpB region B contains 224 bp of MgPar2 region B; not shown is the MgPar7-specific sequence change of 3 bp in region EF. The sequence of MgPar5 of revertant C11-R was not determined, as indicated by the faded gray rectangle; similarly, the MgPar2 locus of C11-R was not analyzed (not shown).