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Summary: The appearance of new mutations is determined by the equilibrium between DNA error formation and repair. In bacteria like Escherichia coli, stresses are thought shift this balance towards increased mutagenesis. Recent findings, however, suggest a very uneven relationship between stress and mutations. Only a subset of stressful environments increase the net rate of mutation and different forms of nutritional stress (such as oxygen, carbon or phosphorus limitations) result in markedly different mutation rates after similar reductions in growth rate. Moreover, different stresses result in altered mutational spectra, with some increasing transposition and others increasing indel formation. Single‐base substitution rates are lower with some stresses than in unstressed bacteria. Indeed, changes to the mix of mutations with stress are more widespread than a marked increase in net mutation rate. Much remains to be learned on how environments have unique mutational signatures and why some stresses are more mutagenic than others. Even beyond stress‐induced genetic variation, the fundamental unresolved question in the stress–mutation relationship is the adaptive value of different types of mutations and mutation rates; is transposition, for example, more advantageous under anaerobic conditions? It remains to be investigated whether stress‐specific genetic variation impacts on evolvability differentially in distinct environments. [ABSTRACT FROM AUTHOR]

Summary: Genetic variation in bacterial populations is remarkably sensitive to environmental influences, including simple, nutritional differences. Not only the rate but also the kind of mutational changes is biased by the nutritional state of bacteria. Here we investigate the mutational consequences of a universal variable for free‐living bacteria, namely the growth rate. By controlling growth in chemostats, the rate and mix of mutations was investigated for populations of Escherichia coli subject to different specific growth rates. Both aerobic and anaerobic cultures were compared with see if growth rate is a factor in the commonest respiratory conditions for E. coli. We find mutation rates are raised markedly with decreasing growth rate. Base pair substitutions and 1‐bp insertions and deletions increase with reduced growth rate, but less so in anaerobic cultures. Insertion sequence movements are particularly sensitive to growth rate, with IS2 being optimal at intermediate growth rates whereas IS1 and IS150 movements are highest at the slowest tested growth rate. A comprehensive comparison of growth rate effects, as well as six other environmental factors, provides the most complete picture yet of the range of mutational signatures in bacterial genetic variation. [ABSTRACT FROM AUTHOR]

The relationship between bacterial drug resistance and growth fitness is a contentious topic, but some antibiotic resistance mutations clearly have a fitness cost in the laboratory. Whether these costs translate into deleterious effects in natural habitats is less certain however. Previously, fitness effects of resistance mutations were mostly characterized in nutrient-rich, fast-growth conditions, which bacteria rarely encounter in natural habitats. Carbon, phosphate, iron or oxygen limitations are conditions met by bacterial pathogens in various compartments of the human body. Here, we measured the fitness of four different rpoB mutations commonly found in rifampicin-resistant bacterial isolates. The fitness properties and the emergence of these and other alleles were studied in Escherichia coli populations growing under nutrient excess and in four different nutrient-limited states. Consistent with previous findings, all four mutations exhibited deleterious fitness effects under nutrient-rich conditions. In stark contrast, we found positive or neutral fitness effects under nutrient-limited conditions. Two particular rpoB alleles had a remarkable fitness increase under phosphate limitation and these alleles arose to high frequencies specifically under phosphate limitation. These findings suggest that it is not meaningful to draw general conclusions on fitness costs without considering bacterial microenvironments in humans and other animals. [ABSTRACT FROM AUTHOR]

Here, we propose that the heterogeneity of mutational types in populations underpins alternative pathways of evolutionary adaptation. Point mutations, deletions, insertions, transpositions and duplications cause different biological effects and provide distinct adaptive possibilities. Experimental evidence for this notion comes from the mutational origins of adaptive radiations in large, clonal bacterial populations. Independent sympatric lineages with different phenotypes arise from distinct genetic events including gene duplication, different insertion sequence movements and several independent point mutations. The breadth of the mutational spectrum in the ancestral population should be viewed as a form of bet-hedging, reducing the risk of evolutionary dead ends and complementing the phenotypic and epigenetic heterogeneities that improve the survival capabilities of a population. Different mutational events arise from distinct cellular processes and are subject to separate environmental impacts, so the availability of any particular type of mutation may constrain or promote adaptive pathways in populations. [ABSTRACT FROM AUTHOR]

Adaptation of environmental bacteria to laboratory conditions can lead to modification of important traits, what we term domestication. Little is known about the rapidity and reproducibility of domestication changes, the uniformity of these changes within a species or how diverse these are in a single culture. Here, we analysed phenotypic changes in nutrient-rich liquid media or on agar of four Escherichia coli strains newly isolated through minimal steps from different sources. The laboratory-cultured populations showed changes in metabolism, morphotype, fitness and in some phenotypes associated with the sigma factor RpoS. Domestication events and phenotypic diversity started to emerge within 2-3 days in replicate subcultures of the same ancestor. In some strains, increased amino acid usage and higher fitness under nutrient limitation resembled those in mutants with the GASP (growth advantage in stationary phase) phenotype. The domestication changes are not uniform across a species or even within a single domesticated population. However, some parallelism in adaptation within repeat cultures was observed. Differences in the laboratory environment also determine domestication effects, which differ between liquid and solid media or with extended stationary phase. Important lessons for the handling and storage of organisms can be based on these studies. [ABSTRACT FROM AUTHOR]

Bacteria can exhibit high levels of resistance to one or more environmental stresses such as temperature, osmolarity, radiation, pH, starvation, as well as resistance to noxious chemicals and antibiotics. Yet evolution has not optimized stress resistance in all bacteria to all stresses. Even within a species like Escherichia coli, stress resistance is not constant between strains, suggesting that selection for stress resistance is under counterselection in some environments. The tradeoffs associated with stress resistance in E. coli are due to more than the direct cost of resistance mechanisms. A significant indirect cost is that high stress resistance is associated with a reduced ability to compete for poor growth substrates like acetate or even good substrates like glucose at suboptimal concentrations. High stress resistance also decreases the ability to use inorganic nutrients like phosphate. This tradeoff between self-preservation and nutritional competence, called the SPANC balance, is likely to be the major selective influence in natural populations. Another cost of high stress resistance in E. coli is an elevated mutation rate and the increased generation of deleterious mutations. Directional adaptations in SPANC balance and mutation rate are environment-dependent. The most common variations in SPANC are due to polymorphisms in the levels of global regulators RpoS and ppGpp between different strains. High levels favor stress resistance, and low levels allow better nutrition. The intimate association of RpoS/ppGpp with stress resistance and SPANC balancing influences numerous cellular processes and bacterial properties, including virulence. [ABSTRACT FROM AUTHOR]

Stress protection is an important but costly contributor to bacterial survival. Two distinct forms of environmental protection share a common cost and a significant species-wide variability. Porin-mediated outer membrane permeability and the RpoS-controlled general stress response both involve a trade-off betweenelf-reservationndutritionalompetence, called the SPANC balance. Interestingly, differentEscherichia colistrains exhibit distinct settings of the SPANC balance. It is tilted towards high stress resistance and a restricted diet in some isolates whereas others have broader nutritional capability and better nutrient affinity but lower levels of resistance. Growth- or stress-related selective pressures working in opposite directions (antagonistic pleiotropy) result in polymorphisms affecting porins and RpoS. Consequently, these important cellular components are present at distinct concentrations in different isolates. A generalized hypothesis to explain bacterial adaptation, based on the SPANC investigations, is offered. A holistic approach to bacterial adaptation, involving a gamut of regulation and mutation, is likely to be the norm in broadening the capabilities of a species. Indeed, there is unlikely to be a standard regulatory setting typical for all members of a species. Gene regulation provides a limited fine control for maintaining the right level of adaptation in a particular niche but mutational changes provide the coarse control for adaptation between the species-wide environments of free-living bacteria. [ABSTRACT FROM AUTHOR]

SummaryBacteria are sometimes neither starving nor under nutrient-excess conditions. When growing with suboptimal levels of nutrients, hungry bacteria express appropriate cellular responses. This review discusses approaches to defining the hunger response in both molecular and growth kinetic terms. The gene expression changes unique to hunger conditions are described, using Escherichia coli as the primary example. Metabolite changes with hunger and starvation and the differing role of the stationary phase regulator RpoS also lead to the hypothesis proposed in this review that bacteria undertake distinct approaches to hunger and starvation. Indeed, an understanding of the difference between hunger and starvation and the incompatibility between hunger and starvation responses explains some of the paradoxical mutational adaptations, such as rpoS inactivation, found in natural populations. [ABSTRACT FROM AUTHOR]

The mutational adaptation of E. coli to low glucose concentrations was studied in chemostats over 280 generations of growth. All members of six independent populations acquired increased fitness through the acquisition of mutations at the mgl locus, increasing the binding protein-dependent transport of glucose. These mutations provided a strong fitness advantage (up to 10-fold increase in glucose affinity) and were present in most isolates after 140 generations. mgl constitutivity in some isolates was caused by base substitution, short duplication, small deletion and IS1 insertion in the 1041 bp gene encoding the repressor of the mgl system, mglD (galS). But an unexpectedly large proportion of mutations were located in the short mgl operator sequence (mglO), and the majority of mutations were in mglO after 280 generations of selection. The adaptive mglO substitutions in several independent populations were at exactly the positions conserved in the two 8 bp half-sites of the mgl operator, with the nature of the base changes also completely symmetrical. Either mutations were directed to the operator or the particular operator mutations had a selective advantage under glucose limitation. Indeed, isolates carrying mglO mutations showed greater rates of transport for glucose and galactose at low concentrations than those carrying mglD null mutations. mglO mutations avoid cross-talk by members of the GalR–LacI repressor family, reducing transporter expression and providing a competitive advantage in a glucose-limited environment. Another interesting aspect of these results was that each adapted population acquired multiple mgl alleles, with several populations containing at least six different mgl-regulatory mutations co-existing after 200 generations. The diversity of mutations in the mglO/mglD region, generally in combination with mutations at other loci regulating glucose uptake (malT, mlc, ptsG), provided evidence for multiple clones in each population.... [ABSTRACT FROM AUTHOR]

The multicomponent glucose transport system of Escherichia coli was used to study the polygenic basis of increased fitness in prolonged nutrient-limited, continuous cultures. After 280 generations of glucose-limited growth, nearly all bacteria in four independent chemostat populations exhibited increased glucose transport and contained multiple, stable mutations. Fitter bacteria increased outer membrane permeability for glucose through overexpression of the LamB glycoporin. Three classes of mutation influenced LamB levels as well as regulation of other mal genes. Low-level mal/lamB constitutivity resulted from mlc mutations acquired in all populations as well as changes at another uncharacterized locus. Larger increases in transporter content resulted from widespread acquisition of a regulatory malT–con mutation in fit isolates. The malT mutations sequenced from 67 adapted isolates were all single base substitutions resulting in amino acid replacements in the N-terminal third of the MalT activator protein. Analysis of malT–con sequences revealed a mutational spectrum distinct from that found in plate-selected malT mutants, suggesting that mutational pathways were affected by environmental factors. A second major finding was the remarkable allele diversity in malT within a population derived from a single clone, with at least 11 different alleles co-existing in a population. The multiplicity of alleles (as well as those found in adaptive mgl changes in the accompanying study) suggest that the periodic selection events observed previously in such populations are not a major factor in reducing genetic diversity. A simple model is presented for the generation of genetic heterogeneity in bacterial populations undergoing polygenic selection. [ABSTRACT FROM AUTHOR]

1. Escherichia coli can accumulate 14C-labelled (α1 → 4)-linked D-glucose oligomers up to maltoheptaose. Longer maltodextrins are not transported and are not utilized as carbon sources. 2. Maltodextrins too large to be transported are nevertheless bound by the outer envelope of intact E. coli. This binding is saturable (Kd for maltodecaose = 3-4 µM) and the binding sites are inducible by maltose. Each bacterium has approximately 30 000 sites when fully induced. 3. Using mutants devoid of various components of the maltose transport system, the highaffinity binding of maltodextrins by intact bacteria has been shown to be dependent on the presence of both λ receptor (an outer membrane protein) and periplasmic maltose binding protein. 4. The same binding sites are accessible to both utilizable and non-utilizable maltodextrins. Maltodecapentaose is a competitive inhibitor of maltose transport (Ki 1.5-2.5 µM). 5. These results show that the periplasmic maltose binding protein is readily accessible to substrates of at least 2500 molecular weight. The inability to transport dextrins larger than maltoheptaose is, therefore, due to the inability of E. coli to transfer large substrates from the binding protein to the cytoplasm and not to lack of access through the outer membrane. [ABSTRACT FROM AUTHOR]

1. Accumulation of substrate via the maltose transport system of Escherichia coli is incompletely inhibited by uncouplers of oxidative phosphorylation. The extent of this inhibition is reduced upon introduction of a mutation in the Mg2+ Ca2+-dependent ATPase. This suggests that lack of importance of the proton motive force for accumulation of substrate. 2. Starved cells of the ATPase mutant show a reduced rate of maltose uptake. This low rate was strongly stimulated by the glycolytic carbon source D-fructose but not by oxidisable energy sources such as D-lactate or ascorbate plus phenazine methosulphate. These findings are also in agreement with the lack of importance of the proton motive force in maltose accumulation. 3. Incubation of wild-type as well as ATPase-less cells with high concentrations of arsenate, that is known to deplete the intracellular level of ATP in E. coli, did not significantly inhibit the uptake of maltose. This suggests that the level of intracellular ATP is not directly the energy source for maltose uptake. 4. The exit of maltose, in both the wild-type and the ATPase-less strains, is inhibited by uncouplers of oxidative phosphorylation. Azide, dinitrophenol, and carbonyl cyanide chlorophenyl-hydrazone inhibited the downhill exit of maltose and also the exchange of maltose; arsenate did not. This suggests a requirement for the proton motive force in transport of maltose in the outward direction, and contrasts from the properties of the entry reaction. These results are consistent with the view that entry and exit of maltose are mediated via different carriers and energized by different modes. [ABSTRACT FROM AUTHOR]

LamB glycoporin has an important general role in carbohydrate uptake during growth at low extracellular sugar concentrations. lamB and mal regulon induction during glucose starvation and glucose-limited continuous culture was investigated using lacZ fusions. A low-level burst of lamB induction occurred upon entry into glucose starvation-induced stationary phase but returned to basal levels during continued nutrient deprivation. Glucose-limited continuous culture elicited much higher expression of transporter genes in the mal regulon, as well as [14C]-maltose-transport activity; malEFG and malKlamB operons in glucose-limited chemostats were expressed to close to half of the level of maltose-induced batch cultures. Limitation-induced expression was dependent on both Crp-cAMP and MaIT activation but was independent of RpoS function. As expected for a gene with a Crp-controiled promoter, maIT expression was maximal under conditions which elicited the highest cAMP levels, but lamB induction did not behave in a corresponding fashion. Rather, maximal lamB induction occurred at rapid but suboptimal growth rates with micromolar or submicromolar medium glucose. Maximal transport and lamB induction coincided with increased endogenous maltotriose (inducer) concentrations during growth on glucose. Hence regulation of glycoporin and the maltose-transport system is not a starvation- or stationary-phase response but facilitates the adaptation of Escherichia coli to low-nutrient environments through endoinduction. [ABSTRACT FROM AUTHOR]