Limits...
Mutability and importance of a hypermutable cell subpopulation that produces stress-induced mutants in Escherichia coli.

Gonzalez C, Hadany L, Ponder RG, Price M, Hastings PJ, Rosenberg SM - PLoS Genet. (2008)

Bottom Line: First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state.Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac(+) mutation independently of the HMS.We consider a model in which HMS differentiation is controlled by stress responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
In bacterial, yeast, and human cells, stress-induced mutation mechanisms are induced in growth-limiting environments and produce non-adaptive and adaptive mutations. These mechanisms may accelerate evolution specifically when cells are maladapted to their environments, i.e., when they are are stressed. One mechanism of stress-induced mutagenesis in Escherichia coli occurs by error-prone DNA double-strand break (DSB) repair. This mechanism was linked previously to a differentiated subpopulation of cells with a transiently elevated mutation rate, a hypermutable cell subpopulation (HMS). The HMS could be important, producing essentially all stress-induced mutants. Alternatively, the HMS was proposed to produce only a minority of stress-induced mutants, i.e., it was proposed to be peripheral. We characterize three aspects of the HMS. First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state. This implies that these mutants are not necessarily an evolutionary dead end, and could contribute to adaptive evolution. Second, we show that stress-induced Lac(+) mutants, with and without evidence of descent from the HMS, have similar Lac(+) mutation sequences. This provides evidence that HMS-descended and most stress-induced mutants form via a common mechanism. Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac(+) mutation independently of the HMS. This and the previous finding support the hypothesis that the HMS underlies most stress-induced mutants, not just a minority of them, i.e., it is important. We consider a model in which HMS differentiation is controlled by stress responses. Differentiation of an HMS potentially limits the risks of mutagenesis in cell clones.

Show MeSH

Related in: MedlinePlus

Lac+ Mutation Sequences in HMS-Descended Cells.The sequences of stress-induced Lac+ frameshift-reversion mutations are nearly all -1 deletions in small mononucleotide repeats at the positions shown. Those from cells carrying chromosomal “secondary” mutations, detected in our screens, (•, this study) are indistinguishable from stress-induced Lac+ frameshift reversions from cells without detected secondary mutations (X, data from [12],[13]). The 30 new mutants sequenced (•) were identified in a previous screen for Lac+ mutants with chromosomal loss-of-function mutations [29] conferring the following phenotypes: Mal− (15 mutants); Xyl− (10 mutants); minimal temperature sensitive (TS), which grow on minimal medium at 37° but not at 42° (1 mutant); Mal− Xyl− double mutants (3 mutants); and Mal− minimal TS (1 mutant).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2543114&req=5

pgen-1000208-g001: Lac+ Mutation Sequences in HMS-Descended Cells.The sequences of stress-induced Lac+ frameshift-reversion mutations are nearly all -1 deletions in small mononucleotide repeats at the positions shown. Those from cells carrying chromosomal “secondary” mutations, detected in our screens, (•, this study) are indistinguishable from stress-induced Lac+ frameshift reversions from cells without detected secondary mutations (X, data from [12],[13]). The 30 new mutants sequenced (•) were identified in a previous screen for Lac+ mutants with chromosomal loss-of-function mutations [29] conferring the following phenotypes: Mal− (15 mutants); Xyl− (10 mutants); minimal temperature sensitive (TS), which grow on minimal medium at 37° but not at 42° (1 mutant); Mal− Xyl− double mutants (3 mutants); and Mal− minimal TS (1 mutant).

Mentions: In models in which the HMS is predicted to produce only a 10% minority of the Lac+ stress-induced mutants, the mutations that occur in the HMS and give rise to Lac+ phenotype are proposed to occur via a different molecular mechanism from that that generates the 90% majority of stress-induced Lac+ mutations [30],[32]. If true, those Lac+ mutations that arise from HMS cells might be predicted to display different reversion-mutation sequences from the majority of stress-induced Lac+ mutations. We examined the Lac+ mutation sequences from stress-induced mutants that demonstrably descended from the HMS, as seen by their carrying an unselected “secondary” chromosomal mutation, and compared these with the published sequences of stress-induced Lac+ mutations [12],[13]. We sequenced a 250bp region spanning the +1 frameshift mutation of the lacI-lacZ (EG10525 and EG10527) fusion gene from 30 independent Lac+ point-mutant isolates carrying secondary mutations. We find that the mutation sequence profile is indistinguishable from those previously reported for stress-induced mutants [12],[13]: dominated by -1 deletions in small mononucleotide repeats with a hotspot at the position of the initial lac frameshift allele (Figure 1). These data support the hypothesis that the mechanism of mutagenesis in the HMS cells is similar to or the same as the stress-induced mutagenesis mechanism that generates all or most Lac+ point mutations. This distinctive mutation spectrum differs from spontaneous generation-dependent reversions of this lac allele, which are more heterogeneous [12],[13]. Summarized in Table S2, these include about half -1 deletions at mononucleotide repeats, and half carrying -1's not at repeats, 2–8 bp insertions, and large insertions and deletions. Instead, the stress-induced Lac+ frameshift-reversion sequences resemble the frameshift component of the error spectrum of DinB/Pol IV [36],[37] which is responsible for ≥85% of Lac+ point mutations in this assay system [24].


Mutability and importance of a hypermutable cell subpopulation that produces stress-induced mutants in Escherichia coli.

Gonzalez C, Hadany L, Ponder RG, Price M, Hastings PJ, Rosenberg SM - PLoS Genet. (2008)

Lac+ Mutation Sequences in HMS-Descended Cells.The sequences of stress-induced Lac+ frameshift-reversion mutations are nearly all -1 deletions in small mononucleotide repeats at the positions shown. Those from cells carrying chromosomal “secondary” mutations, detected in our screens, (•, this study) are indistinguishable from stress-induced Lac+ frameshift reversions from cells without detected secondary mutations (X, data from [12],[13]). The 30 new mutants sequenced (•) were identified in a previous screen for Lac+ mutants with chromosomal loss-of-function mutations [29] conferring the following phenotypes: Mal− (15 mutants); Xyl− (10 mutants); minimal temperature sensitive (TS), which grow on minimal medium at 37° but not at 42° (1 mutant); Mal− Xyl− double mutants (3 mutants); and Mal− minimal TS (1 mutant).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2543114&req=5

pgen-1000208-g001: Lac+ Mutation Sequences in HMS-Descended Cells.The sequences of stress-induced Lac+ frameshift-reversion mutations are nearly all -1 deletions in small mononucleotide repeats at the positions shown. Those from cells carrying chromosomal “secondary” mutations, detected in our screens, (•, this study) are indistinguishable from stress-induced Lac+ frameshift reversions from cells without detected secondary mutations (X, data from [12],[13]). The 30 new mutants sequenced (•) were identified in a previous screen for Lac+ mutants with chromosomal loss-of-function mutations [29] conferring the following phenotypes: Mal− (15 mutants); Xyl− (10 mutants); minimal temperature sensitive (TS), which grow on minimal medium at 37° but not at 42° (1 mutant); Mal− Xyl− double mutants (3 mutants); and Mal− minimal TS (1 mutant).
Mentions: In models in which the HMS is predicted to produce only a 10% minority of the Lac+ stress-induced mutants, the mutations that occur in the HMS and give rise to Lac+ phenotype are proposed to occur via a different molecular mechanism from that that generates the 90% majority of stress-induced Lac+ mutations [30],[32]. If true, those Lac+ mutations that arise from HMS cells might be predicted to display different reversion-mutation sequences from the majority of stress-induced Lac+ mutations. We examined the Lac+ mutation sequences from stress-induced mutants that demonstrably descended from the HMS, as seen by their carrying an unselected “secondary” chromosomal mutation, and compared these with the published sequences of stress-induced Lac+ mutations [12],[13]. We sequenced a 250bp region spanning the +1 frameshift mutation of the lacI-lacZ (EG10525 and EG10527) fusion gene from 30 independent Lac+ point-mutant isolates carrying secondary mutations. We find that the mutation sequence profile is indistinguishable from those previously reported for stress-induced mutants [12],[13]: dominated by -1 deletions in small mononucleotide repeats with a hotspot at the position of the initial lac frameshift allele (Figure 1). These data support the hypothesis that the mechanism of mutagenesis in the HMS cells is similar to or the same as the stress-induced mutagenesis mechanism that generates all or most Lac+ point mutations. This distinctive mutation spectrum differs from spontaneous generation-dependent reversions of this lac allele, which are more heterogeneous [12],[13]. Summarized in Table S2, these include about half -1 deletions at mononucleotide repeats, and half carrying -1's not at repeats, 2–8 bp insertions, and large insertions and deletions. Instead, the stress-induced Lac+ frameshift-reversion sequences resemble the frameshift component of the error spectrum of DinB/Pol IV [36],[37] which is responsible for ≥85% of Lac+ point mutations in this assay system [24].

Bottom Line: First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state.Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac(+) mutation independently of the HMS.We consider a model in which HMS differentiation is controlled by stress responses.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, United States of America.

ABSTRACT
In bacterial, yeast, and human cells, stress-induced mutation mechanisms are induced in growth-limiting environments and produce non-adaptive and adaptive mutations. These mechanisms may accelerate evolution specifically when cells are maladapted to their environments, i.e., when they are are stressed. One mechanism of stress-induced mutagenesis in Escherichia coli occurs by error-prone DNA double-strand break (DSB) repair. This mechanism was linked previously to a differentiated subpopulation of cells with a transiently elevated mutation rate, a hypermutable cell subpopulation (HMS). The HMS could be important, producing essentially all stress-induced mutants. Alternatively, the HMS was proposed to produce only a minority of stress-induced mutants, i.e., it was proposed to be peripheral. We characterize three aspects of the HMS. First, using improved mutation-detection methods, we estimate the number of mutations per genome of HMS-derived cells and find that it is compatible with fitness after the HMS state. This implies that these mutants are not necessarily an evolutionary dead end, and could contribute to adaptive evolution. Second, we show that stress-induced Lac(+) mutants, with and without evidence of descent from the HMS, have similar Lac(+) mutation sequences. This provides evidence that HMS-descended and most stress-induced mutants form via a common mechanism. Third, mutation-stimulating DSBs introduced via I-SceI endonuclease in vivo do not promote Lac(+) mutation independently of the HMS. This and the previous finding support the hypothesis that the HMS underlies most stress-induced mutants, not just a minority of them, i.e., it is important. We consider a model in which HMS differentiation is controlled by stress responses. Differentiation of an HMS potentially limits the risks of mutagenesis in cell clones.

Show MeSH
Related in: MedlinePlus