Formation of an F' Plasmid by Recombination between Imperfectly Repeated Chromosomal Rep Sequences: a Closer Look at an Old Friend(F'128 pro lac)
Department of Biology, University of Utah, Salt Lake City, Utah 84122g$si, 百拇医药
Received 1 August 2002/ Accepted 28 October 2002g$si, 百拇医药
ABSTRACTg$si, 百拇医药
Plasmid F'128 was formed by an exchange between chromosomal Rep sequences that placed lac near dinB between many pairs of Rep sequences. Plasmid F'128 is critical for selection-enhanced lac reversion (adaptive mutation), which requires prior lac amplification. The structure of F'128 supports the idea that amplification is initiated by Rep-Rep recombination and that general mutagenesis requires coamplification of dinB (error-prone polymerase) with lac.g$si, 百拇医药
TEXTg$si, 百拇医药
Plasmid F'128 (proAB lac) is a type II F' plasmid (37) formed by recombination between chromosomal sequences that flank the F plasmid insertion site. F'128 was excised from Escherichia coli Hfr P804 and was shown genetically to include the entire lac operon and the nearby proA and proB genes but not proC (24). The F'128 plasmid was widely used to study the lac operon (8, 30, 45, 49), mutation and mutagen specificity (9, 29), deletion and inversion formation (1, 38, 42), gene amplification (43, 48), and mechanisms of F-plasmid integration (10, 20).
More recently, F'128 has been used in experiments interpreted as indicating that bacteria elevate their general mutation rate in response to selective stress (adaptive mutation) (6, 7, 12, 36, 44). Selection-enhanced reversion requires that the target lac operon be located on a conjugative plasmid (18, 33, 34, 40) with an expressed tra (transfer) operon (14, 15). General mutagenesis accompanies reversion only when lac is on the particular plasmid F'128 and is located cis to the dinB gene (E. S. Slechta, K. Bunny, E. Kofoid, K. Savaraman, S. Gerum, D. I. Andersson, and J. R. Roth, unpublished results). It has been claimed that general mutagenesis is preferentially directed toward the whole F'128 plasmid (13). The role of F'128 is the least well understood aspect of the adaptive-mutation phenomenology.pa., 百拇医药
The amplification-mutagenesis model (3, 22) proposes that selection has no direct effect on mutation but favors growth of cells with a lac amplification. The F' plasmid contributes to reversion by stimulating lac duplication and amplification (40). This alone does not explain why F'128 is specifically required for general mutagenesis, why only a subset of lac revertants appear to be mutagenized (35), or why general mutagenesis might be more intense on F'128 than in the chromosome (13). The structure of F'128 reported here suggests answers to these questions.
Original identification of the F'128 integration site. Previous work (10) showed that the Hfr strain, from which F'128 was formed, arose by recombination between two IS3 sequences, one in the F plasmid and one in the chromosome. Genetic results demonstrated that the F'128 plasmid carries chromosomal genes from both sides of this F integration site and thus was excised from the Hfr chromosome by recombination between chromosomal sequences (24).n, http://www.100md.com
Restriction map for F'128. A restriction map of F'128 was assembled based on available sequence data and pulsed-field gel electrophoresis following digestion with BlnI, NotI, SfiI, or XbaI, assuming that the F plasmid was integrated as described previously (10). This map allowed identification of chromosomal regions within which recombination must have occurred to generate the final plasmid. The sequences of these general regions were examined for repeated elements that might have supported this recombination.
Identification of sequence repeats in regions containing the excision point. Comparison of the two identified regions by using FASTA (32) revealed two pairs of extensive but imperfect repeats. A sequence just clockwise of the mhp operon in the E. coli chromosome was similar to two different sequences located immediately counterclockwise of the dinB gene . All three tracts turned out to be groups of Rep (repetitive extragenic palindrome) elements (23). Such clusters are also called bacterial interspersed mosaic elements (17). Individual Rep elements are related, imperfectly palindromic 33- to 40-nucleotide sequences that have been placed in three subclasses, Y, Z1, and Z2 (4, 16). These elements frequently appear in clusters that have been designated bacterial interspersed mosaic elements (BIMEs).9, 百拇医药
fig.ommitted9, 百拇医药
Sequence repeats flanking lac in the E. coli chromosome. Triangle, insertion site of the F plasmid, at which two IS3 elements recombined to integrate F and form Hfr P804 (10). The sequences designated Rep5.3, Rep5.4, and Rep8.1 are clusters of Rep sequences whose order and orientation are indicated in parentheses; Y and Z2 are two of the three general types of Rep elements (4). Arrows above the map indicate primers tested for PCR amplification of F'128 junction points formed during excision. The pair of direct repeats responsible for F'128 excision are indicated below the map. The three Rep clusters were designated R17, R19, and R32 in a previous discussion of these elements (36a).
The positions of the potential recombination sites are diagrammed in and were designated based on their approximate position in minutes and the nature and orientation of their subelements. Clusters Rep5.3 and Rep5.4 lie counterclockwise of dinB in the E. coli genome. Cluster Rep5.3 is of the form ()( (< and > symbols indicate the orientations of the subelements) and lies immediately counterclockwise of yafJ. Cluster Rep5.4 has the structure (Z2>)()( counterclockwise of fhiA. On the opposite (clockwise) side of lac, cluster Rep8.1 has the same structure as Rep5.4 [(Z2>)()( and lies immediately clockwise of the mhp and yaiL genes. If F' plasmid excision occurred by simple Rep-Rep recombination, it would require an exchange between Rep8.1 (on the right) and either Rep5.4 or Rep5.3 on the left .g@y, http://www.100md.com
Identification of the excision point. PCR amplification across both the potential hybrid junctions within F'128 was attempted. A very strong signal was found with primers in yafJ and yaiL. The sequence of the product corresponded to a Rep5.3-Rep8.1 hybrid with a crossover within a 9-bp block of perfect alignment . The region including the exchange is similar in size to those that support formation of some deletions (11, 47) and duplications (48). The small extent of perfect homology is probably compensated for by the extensive imperfect pairing of nearby sequences.
fig.ommitted0{9eu'{, http://www.100md.com
Crossover event that formed F'128. This plasmid formed by recombination between chromosomal regions flanking the F insertion site. The actual exchange is within a 9-bp perfect repeat flanked by extensive imperfect repeats. The positions of these sequences are indicated by base pair numbers in the E. coli genome sequence and by genetic map locations in minutes.0{9eu'{, http://www.100md.com
A weak PCR signal was generated by primers in fhiA and yaiL, implying that a few cells carry plasmids with the Rep5.4-Rep8.1 junction. It seems likely that the two Rep clusters that remain in the final plasmid after an exchange between Rep5.3 and Rep8.1 can recombine occasionally to generate a deletion that removes about 4 kb and that brings the fhiA and yaiL sequences close together. The formation of F'128 and of this deletion provides evidence that Rep elements can recombine (see below). The whole process of F'128 formation is diagrammed in.0{9eu'{, http://www.100md.com
fig.ommitted0{9eu'{, http://www.100md.com
Formation and final structure of F'128. Filled arrows, genes; open arrows, insertion sequences. Gray arrows designate nested or recombinant insertion sequences. The arc inside the map at the right indicates the extent of pOX38, a minimal F plasmid derivative still capable of both vegetative replication and conjugation (19).+4, 百拇医药
The event that formed F'128 brought lac close to dinB. The dinB gene carried by F'128 encodes an SOS-induced error-prone polymerase (25) that is thought to be responsible for general mutagenesis (adaptive mutation) during lac starvation (28, 41, 44). In the chromosome, lac and dinB genes are separated by over 100 kb. However, on F'128 they are separated by only 16.5 kb. On lactose medium, cells with a leaky lac mutation can grow if they amplify their lac region. The size of the amplified region is generally between 10 and 40 kb (3, 21, 22, 43, 48). The proximity of lac and dinB on F'128 makes it likely that these two genes are at least occasionally coamplified during growth under selection. If dinB amplification is a prerequisite for mutagenesis, then a problem regarding DinB-dependent mutagenesis could be resolved.
SOS induction of a single dinB gene does not cause mutagenesis in either Salmonella enterica (27, 31) or E. coli (26). Mutagenesis by DinB has been seen only when the enzyme is overproduced from plasmids (25). This overproduction could be provided by coamplification of dinB with the nearby lac operon (Slechta et al., unpublished results).5?, 百拇医药
Rep sequences are abundant on F'128. The frequency of Rep elements in the chromosomal region carried by F'128 is about fourfold higher than that in the chromosome as whole. (Two of the Rep sequences recombined to form the plasmid.) The frequency of Rep elements near the lac operon is particularly high, and many flanking pairs are oriented so as to support lac duplication (arcs in ). Roughly 10% of predicted Rep-mediated duplications (inside arcs in include dinB as well as lac. This may explain why only about 10% of Lac+ revertants experience heavy mutagenesis while 90% experience little or none (35). We suggest that the mutagenized lac revertants (10%) arise within clones growing with a lac amplification that includes dinB. Thus most revertants (90%) arise in strains whose lac amplification lacks dinB and that are not mutagenized; in these clones, reversion is enhanced only by an increase in lac copy number. Preliminary results support this possibility (Slechta et al., unpublished results).
fig.ommitted$)sks, http://www.100md.com
Distribution of Rep elements on F'128 The three classes of Rep elements (Y, Z1, and Z2) are indicated wherever they occur in F'128. While each is an imperfect palindrome, the imperfections are such that a polarity can be assigned to each type. Arrowhead, orientation of each element. All elements with arrowheads pointing away from the center of the circle are in the same orientation; those with arrowheads pointing toward the center are in the opposite orientation. Note that no Rep elements are found within F-plasmid sequences.$)sks, http://www.100md.com
Duplications and deletions have previously been shown to form by recombination between Rep elements (2, 39, 46). In the most extensive study, a Rep element between the hisD and hisG genes of the S. enterica histidine operon recombined with a series of distant Rep elements to form a duplication which places the hisD gene adjacent to a foreign promoter at each duplication join point (39). It is proposed here that the same event generates the lac duplications on F'128 that initiate reversion under selection.
Coamplification of tetA and lac may explain the apparent direction of mutation toward F'128. While selective stress causes very little mutagenesis of chromosomal genes independent of lac reversion (5, 44), the claim that it strongly mutagenizes the entire F'128 plasmid has been made (13). This was supported by the observation that starvation for lactose enhanced the reversion of a tetA frameshift mutation in a Tn10 element on F'128 thought to be located too far from lac to be included in any lac amplification. During starvation for lactose, a population carrying both the lac and tetA mutations on F'128 was seen to accumulate tetracycline-resistant (tetA+) mutants that had not reverted to lac+ (13).z$, 百拇医药
We sequenced the Tn10 insertion used in this experiment and found that it lies within the mhpC gene (bp 132), very close to lac. Another Tn10 insertion in this gene (bp 782) is known to be included in lac amplifications that arise under selection (18). Many pairs of Rep elements flank the mhpC-lac gene pair . It seems likely that tetA reversion was enhanced because many clones grew with a lac amplification that included the mhpC::Tn10 element (and perhaps dinB as well). For such clones, tetA reversion frequency would be enhanced by multiple tetA copies and possibly by mutagenic overexpression of dinB+. This reversion can occur in clones that have not yet experienced Lac+ reversion.
Summary. The structure of F'128 suggests answers for several questions regarding the adaptive-mutation controversy. Why does selection-enhanced reversion require that the lac gene be on a conjugative plasmid (34)? Why must the plasmid genes for conjugative transfer (tra) be expressed for optimal lac reversion (14, 15)? Why does selection-induced general mutagenesis require that the lac mutation be located on the specific plasmid F'128 (Slechta et al., unpublished results)? How can DinB cause general mutagenesis in only some of the revertant clones (35)? We propose that the transfer origin of conjugative plasmids generates DNA ends that simulate lac amplification (40). The Rep sequences that flank lac on F'128 may allow frequent amplification. General SOS mutagenesis may rely on the proximity (and coamplification) of the dinB and lac genes. These special features of F'128 may explain why the apparent directed mutation and the induced general mutagenesis seen in the Cairns system are not observed in other genetic systems.
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Received 1 August 2002/ Accepted 28 October 2002g$si, 百拇医药
ABSTRACTg$si, 百拇医药
Plasmid F'128 was formed by an exchange between chromosomal Rep sequences that placed lac near dinB between many pairs of Rep sequences. Plasmid F'128 is critical for selection-enhanced lac reversion (adaptive mutation), which requires prior lac amplification. The structure of F'128 supports the idea that amplification is initiated by Rep-Rep recombination and that general mutagenesis requires coamplification of dinB (error-prone polymerase) with lac.g$si, 百拇医药
TEXTg$si, 百拇医药
Plasmid F'128 (proAB lac) is a type II F' plasmid (37) formed by recombination between chromosomal sequences that flank the F plasmid insertion site. F'128 was excised from Escherichia coli Hfr P804 and was shown genetically to include the entire lac operon and the nearby proA and proB genes but not proC (24). The F'128 plasmid was widely used to study the lac operon (8, 30, 45, 49), mutation and mutagen specificity (9, 29), deletion and inversion formation (1, 38, 42), gene amplification (43, 48), and mechanisms of F-plasmid integration (10, 20).
More recently, F'128 has been used in experiments interpreted as indicating that bacteria elevate their general mutation rate in response to selective stress (adaptive mutation) (6, 7, 12, 36, 44). Selection-enhanced reversion requires that the target lac operon be located on a conjugative plasmid (18, 33, 34, 40) with an expressed tra (transfer) operon (14, 15). General mutagenesis accompanies reversion only when lac is on the particular plasmid F'128 and is located cis to the dinB gene (E. S. Slechta, K. Bunny, E. Kofoid, K. Savaraman, S. Gerum, D. I. Andersson, and J. R. Roth, unpublished results). It has been claimed that general mutagenesis is preferentially directed toward the whole F'128 plasmid (13). The role of F'128 is the least well understood aspect of the adaptive-mutation phenomenology.pa., 百拇医药
The amplification-mutagenesis model (3, 22) proposes that selection has no direct effect on mutation but favors growth of cells with a lac amplification. The F' plasmid contributes to reversion by stimulating lac duplication and amplification (40). This alone does not explain why F'128 is specifically required for general mutagenesis, why only a subset of lac revertants appear to be mutagenized (35), or why general mutagenesis might be more intense on F'128 than in the chromosome (13). The structure of F'128 reported here suggests answers to these questions.
Original identification of the F'128 integration site. Previous work (10) showed that the Hfr strain, from which F'128 was formed, arose by recombination between two IS3 sequences, one in the F plasmid and one in the chromosome. Genetic results demonstrated that the F'128 plasmid carries chromosomal genes from both sides of this F integration site and thus was excised from the Hfr chromosome by recombination between chromosomal sequences (24).n, http://www.100md.com
Restriction map for F'128. A restriction map of F'128 was assembled based on available sequence data and pulsed-field gel electrophoresis following digestion with BlnI, NotI, SfiI, or XbaI, assuming that the F plasmid was integrated as described previously (10). This map allowed identification of chromosomal regions within which recombination must have occurred to generate the final plasmid. The sequences of these general regions were examined for repeated elements that might have supported this recombination.
Identification of sequence repeats in regions containing the excision point. Comparison of the two identified regions by using FASTA (32) revealed two pairs of extensive but imperfect repeats. A sequence just clockwise of the mhp operon in the E. coli chromosome was similar to two different sequences located immediately counterclockwise of the dinB gene . All three tracts turned out to be groups of Rep (repetitive extragenic palindrome) elements (23). Such clusters are also called bacterial interspersed mosaic elements (17). Individual Rep elements are related, imperfectly palindromic 33- to 40-nucleotide sequences that have been placed in three subclasses, Y, Z1, and Z2 (4, 16). These elements frequently appear in clusters that have been designated bacterial interspersed mosaic elements (BIMEs).9, 百拇医药
fig.ommitted9, 百拇医药
Sequence repeats flanking lac in the E. coli chromosome. Triangle, insertion site of the F plasmid, at which two IS3 elements recombined to integrate F and form Hfr P804 (10). The sequences designated Rep5.3, Rep5.4, and Rep8.1 are clusters of Rep sequences whose order and orientation are indicated in parentheses; Y and Z2 are two of the three general types of Rep elements (4). Arrows above the map indicate primers tested for PCR amplification of F'128 junction points formed during excision. The pair of direct repeats responsible for F'128 excision are indicated below the map. The three Rep clusters were designated R17, R19, and R32 in a previous discussion of these elements (36a).
The positions of the potential recombination sites are diagrammed in and were designated based on their approximate position in minutes and the nature and orientation of their subelements. Clusters Rep5.3 and Rep5.4 lie counterclockwise of dinB in the E. coli genome. Cluster Rep5.3 is of the form (
Identification of the excision point. PCR amplification across both the potential hybrid junctions within F'128 was attempted. A very strong signal was found with primers in yafJ and yaiL. The sequence of the product corresponded to a Rep5.3-Rep8.1 hybrid with a crossover within a 9-bp block of perfect alignment . The region including the exchange is similar in size to those that support formation of some deletions (11, 47) and duplications (48). The small extent of perfect homology is probably compensated for by the extensive imperfect pairing of nearby sequences.
fig.ommitted0{9eu'{, http://www.100md.com
Crossover event that formed F'128. This plasmid formed by recombination between chromosomal regions flanking the F insertion site. The actual exchange is within a 9-bp perfect repeat flanked by extensive imperfect repeats. The positions of these sequences are indicated by base pair numbers in the E. coli genome sequence and by genetic map locations in minutes.0{9eu'{, http://www.100md.com
A weak PCR signal was generated by primers in fhiA and yaiL, implying that a few cells carry plasmids with the Rep5.4-Rep8.1 junction. It seems likely that the two Rep clusters that remain in the final plasmid after an exchange between Rep5.3 and Rep8.1 can recombine occasionally to generate a deletion that removes about 4 kb and that brings the fhiA and yaiL sequences close together. The formation of F'128 and of this deletion provides evidence that Rep elements can recombine (see below). The whole process of F'128 formation is diagrammed in.0{9eu'{, http://www.100md.com
fig.ommitted0{9eu'{, http://www.100md.com
Formation and final structure of F'128. Filled arrows, genes; open arrows, insertion sequences. Gray arrows designate nested or recombinant insertion sequences. The arc inside the map at the right indicates the extent of pOX38, a minimal F plasmid derivative still capable of both vegetative replication and conjugation (19).+4, 百拇医药
The event that formed F'128 brought lac close to dinB. The dinB gene carried by F'128 encodes an SOS-induced error-prone polymerase (25) that is thought to be responsible for general mutagenesis (adaptive mutation) during lac starvation (28, 41, 44). In the chromosome, lac and dinB genes are separated by over 100 kb. However, on F'128 they are separated by only 16.5 kb. On lactose medium, cells with a leaky lac mutation can grow if they amplify their lac region. The size of the amplified region is generally between 10 and 40 kb (3, 21, 22, 43, 48). The proximity of lac and dinB on F'128 makes it likely that these two genes are at least occasionally coamplified during growth under selection. If dinB amplification is a prerequisite for mutagenesis, then a problem regarding DinB-dependent mutagenesis could be resolved.
SOS induction of a single dinB gene does not cause mutagenesis in either Salmonella enterica (27, 31) or E. coli (26). Mutagenesis by DinB has been seen only when the enzyme is overproduced from plasmids (25). This overproduction could be provided by coamplification of dinB with the nearby lac operon (Slechta et al., unpublished results).5?, 百拇医药
Rep sequences are abundant on F'128. The frequency of Rep elements in the chromosomal region carried by F'128 is about fourfold higher than that in the chromosome as whole. (Two of the Rep sequences recombined to form the plasmid.) The frequency of Rep elements near the lac operon is particularly high, and many flanking pairs are oriented so as to support lac duplication (arcs in ). Roughly 10% of predicted Rep-mediated duplications (inside arcs in include dinB as well as lac. This may explain why only about 10% of Lac+ revertants experience heavy mutagenesis while 90% experience little or none (35). We suggest that the mutagenized lac revertants (10%) arise within clones growing with a lac amplification that includes dinB. Thus most revertants (90%) arise in strains whose lac amplification lacks dinB and that are not mutagenized; in these clones, reversion is enhanced only by an increase in lac copy number. Preliminary results support this possibility (Slechta et al., unpublished results).
fig.ommitted$)sks, http://www.100md.com
Distribution of Rep elements on F'128 The three classes of Rep elements (Y, Z1, and Z2) are indicated wherever they occur in F'128. While each is an imperfect palindrome, the imperfections are such that a polarity can be assigned to each type. Arrowhead, orientation of each element. All elements with arrowheads pointing away from the center of the circle are in the same orientation; those with arrowheads pointing toward the center are in the opposite orientation. Note that no Rep elements are found within F-plasmid sequences.$)sks, http://www.100md.com
Duplications and deletions have previously been shown to form by recombination between Rep elements (2, 39, 46). In the most extensive study, a Rep element between the hisD and hisG genes of the S. enterica histidine operon recombined with a series of distant Rep elements to form a duplication which places the hisD gene adjacent to a foreign promoter at each duplication join point (39). It is proposed here that the same event generates the lac duplications on F'128 that initiate reversion under selection.
Coamplification of tetA and lac may explain the apparent direction of mutation toward F'128. While selective stress causes very little mutagenesis of chromosomal genes independent of lac reversion (5, 44), the claim that it strongly mutagenizes the entire F'128 plasmid has been made (13). This was supported by the observation that starvation for lactose enhanced the reversion of a tetA frameshift mutation in a Tn10 element on F'128 thought to be located too far from lac to be included in any lac amplification. During starvation for lactose, a population carrying both the lac and tetA mutations on F'128 was seen to accumulate tetracycline-resistant (tetA+) mutants that had not reverted to lac+ (13).z$, 百拇医药
We sequenced the Tn10 insertion used in this experiment and found that it lies within the mhpC gene (bp 132), very close to lac. Another Tn10 insertion in this gene (bp 782) is known to be included in lac amplifications that arise under selection (18). Many pairs of Rep elements flank the mhpC-lac gene pair . It seems likely that tetA reversion was enhanced because many clones grew with a lac amplification that included the mhpC::Tn10 element (and perhaps dinB as well). For such clones, tetA reversion frequency would be enhanced by multiple tetA copies and possibly by mutagenic overexpression of dinB+. This reversion can occur in clones that have not yet experienced Lac+ reversion.
Summary. The structure of F'128 suggests answers for several questions regarding the adaptive-mutation controversy. Why does selection-enhanced reversion require that the lac gene be on a conjugative plasmid (34)? Why must the plasmid genes for conjugative transfer (tra) be expressed for optimal lac reversion (14, 15)? Why does selection-induced general mutagenesis require that the lac mutation be located on the specific plasmid F'128 (Slechta et al., unpublished results)? How can DinB cause general mutagenesis in only some of the revertant clones (35)? We propose that the transfer origin of conjugative plasmids generates DNA ends that simulate lac amplification (40). The Rep sequences that flank lac on F'128 may allow frequent amplification. General SOS mutagenesis may rely on the proximity (and coamplification) of the dinB and lac genes. These special features of F'128 may explain why the apparent directed mutation and the induced general mutagenesis seen in the Cairns system are not observed in other genetic systems.
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