Expansion of a chromosomal repeat in Escherichia coli: roles of replication, repair, and recombination functions
© Poteete; licensee BioMed Central Ltd. 2009
Received: 11 November 2008
Accepted: 23 February 2009
Published: 23 February 2009
Previous studies of gene amplification in Escherichia coli have suggested that it occurs in two steps: duplication and expansion. Expansion is thought to result from homologous recombination between the repeated segments created by duplication. To explore the mechanism of expansion, a 7 kbp duplication in the chromosome containing a leaky mutant version of the lac operon was constructed, and its expansion into an amplified array was studied.
Under selection for lac function, colonies bearing multiple copies of the mutant lac operon appeared at a constant rate of approximately 4 to 5 per million cells plated per day, on days two through seven after plating. Expansion was not seen in a recA strain; null mutations in recBCD and ruvC reduced the rate 100- and 10-fold, respectively; a ruvC recG double mutant reduced the rate 1000-fold. Expansion occurred at an increased rate in cells lacking dam, polA, rnhA, or uvrD functions. Null mutations of various other cellular recombination, repair, and stress response genes had little effect upon expansion. The red recombination genes of phage lambda could substitute for recBCD in mediating expansion. In the red-substituted cells, expansion was only partially dependent upon recA function.
These observations are consistent with the idea that the expansion step of gene amplification is closely related, mechanistically, to interchromosomal homologous recombination events. They additionally provide support for recently described models of RecA-independent Red-mediated recombination at replication forks.
Expression of a chromosomal gene in Escherichia coli can be elevated by gene amplification. The mechanism of this amplification is thought to consist of two steps, duplication and expansion. Duplication is rare, largely recA-independent, and occurs between microhomologies in the chromosome as a replication accident. Expansion is frequent, recA-dependent, and thought to result from unequal crossing-over events between the duplicated segments [1–3].
Recent investigations of gene amplification in E. coli have focused on amplification of plasmid-borne genes. A phenotypically leaky F'-borne mutation, ϕ(lacIX13-lacZ), gives rise to Lac+ revertants bearing amplified arrays of 40–80 copies of the lac region . Lac+ revertants of F'lac bearing the +1 frameshift allele ϕ(lacI33-lacZ), extensively employed in studies of adaptive mutation, consist mainly of one-base deletions in runs of iterated bases [5, 6], but clones bearing amplified arrays appear at a lower rate as well [7, 8]. Properties of lac amplification have generally supported the duplication-expansion model. (i) An engineered duplication of the frameshift mutant lac locus amplifies at a greatly elevated frequency , as predicted by the idea that duplication is the rate-limiting step (and as had been seen in the case of chromosomal ampC ). (ii) Amplification is dependent upon recBCD and ruvABC, as well as recA, indicating an important role for homologous recombination .
Expansion of a pre-existing repeat has also been studied primarily on plasmids. In one study, a pBR322 derivative was constructed with two directly repeated tetA genes, each bearing an inactivating mutation, but arranged in such a way that a single unequal crossover would generate an array of three copies, one of which was a functioning gene. In this system, expansion was reduced only five-fold in a recA mutant; expansion was elevated in strains bearing mutations in dnaQ, dnaE, dnaB, or dnaN .
Expansion of a pre-existing duplication was compared with amplification of a single copy of F'-borne ϕ(lacI33-lacZ) in another study . Expansion was found to be increased in a polA mutant, and unaffected by overexpression of xonA, while amplification from a single copy was inhibited by both of these conditions. It was concluded that the amplification defects caused by the polA mutant and by xonA overproduction were in duplication, not expansion.
Expansion and survival tests
Lac+ clone expansion test
No. positive/no. tested
Survival on lactose minimal medium relative to wild typeb
0.9 ± 0.1
0.9 ± 0.1
1.2 ± 0.4
0.6 ± 0.2
wild type single copy
Null mutations in recBCD and ruvC reduced Lac+ colony formation 100-fold and 10-fold, respectively. Other mutations eliminating single recombination functions, recF, recG, recN, recQ, recR, and ruvAB, had little overall effect. A ruvC recG double mutant was also tested. Like the recA mutation, and as in other homologous recombination events , it generated Lac+ colonies approximately 1000-fold less efficiently than wild type.
A disruption of the E. coli yfgL gene was reported to confer a strong recombination/repair deficiency phenotype . As shown in Fig. 4, however, a yfgL null mutation constructed for this study has little or no effect on expansion. It also confers no UV-sensitivity or transductional recombination phenotype, in either an MG1655 or an AB1157 strain background (not shown); others have found no recombination/repair phenotype associated with a yfgL null as well .
To test the hypothesis that the deficiencies of recA, recBCD, ruvC, and ruvC recG mutants in Lac+ colony formation are due to their inability to expand the duplication, two alternative explanations were considered and ruled out. (i) Lac+ revertants of these mutants could grow much more slowly than Lac+ revertants of wild type. Lac+ colonies were restreaked on minimal lactose plates on the days they arose. In the cases of wild type, recA, recBCD, and ruvC, all of the Lac+ clones formed colonies visible to the unaided eye by 24 hours after restreaking, independent of the day on which they arose. In the case of the ruvC recG double mutant, none of the 8 tested Lac+ revertants formed visible colonies by 24 hours, but all did so by 48 hours. However, counting the ruvC recG colonies after 8 days instead of 7 only increased the median from 0.029 to 0.056 colonies per million viable cells plated (data not shown). Thus, slow growth of revertants can account for only a small part of the deficiencies of the recombination mutants in forming Lac+ colonies. (ii) The mutants could be proficient at expansion, but deficient at survival on the selection plates. Survival of the mutants on lactose minimal medium was tested as described in the methods section. The results (Table 1) indicate that none of the mutants has a substantial survival defect relative to wild type.
The Lac+ revertants of the deficient mutants consist of varying populations of expanded and mutated clones (Table 1). In the case of recA, none of 10 tested clones had an expanded lac array. The frequency of expanded arrays among recBCD revertants was 9 out of 10; ruvC was 10 out of 10, recG was 18 out of 18, and ruvC recG was 6 out of 14.
The Lac+ reversion phenotype of a recG null mutant is more complex than the data in Fig. 4 suggest. The mutant strain's rate of colony formation tends to increase sharply late in the experiment, with the new colonies tending to appear as satellites of older colonies (not shown). Factors influencing the timing and extent of this satellite-based population explosion include plating density, but are otherwise unknown. The recG mutant data shown in Fig. 4 are from selected experiments, in which the plating density was low, and satellitism was not as strongly evident as in other experiments. Satellitism of this sort suggests that something produced by the older colonies on the plate stimulates recombination, perhaps via a genotoxic effect. It is consistent with the finding that overexpression of recG protects E. coli against weak organic acids .
The effects on expansion of varying RecA activity were tested by plating mutants affecting recA expression. The uninducible lexA3 mutation has been reported to reduce the frequency of a number of different homologous recombination events ; it also reduced Lac+ colony formation approximately five-fold. The SOS-constitutive lexA71::Tn5 mutation (in a sulA null background, to suppress its lethality) had no significant effect; neither did the recAo281 operator constitutive allele.
A number of replication genes were tested for roles in expansion. A null mutation in polA increased the rate of expansion 5-fold; null mutations in the other non-essential DNA polymerase-encoding genes polB, dinB, and umuDC, had little or no effect. Loss of the replication restart function priA caused a small decrease in expansion efficiency, while loss of rnhA caused a nearly 7-fold increase.
A strain lacking dam function exhibited a 35-fold elevated rate of Lac+ colony formation. Apparently, part of this elevated rate is due to the double-strand breaks which occur as the result of mis-directed mismatch repair in dam mutants [21, 22]. As shown in Fig. 4, a dam mutH double mutant exhibited an intermediate rate between those of wild type and the dam single mutant. The mutH null allele by itself had little or no effect.
Other DNA repair functions were tested for effects on expansion as well. A uvrD null mutant formed Lac+ colonies at a 10-fold elevated rate, while mutM, mutY, a mutM mutY double, and an ada null mutation had no significant effects.
The question of whether expansion of a chromosomal repeat occurs as part of a stress response, like amplification starting from a single episomal copy , was explored by testing null mutations in rpoS and relA. As shown in Fig. 4, these mutations had little or no effect on Lac+ colony formation.
Expansion mediated by the Red recombination system of phage λ was studied in a series of strains in which the phage red genes replace the recC-ptr-recB-recD gene cluster in the E. coli chromosome (designated "red+" in Fig. 4). Replacing RecBCD with Red has no effect on the rate of Lac+ colony formation, but it changes the extent to which expansion is dependent upon other recombination functions. In the red-substituted background, a recA null mutation reduces Lac+ colony formation only 35-fold. Among the recA revertants, 8 of 10 that were tested contained expanded lac arrays (Table 1), showing that Red, unlike RecBCD, can promote expansion in the absence of RecA. Red-mediated expansion is reduced by a recF null mutation, and elevated slightly by a recG null; these mutant effects are seen in Red-mediated gene replacement events as well . The rnhA null mutation has a stronger effect in the red-substituted background than in wild type, elevating the rate of expansion 45-fold.
Discussion and conclusion
The genetic requirements of homologous recombination in E. coli vary with the particular event examined, but some features of chromosomal events are nearly general: dependence on recA and recBCD, and mild or no dependence upon a variety of other recombination functions whose roles are revealed mainly in the absence of recBCD function . Expansion by duplicated chromosomal ϕ(lacI33-lacZ) fits this general pattern. Similarly, mutations with known hyper-rec phenotypes – polA, dam, uvrD, and rnhA [20, 25, 26] – also cause an elevated rate of expansion. These observations support the idea that expansion is best understood as a homologous recombination event or series of events.
The Red recombination system of phage λ promotes RecA-independent recombination between chromosomes if at least one of the chromosomes is replicating, and RecA-dependent recombination between non-replicating chromosomes [27–30]. The RecA-independent expansion seen in red-substituted bacteria suggests that at least some of the Red-mediated recombination events involved in expansion take place at replication forks [30, 31].
The involvement of replication in recombination events leading to expansion is additionally suggested by the increased rates of expansion of the rnhA and dam mutants. Replication in both these mutants escapes cell cycle regulation. In an rnhA mutant, unsynchronized DNA replication initiates at multiple sites in the chromosome . In a dam mutant, initiation is confined to oriC but is not regulated . Elevated recombination frequencies in both of these mutants may be due, at least in part, to an increased occurrence of double strand breaks. Both exhibit greatly reduced viability in the absence of RecBCD function, possibly because RecBCD is needed to repair the excess double strand breaks [34, 35]. Eliminating the mismatch repair endonuclease MutH in a dam mutant prevents the double strand breaks which result from mis-directed mismatch repair, but does not bring expansion down to the wild type level; unregulated replication itself is a possible cause of the residual excess expansion in the dam mutH double mutant. The mechanism by which improperly regulated replication forks provoke more recombination events than normally regulated replication forks is unknown, but there are a number of possible explanations. They might be more prone to breaking down or to running into each other, or just more numerous in the cell.
Duplication of ϕ(lacI33-lacZ)-lacY
Reference, source, or construction
polAΔfrt-kan/F' polA+ camR
M. Marinus; polA allele 
derivative of KM32 
substitution of Tn903 aph for Tn10 tetRA in sulAΔtet 
FC691 × P1•TP547
TP730 × pTP822 linear 
TP832 × cat15,16 pcr of Tn9c
MG1655 × P1•TP872
TP732 × pTP1061 lineare
TP889 × P1•TP922
TP922 × Toc1,2 pcr of Tn9
TP798 × din7,8 pcr of Tn10
TP889 was transduced with a P1 lysate of an unnamed intermediate strain, TP798 × LAT2,3 pcr of Tn21 aadA
TP929 × P1•TP942
TP1004 × P1•TP1000
TP1004 × P1•TP796
TP1004 × P1•TP577
TP1004 × P1•TP538
TP1004 × P1•TP664
TP1004 × P1•TP643
TP1004 × P1•TP838
TP1004 × P1•TP540
TP1004 × P1•TP797
TP1004 × P1•TP645
Spontaneous chloramphenicol-sensitive TP1004 derivative
TP798 × recJ1,2 pcr of Tn10
TP798 × rnhA1,2 pcr of Tn10
TP798 × polB1,2 pcr of Tn10
TP1004 × P1•TP1031
TP1004 × P1•TP1032
TP1004 × P1•TP1033
TP832 × P1•TP942
TP1004 × P1•GM3819
TP1004 × P1•TP605
TP1004/pKM208 × uvrD1,2 pcr of Tn10
(lacIZ33Y)2-cat sulAΔtet lexA71::Tn5
TP1043 × P1•MV2104
TP1004/pKM208  × umDC1,2 pcr of Tn10
TP1004/pKM208 × malF1,2 pcr of TP997
TP1004/pKM208 × rpoS1,2 pcr of Tn10
TP1004/pKM208 × ADA1,2 pcr of Tn903 aph
TP1004/pKM208 × priA1,2 pcr of Tn903 aph
TP1004/pKM208 × relA1,2 pcr of Tn903 aph
(lacIZ33Y)2-cat sulAΔtet priAΔkan
TP1043 × P1•1054
red-amp (lacIZ33Y)2-cat recAΔtet
TP1038 × P1•796
red-amp (lacIZ33Y)2-cat recFΔtet
TP1038 × P1•577
red-amp (lacIZ33Y)2-cat recGΔtet
TP1038 × P1•538
red-amp (lacIZ33Y)2-cat rnhAΔtet
TP1038 × P1•1035
TP1022 × P1•796
TP1004/pKM208 × recA3,4 pcr of Tn903 aph
TP1004/pKM208 × mutH1,2 pcr of 1016
TP1004/pKM208 × mutM1,2 pcr of Tn10
TP1004/pKM208 × mutY1,2 pcr of Tn903 aph
(lacIZ33Y)2-cat mutMΔtet mutYΔkan
TP1065 × P1TP1066
TP889 × P1•TP796
TP889 × P1•TP838
TP889 × P1•TP797
TP1004 × P1•JW3835
TP1004/pKM208 × yfgL1,2 pcr of Tn903 aph
TP1050 × P1•MV1154, selection for Mal+, screen for UV-sensitivity
(lacIZ33Y)2-cat damΔkan mutHΔtet
TP1064 × P1•GM3819
(lacIZ33Y)2-cat srl300::Tn10 recAo281
TP1063 × P1•MV1132
(lacIZ33Y)2-cat ruvCΔtet recGΔkan
TP1015 × P1•TP539
TP1095 × P1•TP1063, selection for Srl+, screen for kanamycin sensitivity and the Sph site created by the recAo281 mutation 
Strains to be tested for reversion to Lac+ were grown to saturation in M9 0.1% glycerol minimal medium at 37°C, and plated on M9 0.1% lactose plates at 37°C. M9 minimal media, supplemented with thiamine at 5 μg/ml, were as described . In most cases, viable duplication-positive titers were determined by plating on LB agar supplemented with chloramphenicol at 10 μg/ml, which permits colony formation only by bacteria retaining the cat gene between the duplicated segments. Strains with poor viability in rich media (ruvC recG, polA, priA) were titered on M9 glucose plates; retention of the duplication in these cases was assessed by testing the chloramphenicol resistance of individual colonies from the titer plates. Lactose minimal plates were inoculated with 1–2 × 109 cells, either of the strain to be tested by itself, or, in most cases, of the strain to be tested plus a non-reverting, lac deletion-bearing scavenger strain . The rates of appearance of Lac+ colonies shown in Figures 2, 3, and 4 are calculated as Lac+ colonies per million chloramphenicol-resistant viable cells plated (or per million viable cells, in the cases of the single copy strains).
Revertant colonies appearing on the M9 lactose plates were streaked on M9 lactose plates, which were incubated at 37°C until visible colonies formed. Heavy inocula constituting the bulk of the growth from the streaks were then scraped from the plate and grown to saturation in 5 ml M9 0.1% lactose minimal medium at 37°C. (Some of the revertant colonies tested, in the wild type background only, were inoculated directly from the selection plates into liquid lactose minimal medium; but all revertants in the wild type background tested positive for expanded arrays, regardless of the variation in culture methods). DNA was extracted by the use of a procedure involving freezing and thawing, lysozyme digestion, extraction with a phenol/chloroform/isoamyl alcohol mixture, extraction with ether, and precipitation with ethanol . Portions were digested with EcoR1 and RNase, and subjected to electrophoresis in an agarose gel, followed by ethidium bromide staining. For quantitation of the repeat-specific bands, standards consisting of HindIII-digested phage lambda DNA of known concentration were included in the gel, in separate lanes. Total DNA in the sample was quantitated by spotting RNase-treated samples on an agarose slab containing ethidium bromide at 1 μg/ml, along with standards of known concentration. Band and spot intensities were measured by the use of digital photography and Kodak 1D software.
Mutations to be tested for their effects on survival on lactose minimal medium were crossed into a strain bearing a deletion of the lac operon. Cultures were grown to saturation in M9 0.1% glycerol as in the Lac+ reversion test, and deposited on the surfaces of 0.6 ml M9 lactose agar plugs at the bottom of 12 × 75 mm plastic tubes, at approximately the same plating density (relative to volume of medium) as the bacteria in the Lac+ reversion test. The tubes were incubated at 37°C. Cells were suspended by vortexing in 2 ml of buffer, and titered on M9 glucose plates, on days 0 and 7. The ratio of titer on day 7 to titer on day 0 for the wild type control was 0.83 ± 0.12 (mean ± standard error from six measurements).
I thank Patricia Foster, Michael Volkert, Martin Marinus, and Susan Lovett for helpful discussions; Michael Volkert and Martin Marinus for strains; and Rosemary Proff, Dery Miller, Shilpa Nadimpalli, Bach Nguyen, Juliet Zhang, Aiden Galarza, and Matthew Lim for technical assistance. This research was supported by US National Science Foundation grant MCB-0234991, and by a grant from the University of Massachusetts Healey Endowment.
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