The tandem inversion duplication in Salmonella enterica: selection drives unstable precursors to final mutation types

Abstract

During growth under selection, mutant types appear that are rare in unselected populations. Stress-induced mechanisms may cause these structures or selection may favor a series of standard events that modify common preexisting structures. One such mutation is the short junction (SJ) duplication with long repeats separated by short sequence elements: AB*(CD)*(CD)*E (* = a few bases). Another mutation type, described here, is the tandem inversion duplication (TID), where two copies of a parent sequence flank an inverse-order segment: AB(CD)(E'D'C'B')(CD)E. Both duplication types can amplify by unequal exchanges between direct repeats (CD), and both are rare in unselected cultures but common after prolonged selection for amplification. The observed TID junctions are asymmetric (aTIDs) and may arise from a symmetrical precursor (sTID)-ABCDE(E'D'C'B'A')ABCDE-when sequential deletions remove each palindromic junction. Alternatively, one deletion can remove both sTID junctions to generate an SJ duplication. It is proposed that sTID structures form frequently under all growth conditions, but are usually lost due to their instability and fitness cost. Selection for increased copy number helps retain the sTID and favors deletions that remodel junctions, improve fitness, and allow higher amplification. Growth improves with each step in formation of an SJ or aTID amplification, allowing selection to favor completion of the mutation process.

Figure 1.—

Formation of SJ duplications under selection. The short arrows between b–c and d–e in the parent sequence are repeats of 3–12 bp. The boxes are the 1.25-kb elements IS3A and IS3C that flank lac on the F′₁₂₈ plasmid. The duplicated region between IS3 copies is ∼125 kb, and duplications of this type are carried by 0.1% of cells prior to selection. Deletion between identical short sequences leaves a smaller duplication, typically ∼20 kb. Shortening reduces the reversion rate and fitness cost of a duplication and thereby allows higher amplification (Kugelberg et al. 2006).

Figure 2.—

Proposal for the formation of symmetrical TID structures. Short quasi-palindromic sequences are frequent and can support formation of snap-back structures that initiate repair synthesis, which can switch templates and be redirected toward the fork. The resulting branched structure can be broken at arrow and repaired or can be replicated to produce the sTID structure proposed to initiate the aTID structures described here. These events may also underlie recombination-independent formation of simple duplications.

Figure 3.—

Conversion of an sTID precursor to an aTID duplication. The sTID forms and is lost frequently. Asterisks in the parent sequence indicate small palindromic sequences that promote sTID formation. Prolonged selection for higher expression (e.g., lac, the black dot) favors retention of the sTID and sequential accumulation of deletions that render junctions asymmetric (forming the aTID). As fitness cost is reduced, higher amplification allows faster growth and ultimately provides sufficient lac targets so that point mutations can stably alter the gene under selection. Short colored arrows are repeated oligonucleotide sequences present in the parent that serve as endpoints for deletions and junctions of the final aTID duplication. Uninvolved repeats are omitted after the first two lines.

Figure 4.—

Structure of the F′₁₂₈ plasmid on which rearrangements occur. The base numbering conventions are the following: Bases 1–100,413 are derived from the F factor (bold arc of circle). Bases 100414–150235 are from the E. coli chromosome extending from IS3C to include all of the lac operon up to base 150,235, which is immediately adjacent to the promoter-proximal end of the lacZ coding sequence. Bases 150,236–231,638 of the plasmid are also from the E. coli chromosome and extend from lacZ to beginning of F factor sequence (at base 1). The origin and structure of this plasmid have been described in detail (Kofoid et al. 2003).

Figure 5.—

Depth of Illumina reads from the F′₁₂₈ lac plasmid of a strain carrying a TID amplification. The plasmid genotype is diagrammed below the graph with the F factor plasmid sequence indicated by a bold line and the E. coli material by a finer line. Strains are recA and were grown on glycerol with no selection for lac amplification. The read depth for chromosomal genes (not shown) was ∼90-fold. The nearly 200-fold coverage seen for the plasmid (plotted horizonal line) suggests that the strain has several copies of the F′₁₂₈ plasmid. The lac region is amplified ∼4-fold above the rest of the plasmid.

Figure 6.—

Multiple structures are consistent with a single pair of junction sequences. In the examples listed (I–IV), the leftmost (converging) junction is D–C′ (equivalent to C–D′) and the rightmost (diverging) junction is B′A (equivalent to A′B). If the asymmetric inversion junctions are formed by deletions in a symmetrical precursor, then four possible structures could be generated by the various combination of deletions described in the bottom line. The colored arrows indicate repeated sequences present in inverse order in the parent chromosome and how they contribute to aTID formation.

Figure 7.—

Genetic testing of TID amplification structure. To determine whether an aTID junction is in orientation CD′ or DC′, a resistance determinant (tetRA) was added to the sequenced junction of strain TT25771 (see Table 1). The chromosome of the resulting strain is expected to be as diagrammed for strain 1 or strain 2 (circled). In a second transformation cross, a donor Cam^R determinant is introduced with one flanking sequence matching a reference region at the left and the other matching the tetRA sequence. The Cam^R determinant can be inherited only if the orientation of the recipient junction (tetRA or ARtet) matches that of the donor recombining sequence (right side of Cam^R). The donor fragments are designed such that inheritance of Cam^R creates a deletion of the region between the reference sequence and the right side of the Tet^R determinant in the recipient. The recipient strain is recA+ and was grown nonselectively before transformation.

Figure 8.—

Effects of inversions on structure of amplified aTID array. One initial aTID structure is depicted with the changes that are possible due to amplification followed by recombination between inverse-order sequence repeats.

Figure 9.—

A TID amplification with one unmodified symmetrical junction. The diagram at left and the solid arrows at top right indicate a parental quasi-palindromic sequence that might generate snap-back pairing. The events at right describe how the snap-back structure might prime repair synthesis, leading to the TID junction sequences found in strain TT25790 (Table 1). The quasi-palindromic junction is extended without modification. The second junction forms by template switching and is later modified by deletion.

Figure 10.—

The sTID can also contribute to formation of an SJ duplication. The parental sequence is diagrammed at the top (boxed) in which open rectangles designate a copy of IS3 and the solid circle designates the lac operon. An SJ duplication can form when a deletion removes the junction of an IS3 duplication (left middle). An SJ duplication also forms when a single deletion removes both sTID junctions (right middle). Two deletions convert an sTID to an aTID duplication. Some IS3-bounded lac duplications may form through sTID intermediates.