When a particular lac mutant of Escherichia coli starves in the presence of lactose, nongrowing cells appear to direct mutations preferentially to sites that allow growth (adaptive mutation). This observation suggested that growth limitation stimulates mutability. Evidence is provided here that this behavior is actually caused by a standard Darwinian process in which natural selection acts in three sequential steps. First, growth limitation favors growth of a subpopulation with an amplification of the mutant lac gene; next, it favors cells with a lac ؉ revertant allele within the amplified array. Finally, it favors loss of mutant copies until a stable haploid lac ؉ revertant arises and overgrows the colony. By increasing the lac copy number, selection enhances the likelihood of reversion within each developing clone. This sequence of events appears to direct mutations to useful sites. General mutagenesis is a side-effect of growth with an amplification (SOS induction). The F plasmid, which carries lac, contributes by stimulating gene duplication and amplification. Selective stress has no direct effect on mutation rate or target specificity, but acts to favor a succession of cell types with progressively improved growth on lactose. The sequence of events-amplification, mutation, segregation-may help to explain both the origins of some cancers and the evolution of new genes under selection. A selection system developed by John Cairns (1, 2) provided evidence that bacteria might sense growth-limiting stress and direct mutations to sites that enhance growth (adaptive mutation). The behavior of Cairns' system does not contradict classical demonstrations that some mutations arise independent of selection (3, 4). However, the behavior raises the possibility that another fraction of mutations might be induced or even directed by growth limitation. Stress-induced mutations (general or directed) would contradict the neo-Darwinian view that agents of selection play no role in causing mutations, but affect only the relative reproductive success of organisms with different genotypes.Experiments supporting directed mutation used an Escherichia coli strain with a lac deletion on the chromosome and a revertible lacZ (ϩ1) frameshift mutation on an FЈ 128 plasmid (2). During growth, the lac point mutation reverts at a rate of 10 Ϫ8 per cell per division. When 10 8 of these Lac Ϫ cells are starved in the presence of lactose, about 100 revertant colonies accumulate over a period of 6 days, during which the plated population does not grow and does not accumulate unselected mutations (5). This behavior suggested that selection directs mutations to growth-promoting sites (2, 6).Subsequent studies showed that lac ϩ revertants have a 10-to 100-fold higher probability of carrying an unselected mutation than do unselected cells or starved nonrevertant cells (7). Thus, the revertants (but not the starved population as a whole) were generally mutagenized in the process of reversion. Once isolated, the revertants show a normal mutation rate....

Adaptive mutability is the apparent alteration in specificity or rate of mutability seen in bacteria during stress. A model is proposed by which gene amplification during selective growth can give the appearance of adaptive mutability without requiring any change in mutability. The model is based on two assumptions, that a mutant lac locus with residual function allows growth if its copy number is increased, and that true reversion events are made more likely by replication of chromosomes with many copies of the locus. Apparent directed mutability, its recombination requirement, and its apparent independence of cell growth are all accounted for by the model. Evidence is provided for the required residual function and gene amplification.

Hfq is a bacterial RNA chaperone involved in the posttranscriptional regulation of many stress-inducible genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Escherichia coli (UPEC) isolate UTI89 to effectively colonize the bladder and kidneys in a murine urinary tract infection model system. The disruption of hfq did not affect bacterial adherence to or invasion of host cells but did limit the development of intracellular microcolonies by UTI89 within the terminally differentiated epithelial cells that line the lumen of the bladder. In vitro, the hfq mutant was significantly impaired in its abilities to handle the antibacterial cationic peptide polymyxin B and reactive nitrogen and oxygen radicals and to grow in acidic medium (pH 5.0). Relative to the wild-type strain, the hfq mutant also had a substantially reduced migration rate on motility agar and was less prone to form biofilms. Hfq activities are known to impact the regulation of both the stationary-phase sigma factor RpoS ( S ) and the envelope stress response sigma factor RpoE ( E ). Although we saw similarities among hfq, rpoS, and rpoE deletion mutants in our assays, the rpoE and hfq mutants were phenotypically the most alike. Cumulatively, our data indicate that Hfq likely affects UPEC virulence-related phenotypes primarily by modulating membrane homeostasis and envelope stress response pathways.Small noncoding regulatory RNAs (sRNAs) can modulate the translation and stability of specific target mRNAs in prokaryotes and can thereby impact multiple aspects of bacterial cell physiology. In Escherichia coli, more than 60 sRNAs have been conclusively identified, representing 1 to 2% of the number of known protein-encoding genes in this organism (20). Interactions between most sRNA molecules and mRNAs occur through multiple regions of homology of 2 to 8 bp, typically within the 5Ј ends of target transcripts (21). In many cases, these RNA-RNA interactions require Hfq, a protein originally identified as a host factor needed for Q␤ bacteriophage replication (18,19). Hfq assembles into homohexameric rings, which are structurally similar to those formed by the Sm and Sm-like proteins that comprise the core of splicing and mRNA degradation complexes in eukaryotic and archaeal cells (21,52,56). By binding single-stranded AU-rich regions, Hfq can stabilize sRNA molecules as well as stimulate the formation of sRNA-mRNA pairs. In most cases, these Hfq-mediated interactions have an inhibitory effect on either the translation or the stability of the target mRNA.A number of sRNA molecules that bind Hfq are key regulatory elements in bacterial stress responses (20). Among these are sRNAs that help control the expression of the sigma factor RpoS ( S ), a master regulator of the general stress response in E. coli and many other gram-negative bacteria. RpoS, which is also known as the stationary-phase sigma factor, regulates the expression of numerous genes that promote bacterial survival in the face of various environmental stresses, i...

In a particular genetic system, selection stimulates reversion of a lac mutation and causes genome-wide mutagenesis (adaptive mutation). Selection allows rare plated cells with a duplication of the leaky lac allele to initiate clones within which further lac amplification improves growth rate. Growth and amplification add mutational targets to each clone and thereby increase the likelihood of reversion. We suggest that general mutagenesis occurs only in clones whose lac amplification includes the nearby dinB ؉ gene (for error-prone DNA polymerase IV). Thus mutagenesis is not a programmed response to stress but a side effect of amplification in a few clones; it is not central to the effect of selection on reversion.W hen a particular lac mutant of Escherichia coli is plated on selective medium, revertant colonies accumulate over several days (1, 2). Two models assume that mutations arise in the nongrowing population (3). Directed mutation proposes that stress preferentially induces beneficial (i.e., Lac ϩ ) mutations (1, 2). The hypermutable state proposes that stress induces general (genome-wide, undirected) mutagenesis in a subset of cells (Ϸ0.1%), and this mutagenesis produces the Lac ϩ revertants (4-6).An alternative model, amplification mutagenesis, proposes that reversion occurs in cells growing under selection and requires no change in mutability. On selective medium, rare preexisting cells with a lac duplication initiate slow-growing clones within which the growth rate increases progressively as amplification increases the copy number of the partially functional mutant lac allele (7,8). The probability of reversion within each clone increases with the number of target lac copies. After reversion, selection holds the revertant lac ϩ allele and favors cells that stabilize this allele by loss of mutant copies. This model is a specific form of a more general hypothesis proposed by Lenski et al. (9).Genomewide mutagenesis (an Ϸ100-fold increase in rate) is experienced by some revertants. This mutagenesis depends on the error-prone DNA polymerase, DinB (10-12), which may be induced when the SOS regulon is activated by DNA fragments released during segregation of the amplified lac region (8). Three problems complicate understanding how selection might cause general mutagenesis:(i) Induction of SOS does not mutagenize strains with a single wild-type dinB ϩ gene (13-15).(ii) Only 10% of Lac ϩ revertants arising under selection experience general mutagenesis (16,17).(iii) Selection causes mutagenesis only when lac is near the dinB gene (16,(18)(19)(20)(21).Evidence is presented that general mutagenesis occurs only in those developing clones whose amplified lac region includes the nearby dinB ϩ gene. Thus general mutagenesis is not a programmed response to stress in stationary phase but rather a side effect in a subset of developing clones growing under strong selection. Materials and MethodsSupporting Information. More detailed descriptions of methods and complete genotypes of all strains are published as supportin...