Integration of the pSLT Plasmid into the Salmonella Chromosome Results in a Temperature-Sensitive Growth Defect Due to Aberrant DNA Replication.

A mutant of Salmonella enterica serovar Typhimurium was isolated that simultaneously affected two metabolic pathways as follows: NAD metabolism and DNA repair. The mutant was isolated as resistant to a nicotinamide analog and as temperature-sensitive for growth on minimal glucose medium. In this mutant, Salmonella's 94-kb virulence plasmid pSLT had recombined into the chromosome upstream of the NAD salvage pathway gene pncA This insertion blocked most transcription of pncA, which reduced uptake of the nicotinamide analog. The pSLT insertion mutant also exhibited phenotypes associated with induction of the SOS DNA repair system, including an increase in filamentous cells, higher exonuclease III and catalase activities, and derepression of SOS gene expression. Genome sequencing revealed increased read coverage extending out from the site of pSLT insertion. The two pSLT replication origins are likely initiating replication of the chromosome near the normal replication terminus. Too much replication initiation at the wrong site is probably causing the observed growth defects. Accordingly, deletion of both pSLT replication origins restored growth at higher temperatures.IMPORTANCE In studies that insert a second replication origin into the chromosome, both origins are typically active at the same time. In contrast, the integrated pSLT plasmid initiated replication in stationary phase after normal chromosomal replication had finished. The gradient in read coverage extending out from a single site could be a simple but powerful tool for studying replication and detecting chromosomal rearrangements. This technique may be of particular value when a genome has been sequenced for the first time to verify correct assembly.

Selective Inbreeding: Genetic Crosses Drive Apparent Adaptive Mutation in the Cairns-Foster System of Escherichia coli.

The Escherichia coli system of Cairns and Foster employs a lac frameshift mutation that reverts rarely (10-9/cell/division) during unrestricted growth. However, when 108 cells are plated on lactose medium, the nongrowing lawn produces ∼50 Lac+ revertant colonies that accumulate linearly with time over 5 days. Revertants carry very few associated mutations. This behavior has been attributed to an evolved mechanism ("adaptive mutation" or "stress-induced mutagenesis") that responds to starvation by preferentially creating mutations that improve growth. We describe an alternative model, "selective inbreeding," in which natural selection acts during intercellular transfer of the plasmid that carries the mutant lac allele and the dinB gene for an error-prone polymerase. Revertant genome sequences show that the plasmid is more intensely mutagenized than the chromosome. Revertants vary widely in their number of plasmid and chromosomal mutations. Plasmid mutations are distributed evenly, but chromosomal mutations are focused near the replication origin. Rare, heavily mutagenized, revertants have acquired a plasmid tra mutation that eliminates conjugation ability. These findings support the new model, in which revertants are initiated by rare pre-existing cells (105) with many copies of the F'lac plasmid. These cells divide under selection, producing daughters that mate. Recombination between donor and recipient plasmids initiates rolling-circle plasmid over-replication, causing a mutagenic elevation of DinB level. A lac+ reversion event starts chromosome replication and mutagenesis by accumulated DinB. After reversion, plasmid transfer moves the revertant lac+ allele into an unmutagenized cell, and away from associated mutations. Thus, natural selection explains why mutagenesis appears stress-induced and directed.

Selection and Plasmid Transfer Underlie Adaptive Mutation in Escherichia coli.

In the Cairns-Foster adaptive mutation system, a +1 lac frameshift mutant of Escherichia coli is plated on lactose medium, where the nondividing population gives rise to Lac+ revertant colonies during a week under selection. Reversion requires the mutant lac allele to be located on a conjugative F'lac plasmid that also encodes the error-prone DNA polymerase, DinB. Rare plated cells with multiple copies of the mutant F'lac plasmid initiate the clones that develop into revertants under selection. These initiator cells arise before plating, and their extra lac copies allow them to divide on lactose and produce identical F'lac-bearing daughter cells that can mate with each other. DNA breaks can form during plasmid transfer and their recombinational repair can initiate rolling-circle replication of the recipient plasmid. This replication is mutagenic because the amplified plasmid encodes the error-prone DinB polymerase. A new model proposes that Lac+ revertants arise during mutagenic over-replication of the F'lac plasmid under selection. This mutagenesis is focused on the plasmid because the cell chromosome replicates very little. The outer membrane protein OmpA is essential for reversion under selection. OmpA helps cells conserve energy and may stabilize the long-term mating pairs that produce revertants.

Selection-Enhanced Mutagenesis of lac Genes Is Due to Their Coamplification with dinB Encoding an Error-Prone DNA Polymerase.

To test whether growth limitation induces mutations, Cairns and Foster constructed an Escherichia coli strain whose mutant lac allele provides 1-2% of normal ability to use lactose. This strain cannot grow on lactose, but produces ∼50 Lac+ revertant colonies per 108 plated cells over 5 days. About 80% of revertants carry a stable lac+ mutation made by the error-prone DinB polymerase, which may be induced during growth limitation; 10% of Lac+ revertants are stable but form without DinB; and the remaining 10% grow by amplifying their mutant lac allele and are unstably Lac+ Induced DinB mutagenesis has been explained in two ways: (1) upregulation of dinB expression in nongrowing cells ("stress-induced mutagenesis") or (2) selected local overreplication of the lac and dinB+ genes on lactose medium (selected amplification) in cells that are not dividing. Transcription of dinB is necessary but not sufficient for mutagenesis. Evidence is presented that DinB enhances reversion only when encoded somewhere on the F'lac plasmid that carries the mutant lac gene. A new model will propose that rare preexisting cells (1 in a 1000) have ∼10 copies of the F'lac plasmid, providing them with enough energy to divide, mate, and overreplicate their F'lac plasmid under selective conditions. In these clones, repeated replication of F'lac in nondividing cells directs opportunities for lac reversion and increases the copy number of the dinB+ gene. Amplification of dinB+ increases the error rate of replication and increases the number of lac+ revertants. Thus, reversion is enhanced in nondividing cells not by stress-induced mutagenesis, but by selected coamplification of the dinB and lac genes, both of which happen to lie on the F'lac plasmid.

The Promiscuous sumA Missense Suppressor from Salmonella enterica Has an Intriguing Mechanism of Action.

While most missense suppressors have very narrow specificities and only suppress the allele against which they were isolated, the sumA missense suppressor from Salmonella enterica serovar Typhimurium is a promiscuous or broad-acting missense suppressor that suppresses numerous missense mutants. The sumA missense suppressor was identified as a glyV tRNA Gly3(GAU/C) missense suppressor that can recognize GAU or GAC aspartic acid codons and insert a glycine amino acid instead of aspartic acid. In addition to rescuing missense mutants caused by glycine to aspartic acid changes as expected, sumA could also rescue a number of other missense mutants as well by changing a neighboring (contacting) aspartic acid to glycine, which compensated for the other amino acid change. Thus the ability of sumA to rescue numerous missense mutants was due in part to the large number of glycine codons in genes that can be mutated to an aspartic acid codon and in part to the general tolerability and/or preference for glycine amino acids in proteins. Because the glyV tRNA Gly3(GAU/C) missense suppressor has also been extensively characterized in Escherichia coli as the mutA mutator, we demonstrated that all gain-of-function mutants isolated in a glyV tRNA Gly3(GAU/C) missense suppressor are transferable to a wild-type background and thus the increased mutation rates, which occur in glyV tRNA Gly3(GAU/C) missense suppressors, are not due to the suppression of these mutants.

Reinterpreting Long-Term Evolution Experiments: Is Delayed Adaptation an Example of Historical Contingency or a Consequence of Intermittent Selection?

Van Hofwegen et al. demonstrated that Escherichia coli rapidly evolves the ability to use citrate when long selective periods are provided (D. J. Van Hofwegen, C. J. Hovde, and S. A. Minnich, J Bacteriol 198:1022-1034, 2016, http://dx.doi.org/10.1128/JB.00831-15). This contrasts with the extreme delay (15 years of daily transfers) seen in the long-term evolution experiments of Lenski and coworkers. Their idea of "historical contingency" may require reinterpretation. Rapid evolution seems to involve selection for duplications of the whole cit locus that are too unstable to contribute when selection is provided in short pulses.

The Origin of Mutants Under Selection: How Natural Selection Mimics Mutagenesis (Adaptive Mutation).

Selection detects mutants but does not cause mutations. Contrary to this dictum, Cairns and Foster plated a leaky lac mutant of Escherichia coli on lactose medium and saw revertant (Lac(+)) colonies accumulate with time above a nongrowing lawn. This result suggested that bacteria might mutagenize their own genome when growth is blocked. However, this conclusion is suspect in the light of recent evidence that revertant colonies are initiated by preexisting cells with multiple copies the conjugative F'lac plasmid, which carries the lac mutation. Some plated cells have multiple copies of the simple F'lac plasmid. This provides sufficient LacZ activity to support plasmid replication but not cell division. In nongrowing cells, repeated plasmid replication increases the likelihood of a reversion event. Reversion to lac(+) triggers exponential cell growth leading to a stable Lac(+) revertant colony. In 10% of these plated cells, the high-copy plasmid includes an internal tandem lac duplication, which provides even more LacZ activity­sufficient to support slow growth and formation of an unstable Lac(+) colony. Cells with multiple copies of the F'lac plasmid have an increased mutation rate, because the plasmid encodes the error-prone (mutagenic) DNA polymerase, DinB. Without DinB, unstable and stable Lac(+) revertant types form in equal numbers and both types arise with no mutagenesis. Amplification and selection are central to behavior of the Cairns-Foster system, whereas mutagenesis is a system-specific side effect or artifact caused by coamplification of dinB with lac. Study of this system has revealed several broadly applicable principles. In all populations, gene duplications are frequent stable genetic polymorphisms, common near-neutral mutant alleles can gain a positive phenotype when amplified under selection, and natural selection can operate without cell division when variability is generated by overreplication of local genome subregions.

Mechanisms of gene duplication and amplification.

Changes in gene copy number are among the most frequent mutational events in all genomes and were among the mutations for which a physical basis was first known. Yet mechanisms of gene duplication remain uncertain because formation rates are difficult to measure and mechanisms may vary with position in a genome. Duplications are compared here to deletions, which seem formally similar but can arise at very different rates by distinct mechanisms. Methods of assessing duplication rates and dependencies are described with several proposed formation mechanisms. Emphasis is placed on duplications formed in extensively studied experimental situations. Duplications studied in microbes are compared with those observed in metazoan cells, specifically those in genomes of cancer cells. Duplications, and especially their derived amplifications, are suggested to form by multistep processes often under positive selection for increased copy number.

Plasmid copy number underlies adaptive mutability in bacteria.

The origin of mutations under selection has been intensively studied using the Cairns-Foster system, in which cells of an Escherichia coli lac mutant are plated on lactose and give rise to 100 Lac+ revertants over several days. These revertants have been attributed variously to stress-induced mutagenesis of nongrowing cells or to selective improvement of preexisting weakly Lac+ cells with no mutagenesis. Most revertant colonies (90%) contain stably Lac+ cells, while others (10%) contain cells with an unstable amplification of the leaky mutant lac allele. Evidence is presented that both stable and unstable Lac+ revertant colonies are initiated by preexisting cells with multiple copies of the F'lac plasmid, which carries the mutant lac allele. The tetracycline analog anhydrotetracycline (AnTc) inhibits growth of cells with multiple copies of the tetA gene. Populations with tetA on their F'lac plasmid include rare cells with an elevated plasmid copy number and multiple copies of both the tetA and lac genes. Pregrowth of such populations with AnTc reduces the number of cells with multiple F'lac copies and consequently the number of Lac+ colonies appearing under selection. Revertant yield is restored rapidly by a few generations of growth without AnTc. We suggest that preexisting cells with multiple F'lac copies divide very little under selection but have enough energy to replicate their F'lac plasmids repeatedly until reversion initiates a stable Lac+ colony. Preexisting cells whose high-copy plasmid includes an internal lac duplication grow under selection and produce an unstable Lac+ colony. In this model, all revertant colonies are initiated by preexisting cells and cannot be stress induced.

Recombination and annealing pathways compete for substrates in making rrn duplications in Salmonella enterica.

Tandem genetic duplications arise frequently between the seven directly repeated 5.5-kb rrn loci that encode ribosomal RNAs in Salmonella enterica. The closest rrn genes, rrnB and rrnE, flank a 40-kb region that includes the purHD operon. Duplications of purHD arise by exchanges between rrn loci and form at a high rate (10(-3)/cell/division) that remains high in strains blocked for early steps in recombination (recA, recB, and/or recF), but drops 30-fold in mutants blocked for later Holliday junction resolution (ruvC recG). The duplication defect of a ruvC recG mutant was fully corrected by an added mutation in any one of the recA, recB, or recF genes. To explain these results, we propose that early recombination defects activate an alternative single-strand annealing pathway for duplication formation. In wild-type cells, rrn duplications form primarily by the action of RecFORA on single-strand gaps. Double-strand breaks cannot initiate rrn duplications because rrn loci lack Chi sites, which are essential for recombination between two separated rrn sequences. A recA or recF mutation allows unrepaired gaps to accumulate such that different rrn loci can provide single-strand rrn sequences that lack the RecA coating that normally inhibits annealing. A recB mutation activates annealing by allowing double-strand ends within rrn to avoid digestion by RecBCD and provide a new source of rrn ends for use in annealing. The equivalent high rates of rrn duplication by recombination and annealing pathways may reflect a limiting economy of gaps and breaks arising in heavily transcribed, palindrome-rich rrn sequences.

Evidence that a metabolic microcompartment contains and recycles private cofactor pools.

Microcompartments are loose protein cages that encapsulate enzymes for particular bacterial metabolic pathways. These structures are thought to retain and perhaps concentrate pools of small, uncharged intermediates that would otherwise diffuse from the cell. In Salmonella enterica, a microcompartment encloses enzymes for ethanolamine catabolism. The cage has been thought to retain the volatile intermediate acetaldehyde but allow diffusion of the much larger cofactors NAD and coenzyme A (CoA). Genetic tests support an alternative idea that the microcompartment contains and recycles private pools of the large cofactors NAD and CoA. Two central enzymes convert ethanolamine to acetaldehyde (EutBC) and then to acetyl-CoA (EutE). Two seemingly peripheral redundant enzymes encoded by the eut operon proved to be essential for ethanolamine utilization, when subjected to sufficiently stringent tests. These are EutD (acetyl-CoA to acetyl phosphate) and EutG (acetaldehyde to ethanol). Obligatory recycling of cofactors couples the three reactions and drives acetaldehyde consumption. Loss and toxic effects of acetaldehyde are minimized by accelerating its consumption. In a eutD mutant, acetyl-CoA cannot escape the compartment but is released by mutations that disrupt the structure. The model predicts that EutBC (ethanolamine-ammonia lyase) lies outside the compartment, using external coenzyme B12 and injecting its product, acetaldehyde, into the lumen, where it is degraded by the EutE, EutD, and EutG enzymes using private pools of CoA and NAD. The compartment appears to allow free diffusion of the intermediates ethanol and acetyl-PO4 but (to our great surprise) restricts diffusion of acetaldehyde.

Real-time evolution of new genes by innovation, amplification, and divergence.

Gene duplications allow evolution of genes with new functions. Here, we describe the innovation-amplification-divergence (IAD) model in which the new function appears before duplication and functionally distinct new genes evolve under continuous selection. One example fitting this model is a preexisting parental gene in Salmonella enterica that has low levels of two distinct activities. This gene is amplified to a high copy number, and the amplified gene copies accumulate mutations that provide enzymatic specialization of different copies and faster growth. Selection maintains the initial amplification and beneficial mutant alleles but is relaxed for other less improved gene copies, allowing their loss. This rapid process, completed in fewer than 3000 generations, shows the efficacy of the IAD model and allows the study of gene evolution in real time.

Multiple pathways of duplication formation with and without recombination (RecA) in Salmonella enterica.

Duplications are often attributed to "unequal recombination" between separated, directly repeated sequence elements (>100 bp), events that leave a recombinant element at the duplication junction. However, in the bacterial chromosome, duplications form at high rates (10(-3)-10(-5)/cell/division) even without recombination (RecA). Here we describe 1800 spontaneous lac duplications trapped nonselectively on the low-copy F'(128) plasmid, where lac is flanked by direct repeats of the transposable element IS3 (1258 bp) and by numerous quasipalindromic REP elements (30 bp). Duplications form at a high rate (10(-4)/cell/division) that is reduced only about 11-fold in the absence of RecA. With and without RecA, most duplications arise by recombination between IS3 elements (97%). Formation of these duplications is stimulated by IS3 transposase (Tnp) and plasmid transfer functions (TraI). Three duplication pathways are proposed. First, plasmid dimers form at a high rate stimulated by RecA and are then modified by deletions between IS3 elements (resolution) that leave a monomeric plasmid with an IS3-flanked lac duplication. Second, without RecA, duplications occur by single-strand annealing of DNA ends generated in different sister chromosomes after transposase nicks DNA near participating IS3 elements. The absence of RecA may stimulate annealing by allowing chromosome breaks to persist. Third, a minority of lac duplications (3%) have short (0-36 bp) junction sequences (SJ), some of which are located within REP elements. These duplication types form without RecA, Tnp, or Tra by a pathway in which the palindromic junctions of a tandem inversion duplication (TID) may stimulate deletions that leave the final duplication.

Pathways of genetic adaptation: multistep origin of mutants under selection without induced mutagenesis in Salmonella enterica.

In several bacterial systems, mutant cell populations plated on growth-restricting medium give rise to revertant colonies that accumulate over several days. One model suggests that nongrowing parent cells mutagenize their own genome and thereby create beneficial mutations (stress-induced mutagenesis). By this model, the first-order induction of new mutations in a nongrowing parent cell population leads to the delayed accumulation of visible colonies. In an alternative model (selection only), selective conditions allow preexisting small-effect mutants to initiate clones that grow and give rise to faster-growing mutants. By the selection-only model, the delay in appearance of revertant colonies reflects (1) the time required for initial clones to reach a size sufficient to allow the second mutation plus (2) the time required for growth of the improved subclone. We previously characterized a system in which revertant colonies accumulate slowly and contain cells with two mutations, one formed before plating and one after. This left open the question of whether mutation rates increase under selection. Here we measure the unselected formation rate and the growth contribution of each mutant type. When these parameters are used in a graphic model of revertant colony development, they demonstrate that no increase in mutation rate is required to explain the number and delayed appearance of two of the revertant types.

Poxvirus use a "gene accordion" to tune out host defenses.

A multistep process of gene amplification, mutation, and reduction allows poxvirus to overcome host antiviral defenses. The mechanism speeds genetic adaptation and promises to be broadly applicable in many biological settings.

Intestinal inflammation allows Salmonella to use ethanolamine to compete with the microbiota.

Conventional wisdom holds that microbes support their growth in vertebrate hosts by exploiting a large variety of nutrients. We show here that use of a specific nutrient (ethanolamine) confers a marked growth advantage on Salmonella enterica serovar Typhimurium (S. Typhimurium) in the lumen of the inflamed intestine. In the anaerobic environment of the gut, ethanolamine supports little or no growth by fermentation. However, S. Typhimurium is able to use this carbon source by inducing the gut to produce a respiratory electron acceptor (tetrathionate), which supports anaerobic growth on ethanolamine. The gut normally converts ambient hydrogen sulfide to thiosulfate, which it then oxidizes further to tetrathionate during inflammation. Evidence is provided that S. Typhimurium's growth advantage in an inflamed gut is because of its ability to respire ethanolamine, which is released from host tissue, but is not utilizable by competing bacteria. By inducing intestinal inflammation, S. Typhimurium sidesteps nutritional competition and gains the ability to use an abundant simple substrate, ethanolamine, which is provided by the host.

Effect of growth under selection on appearance of chromosomal mutations in Salmonella enterica.

Populations adapt physiologically using regulatory mechanisms and genetically by means of mutations that improve growth. During growth under selection, genetic adaptation can be rapid. In several genetic systems, the speed of adaptation has been attributed to cellular mechanisms that increase mutation rates in response to growth limitation. An alternative possibility is that growth limitation serves only as a selective agent but acts on small-effect mutations that are common under all growth conditions. The genetic systems that initially suggested stress-induced mutagenesis have been analyzed without regard for multistep adaptation and some include features that make such analysis difficult. To test the selection-only model, a simpler system is examined, whose behavior was originally attributed to stress-induced mutagenesis (Yang et al. 2001, 2006). A population with a silent chromosomal lac operon gives rise to Lac+ revertant colonies that accumulate over 6 days under selection. Each colony contains a mixture of singly and doubly mutant cells. Evidence is provided that the colonies are initiated by pre-existing single mutants with a weak Lac+ phenotype. Under selection, these cells initiate slow-growing clones, in which a second mutation arises and improves growth of the resulting double mutant. The system shows no evidence of general mutagenesis during selection. Selection alone may explain rapid adaptation in this and other systems that give the appearance of mutagenesis.

The joys and terrors of fast adaptation: new findings elucidate antibiotic resistance and natural selection.

Experiments of Pränting and Andersson demonstrate how bacteria adapt to the growth limitation caused by antibiotic resistance mutations. The process of adaptation relies on gene copy number changes that arise at high rates, including duplications (10(-4) per cell per generation), amplifications (10(-2) per cell per generation) and mutant copy loss (10(-2) per cell per division). Reversible increases in copy number improve growth by small steps and provide more targets for rare sequence alterations (10(-9) per cell per division) that can stably improve growth. After sequence alteration, selection favours loss of the still mutant gene copies that accelerated adaptation. The results strongly support the amplification-reversion model for fast adaptation and argue against the alternative idea of 'stress-induced mutagenesis'.

The Origin of Mutants under Selection: Interactions of Mutation, Growth, and Selection.

The classical experiments of Luria and Delbrück showed convincingly that mutations exist before selection and do not contribute to the creation of mutations when selection is lethal. In contrast, when nonlethal selections are used,measuring mutation rates and separating the effects of mutation and selection are difficult and require methods to fully exclude growth after selection has been applied. Although many claims of stress-induced mutagenesis have been made, it is difficult to exclude the influence of growth under nonlethal selection conditions in accounting for the observed increases in mutant frequency. Instead, for many of the studied experimental systems the increase in mutant frequency can be explainedbetter by the ability of selection to detect small differences in growth rate caused by common small effect mutations. A verycommon mutant class,found in response to many different types of selective regimensin which increased gene dosage can resolve the problem, is gene amplification. In the well-studiedlac system of Cairns and Foster, the apparent increase in Lac+revertants can be explained by high-level amplification of the lac operon and the increased probability for a reversion mutation to occur in any one of the amplified copies. The associated increase in general mutation rate observed in revertant cells in that system is an artifact caused by the coincidental co-amplification of the nearby dinB gene (encoding the error-prone DNA polymerase IV) on the particular plasmid used for these experiments. Apart from the lac system, similar gene amplification processes have been described for adaptation to toxic drugs, growth in host cells, and various nutrient limitations.

Gut inflammation provides a respiratory electron acceptor for Salmonella.

Salmonella enterica serotype Typhimurium (S. Typhimurium) causes acute gut inflammation by using its virulence factors to invade the intestinal epithelium and survive in mucosal macrophages. The inflammatory response enhances the transmission success of S. Typhimurium by promoting its outgrowth in the gut lumen through unknown mechanisms. Here we show that reactive oxygen species generated during inflammation react with endogenous, luminal sulphur compounds (thiosulphate) to form a new respiratory electron acceptor, tetrathionate. The genes conferring the ability to use tetrathionate as an electron acceptor produce a growth advantage for S. Typhimurium over the competing microbiota in the lumen of the inflamed gut. We conclude that S. Typhimurium virulence factors induce host-driven production of a new electron acceptor that allows the pathogen to use respiration to compete with fermenting gut microbes. Thus the ability to trigger intestinal inflammation is crucial for the biology of this diarrhoeal pathogen.