Patricia L. Foster

Stress-Induced Mutagenesis in Bacteria

ABSTRACT Bacteria spend their lives buffeted by changing environmental conditions. To adapt to and survive these stresses, bacteria have global response systems that result in sweeping changes in gene expression and cellular metabolism. These responses are controlled by master regulators, which include: alternative sigma factors, such as RpoS and RpoH; small molecule effectors, such as ppGpp; gene repressors such as LexA; and, inorganic molecules, such as polyphosphate. The response pathways extensively overlap and are induced to various extents by the same environmental stresses. These stresses include nutritional deprivation, DNA damage, temperature shift, and exposure to antibiotics. All of these global stress responses include functions that can increase genetic variability. In particular, up-regulation and activation of error-prone DNA polymerases, down-regulation of error-correcting enzymes, and movement of mobile genetic elements are common features of several stress responses. The result is that under a variety of stressful conditions, bacteria are induced for genetic change. This transient mutator state may be important for adaptive evolution.

Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by whole-genome sequencing

Knowledge of the rate and nature of spontaneous mutation is fundamental to understanding evolutionary and molecular processes. In this report, we analyze spontaneous mutations accumulated over thousands of generations by wild-type Escherichia coli and a derivative defective in mismatch repair (MMR), the primary pathway for correcting replication errors. The major conclusions are (i) the mutation rate of a wild-type E. coli strain is ∼1 × 10−3 per genome per generation; (ii) mutations in the wild-type strain have the expected mutational bias for G:C > A:T mutations, but the bias changes to A:T > G:C mutations in the absence of MMR; (iii) during replication, A:T > G:C transitions preferentially occur with A templating the lagging strand and T templating the leading strand, whereas G:C > A:T transitions preferentially occur with C templating the lagging strand and G templating the leading strand; (iv) there is a strong bias for transition mutations to occur at 5′ApC3′/3′TpG5′ sites (where bases 5′A and 3′T are mutated) and, to a lesser extent, at 5′GpC3′/3′CpG5′ sites (where bases 5′G and 3′C are mutated); (v) although the rate of small (≤4 nt) insertions and deletions is high at repeat sequences, these events occur at only 1/10th the genomic rate of base-pair substitutions. MMR activity is genetically regulated, and bacteria isolated from nature often lack MMR capacity, suggesting that modulation of MMR can be adaptive. Thus, comparing results from the wild-type and MMR-defective strains may lead to a deeper understanding of factors that determine mutation rates and spectra, how these factors may differ among organisms, and how they may be shaped by environmental conditions.

The coordinated functions of the E. coli MutS and MutL proteins in mismatch repair.

The Escherichia coli MutS and MutL proteins have been conserved throughout evolution, although their combined functions in mismatch repair (MMR) are poorly understood. We have used biochemical and genetic studies to ascertain a physiologically relevant mechanism for MMR. The MutS protein functions as a regional lesion sensor. ADP-bound MutS specifically recognizes a mismatch. Repetitive rounds of mismatch-provoked ADP-->ATP exchange results in the loading of multiple MutS hydrolysis-independent sliding clamps onto the adjoining duplex DNA. MutL can only associate with ATP-bound MutS sliding clamps. Interaction of the MutS-MutL sliding clamp complex with MutH triggers ATP binding by MutL that enhances the endonuclease activity of MutH. Additionally, MutL promotes ATP binding-independent turnover of idle MutS sliding clamps. These results support a model of MMR that relies on two dynamic and redundant ATP-regulated molecular switches.

Error‐prone DNA polymerase IV is controlled by the stress‐response sigma factor, RpoS, in Escherichia coli

An insertion in rpoS, which encodes the general stress response sigma factor σ38, was isolated as an antimutator for ‘stationary‐phase’ or ‘adaptive’ mutation. In the rpoS mutant strain the levels of error‐prone DNA polymerase Pol IV were reduced. Pol IV is encoded by the dinB gene, and the amount of its transcript was also reduced in rpoS mutant cells. In wild‐type cells, the levels of Pol IV increased in late stationary phase and stayed elevated for several days of continuous incubation, whereas in rpoS defective cells Pol IV was not induced and declined during prolonged incubation. Even in cells missing LexA, the repressor of dinB, maximum Pol IV expression required RpoS. These results suggest that induction of Pol IV is part of a cellular response to starvation and other stresses.

Adaptive mutation: implications for evolution

Adaptive mutation is defined as a process that, during nonlethal selections, produces mutations that relieve the selective pressure whether or not other, nonselected mutations are also produced. Examples of adaptive mutation or related phenomena have been reported in bacteria and yeast but not yet outside of microorganisms. A decade of research on adaptive mutation has revealed mechanisms that may increase mutation rates under adverse conditions. This article focuses on mechanisms that produce adaptive mutations in one strain of Escherichia coli, FC40. These mechanisms include recombination‐induced DNA replication, the placement of genes on a conjugal plasmid, and a transient mutator state. The implications of these various phenomena for adaptive evolution in microorganisms are discussed. BioEssays 22:1067–1074, 2000.

Error-Prone Polymerase, DNA Polymerase IV, Is Responsible for Transient Hypermutation during Adaptive Mutation in Escherichia coli

The frequencies of nonselected mutations among adaptive Lac(+) revertants of Escherichia coli strains with and without the error-prone DNA polymerase IV (Pol IV) were compared. This frequency was more than sevenfold lower in the Pol IV-defective strain than in the wild-type strain. Thus, the mutations that occur during hypermutation are due to Pol IV.

Genetic drift, selection and the evolution of the mutation rate

As one of the few cellular traits that can be quantified across the tree of life, DNA-replication fidelity provides an excellent platform for understanding fundamental evolutionary processes. Furthermore, because mutation is the ultimate source of all genetic variation, clarifying why mutation rates vary is crucial for understanding all areas of biology. A potentially revealing hypothesis for mutation-rate evolution is that natural selection primarily operates to improve replication fidelity, with the ultimate limits to what can be achieved set by the power of random genetic drift. This drift-barrier hypothesis is consistent with comparative measures of mutation rates, provides a simple explanation for the existence of error-prone polymerases and yields a formal counter-argument to the view that selection fine-tunes gene-specific mutation rates.

Adaptive Mutation in Escherichia coli

In 1988 John Cairns, Julie Overbaugh, and Stephan Miller published a paper entitled “The origin of mutants,” in which they suggested that bacteria could choose which mutations to make (11). Shortly thereafter, Cairns and I began collaborating on a National Science Foundation-funded project to investigate the genetic basis of what was popularly called “directed mutation” (although at the time we were calling it “selection-dependent mutation,” which now seems as good a name as any). Our plan was to mutagenize an appropriate strain of Escherichia coli and identify and characterize variants defective in selection-dependent mutation. Cairns and coworkers had been investigating a strain called SM195, described in their original paper (11). However, the mechanisms of mutation in this strain had proved to be complicated, and we wanted a new experimental subject. We were setting up to investigate selection-induced activation of the cryptic bgl operon (36), when Jeffrey Miller gave us a Lac− strain, called α45, that he said had a high rate of reversion to Lac+ after plating on minimum lactose medium. The lac allele in this strain is a fusion of lacI to lacZ that eliminates the lac regulatory region as well as several residues of lacI and lacZ; transcription of the fusion is constitutive, initiating at the lacI promoter (7). α45 has a +1 frameshift mutation, lacI33, in the lacI coding region (12). Miller and coworkers have used several similar strains to study various mutational mechanisms (52, 67). As in many of the strains that originated with Jacob and Monod, proAB and lac are deleted from the chromosome and carried on an episome, F′128. This arrangement greatly facilitates genetic manipulations and turned out to be important to adaptive mutation. We mated the episome from α45 into a Δ(lac-pro) recipient that we had made rifampin resistant and named the new strain FC40 (Foster and Cairns #40).

Asymmetric Context-dependent Mutation Patterns Revealed through Mutation-accumulation Experiments

Despite the general assumption that site-specific mutation rates are independent of the local sequence context, a growing body of evidence suggests otherwise. To further examine context-dependent patterns of mutation, we amassed 5,645 spontaneous mutations in wild- type (WT) and mismatch-repair deficient (MMR(-)) mutation-accumulation (MA) lines of the gram-positive model organism Bacillus subtilis. We then analyzed>7,500 spontaneous base-substitution mutations across B. subtilis, Escherichia coli, and Mesoplasma florum WT and MMR(-) MA lines, finding a context-dependent mutation pattern that is asymmetric around the origin of replication. Different neighboring nucleotides can alter site-specific mutation rates by as much as 75-fold, with sites neighboring G:C base pairs or dimers involving alternating pyrimidine-purine and purine-pyrimidine nucleotides having significantly elevated mutation rates. The influence of context-dependent mutation on genome architecture is strongest in M. florum, consistent with the reduced efficiency of selection in organisms with low effective population size. If not properly accounted for, the disparities arising from patterns of context-dependent mutation can significantly influence interpretations of positive and purifying selection.

Error-Prone DNA Polymerase IV Is Regulated by the Heat Shock Chaperone GroE in Escherichia coli

An insertion in the promoter of the operon that encodes the molecular chaperone GroE was isolated as an antimutator for stationary-phase or adaptive mutation. The groE operon consists of two genes, groES and groEL; point mutations in either gene conferred the same phenotype, reducing Lac+ adaptive mutation 10- to 20-fold. groE mutant strains had 1/10 the amount of error-prone DNA polymerase IV (Pol IV). In recG+ strains, the reduction in Pol IV was sufficient to account for their low rate of adaptive mutation, but in recG mutant strains, a deficiency of GroE had some additional effect on adaptive mutation. Pol IV is induced as part of the SOS response, but the effect of GroE on Pol IV was independent of LexA. We were unable to show that GroE interacts directly with Pol IV, suggesting that GroE may act indirectly. Together with previous results, these findings indicate that Pol IV is a component of several cellular stress responses.