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. 2011 Aug;7(8):e1002223.
doi: 10.1371/journal.pgen.1002223. Epub 2011 Aug 25.

Global chromosomal structural instability in a subpopulation of starving Escherichia coli cells

Dongxu Lin  1 , Ian B Gibson, Jessica M Moore, P C Thornton, Suzanne M Leal, P J Hastings
Affiliations

Global chromosomal structural instability in a subpopulation of starving Escherichia coli cells

Dongxu Lin et al. PLoS Genet. 2011 Aug.

Abstract

Copy-number variations (CNVs) constitute very common differences between individual humans and possibly all genomes and may therefore be important fuel for evolution, yet how they form remains elusive. In starving Escherichia coli, gene amplification is induced by stress, controlled by the general stress response. Amplification has been detected only encompassing genes that confer a growth advantage when amplified. We studied the structure of stress-induced gene amplification in starving cells in the Lac assay in Escherichia coli by array comparative genomic hybridization (aCGH), with polymerase chain reaction (pcr) and DNA sequencing to establish the structures generated. About 10% of 300 amplified isolates carried other chromosomal structural change in addition to amplification. Most of these were inversions and duplications associated with the amplification event. This complexity supports a mechanism similar to that seen in human non-recurrent copy number variants. We interpret these complex events in terms of repeated template switching during DNA replication. Importantly, we found a significant occurrence (6 out of 300) of chromosomal structural changes that were apparently not involved in the amplification event. These secondary changes were absent from 240 samples derived from starved cells not carrying amplification, suggesting that amplification happens in a differentiated subpopulation of stressed cells licensed for global chromosomal structural change and genomic instability. These data imply that chromosomal structural changes occur in bursts or showers of instability that may have the potential to drive rapid evolution.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Distribution of structural changes in E. coli genome.
A map of the chromosomal sequence on F′128 bounded by IS3 elements and of the E. coli chromosome showing the positions of some of the structural changes reported here. The chromosomal origin of sequences carried on the F′ is shown as a black bar on the chromosome.
Figure 2
Figure 2. Complex rearrangements on the F′128 revealed by oligonucleotide aCGH and confirmed by PCR and DNA sequencing.
a: Inverted region embedded in the amplification. b: Partially inverted amplification. c: Related deletion embedded in the amplification. d: Deletion independent of amplification. e: Inversion partially overlapping with amplification. Open arrows indicate features described in the text.
Figure 3
Figure 3. Model for the formation of complex structures by template switch events.
The same model of repeated template switch can explain all these events, differing only in the position and orientation of the switch. a: Array CGH scan showing amplification with embedded inverted duplication. The blue, red and black dots correspond to blue, red and black arrows in b. b: Two inverted template switches that generate an inverted triplication (red) an inverted duplication (black) and a direct duplication, followed by unequal crossing-over (NAHR) between direct repeats (red or black) to generate the amplicon as observed. c: Array CGH scan showing partially inverted amplification. The blue, red and black dots correspond to blue, red and black arrows in d. d: Two inverted template switches generate an inverted duplication of part of the red sequence and a direct duplication part of the blue sequence. Unequal crossing-over can occur between blue sequences to generate the amplicon. e: Array CGH scan showing amplification consisting of a partially deleted duplication. The blue, red and black dots correspond to blue, red and black arrows in f. f: Two direct template switches give rise to an embedded deletion. Orientations were determined by sequencing the junctions (Table 1).
Figure 4
Figure 4. Inversion-associated small deletion and insertion mutations.
a: Unidirectional PCR primers to the right of lac give no pcr product. b: when part of the sequence is inverted, a PCR product can be obtained. c: PJH1465; d: PJH1479. The inverted segment is shown in its original orientation to reveal the mutations at each end. When the inverted segments were compared with the original sequence, small deletions and insertions were found on both end points. Blue blocks between mhpE and mhpT, yaiL and yafJ are REP. Colored lines represent genes in the same colors as the colored dots. When the sequences are restored to their genomic positions, small duplications and deletions are revealed (lower parts of c and d). The lengths of micro-duplications and -deletion in base-pairs are given below the diagram.
Figure 5
Figure 5. A model for inversion formation by template-switching giving non-homologous recombination in close to reciprocal positions.
a: Leading-strand DNA synthesis is blocked by a secondary structure (1) in the leading-strand template. b: The stalled 3′-end bypasses blockage by an inverted switch to the lagging-strand template where lagging-strand synthesis has been blocked by a different secondary structure (2). c: Inverted DNA synthesis in lagging-strand stalled for the second time by the reciprocal secondary structure (1R). d: The stalled 3′-end switches to different reciprocal secondary structure (2R) in the leading-strand template. This allows replication to resume. e and f show potential secondary structure near the junctions in mhpA and mhpC respectively of the inversion in PJH1465. Left junction sequence is shown in red, right in blue.
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