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.

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.

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.

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.

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.

Genomic buffering mitigates the effects of deleterious mutations in bacteria.

The relationship between the number of randomly accumulated mutations in a genome and fitness is a key parameter in evolutionary biology. Mutations may interact such that their combined effect on fitness is additive (no epistasis), reinforced (synergistic epistasis) or mitigated (antagonistic epistasis). We measured the decrease in fitness caused by increasing mutation number in the bacterium Salmonella typhimurium using a regulated, error-prone DNA polymerase (polymerase IV, DinB). As mutations accumulated, fitness costs increased at a diminishing rate. This suggests that random mutations interact such that their combined effect on fitness is mitigated and that the genome is buffered against the fitness reduction caused by accumulated mutations. Levels of the heat shock chaperones DnaK and GroEL increased in lineages that had accumulated many mutations, and experimental overproduction of GroEL further increased the fitness of lineages containing deleterious mutations. These findings suggest that overexpression of chaperones contributes to antagonistic epistasis.

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.

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'.

Accumulation of mutants in "aging" bacterial colonies is due to growth under selection, not stress-induced mutagenesis.

Several bacterial systems show behavior interpreted as evidence for stress-induced mutagenesis (adaptive mutation), a postulated process by which nongrowing cells temporarily increase their general mutation rate. Theoretical considerations suggest that periodic stress-induced general mutagenesis would not be advantageous in the long term, due to the high cost of deleterious mutations. Alternative explanations have been tested for very few of the systems used as evidence for stress-induced mutation. In one prominent system, mutants resistant to rifampicin (Rif(R); rpoB; RNA polymerase) accumulate in cell populations that "age" on solid medium with little net growth. Mutant accumulation was initially attributed to stress-induced general mutagenesis in nongrowing cells. Evidence is presented that these Rif(R) mutants accumulate because they grow faster than parent cells during the aging period. Direct tests revealed no increase in the frequency of other mutant types during the aging period.

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.

One pathway can incorporate either adenine or dimethylbenzimidazole as an alpha-axial ligand of B12 cofactors in Salmonella enterica.

Corrinoid (vitamin B12-like) cofactors contain various alpha-axial ligands, including 5,6-dimethylbenzimidazole (DMB) or adenine. The bacterium Salmonella enterica produces the corrin ring only under anaerobic conditions, but it can form "complete" corrinoids aerobically by importing an "incomplete" corrinoid, such as cobinamide (Cbi), and adding appropriate alpha- and beta-axial ligands. Under aerobic conditions, S. enterica performs the corrinoid-dependent degradation of ethanolamine if given vitamin B12, but it can make B12 from exogenous Cbi only if DMB is also provided. Mutants isolated for their ability to degrade ethanolamine without added DMB converted Cbi to pseudo-B12 cofactors (having adenine as an alpha-axial ligand). The mutations cause an increase in the level of free adenine and install adenine (instead of DMB) as an alpha-ligand. When DMB is provided to these mutants, synthesis of pseudo-B12 cofactors ceases and B12 cofactors are produced, suggesting that DMB regulates production or incorporation of free adenine as an alpha-ligand. Wild-type cells make pseudo-B12 cofactors during aerobic growth on propanediol plus Cbi and can use pseudo-vitamin B12 for all of their corrinoid-dependent enzymes. Synthesis of coenzyme pseudo-B12 cofactors requires the same enzymes (CobT, CobU, CobS, and CobC) that install DMB in the formation of coenzyme B12. Models are described for the mechanism and control of alpha-axial ligand installation.

Rebonding of orthodontic brackets. Part II, an XPS and SEM study.

OBJECTIVE: The hypothesis of this two-part study is that adhesive systems for bonding orthodontic brackets (ie, two self-etch primers [Transbond and M-Bond] and a conventional phosphoric acid etch [Rely-a-Bond]) would show a difference with respect to rebonded enamel surface morphology and chemical composition. MATERIALS AND METHODS: This study examined the enamel surface before and after debonding with scanning electron microscopy and the enamel surface chemical composition for the elements Ca, P, O, F, Si, and C using x-ray photoelectron spectroscopy. RESULTS: The etching of the two self-etch groups is less aggressive and less uniform than that of phosphoric acid. The change in the concentration of C indicated that the separation of the bracket from the enamel surface is at the resin-enamel interface for the phosphoric acid-etched adhesive and a mixed mode involving the enamel-resin-bracket interfaces for the self-etching systems. F release appears to occur for Transbond but not for M-Bond. CONCLUSIONS: The results confirm the original hypothesis that differences in adhesive systems are manifested in less aggressive etches and less adhesive left on the enamel surface for the self-etching adhesive systems.

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.

The amplification model for adaptive mutation: simulations and analysis.

It has been proposed that the lac revertants arising under selective conditions in the Cairns experiment do not arise by stress-induced mutagenesis of stationary phase cells as has been previously assumed. Instead, these revertants may arise within growing clones initiated by cells with a preexisting duplication of the weakly functional lac allele used in this experiment. It is proposed that spontaneous stepwise increases in lac copy number (amplification) allow a progressive improvement in growth. Reversion is made more likely primarily by the resultant increase in the number of mutational targets--more cells with more lac copies. The gene amplification model requires no stress-induced variation in the rate or target specificity of mutation and thus does not violate neo-Darwinian theory. However, it does require that a multistep process of amplification, reversion, and amplification segregation be completed within approximately 20 generations of growth. This work examines the proposed amplification model from a theoretical point of view, formalizing it into a mathematical framework and using this to determine what would be required for the process to occur within the specified period. The analysis assumes no stress-induced change in mutation rate and describes only the growth improvement occurring during the process of amplification and subsequent elimination of excess mutant lac copies. The dynamics of the system are described using Monte Carlo simulations and numerical integration of the deterministic equations governing the system. The results imply that the amplification model can account for the behavior of the system using biologically reasonable parameter values and thus can, in principle, explain Cairnsian adaptive mutation.

Evidence that selected amplification of a bacterial lac frameshift allele stimulates Lac(+) reversion (adaptive mutation) with or without general hypermutability.

In the genetic system of Cairns and Foster, a nongrowing population of an E. coli lac frameshift mutant appears to specifically accumulate Lac(+) revertants when starved on medium including lactose (adaptive mutation). This behavior has been attributed to stress-induced general mutagenesis in a subpopulation of starved cells (the hypermutable state model). We have suggested that, on the contrary, stress has no direct effect on mutability but favors only growth of cells that amplify their leaky mutant lac region (the amplification mutagenesis model). Selection enhances reversion primarily by increasing the mutant lac copy number within each developing clone on the selection plate. The observed general mutagenesis is attributed to a side effect of growth with an amplification-induction of SOS by DNA fragments released from a tandem array of lac copies. Here we show that the S. enterica version of the Cairns system shows SOS-dependent general mutagenesis and behaves in every way like the original E. coli system. In both systems, lac revertants are mutagenized during selection. Eliminating the 35-fold increase in mutation rate reduces revertant number only 2- to 4-fold. This discrepancy is due to continued growth of amplification cells until some clones manage to revert without mutagenesis solely by increasing their lac copy number. Reversion in the absence of mutagenesis is still dependent on RecA function, as expected if it depends on lac amplification (a recombination-dependent process). These observations support the amplification mutagenesis model.

Regulating general mutation rates: examination of the hypermutable state model for Cairnsian adaptive mutation.

In the lac adaptive mutation system of Cairns, selected mutant colonies but not unselected mutant types appear to arise from a nongrowing population of Escherichia coli. The general mutagenesis suffered by the selected mutants has been interpreted as support for the idea that E. coli possesses an evolved (and therefore beneficial) mechanism that increases the mutation rate in response to stress (the hypermutable state model, HSM). This mechanism is proposed to allow faster genetic adaptation to stressful conditions and to explain why mutations appear directed to useful sites. Analysis of the HSM reveals that it requires implausibly intense mutagenesis (10(5) times the unselected rate) and even then cannot account for the behavior of the Cairns system. The assumptions of the HSM predict that selected revertants will carry an average of eight deleterious null mutations and thus seem unlikely to be successful in long-term evolution. The experimentally observed 35-fold increase in the level of general mutagenesis cannot account for even one Lac(+) revertant from a mutagenized subpopulation of 10(5) cells (the number proposed to enter the hypermutable state). We conclude that temporary general mutagenesis during stress is unlikely to provide a long-term selective advantage in this or any similar genetic system.

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.