Recombination speeds adaptation by reducing competition between beneficial mutations in populations of Escherichia coli

Identification of the selective forces contributing to the origin and maintenance of sex is a fundamental problem in biology. The Fisher–Muller model proposes that sex is advantageous because it allows beneficial mutations that arise in different lineages to recombine, thereby reducing clonal interference and speeding adaptation. I used the F plasmid to mediate recombination in the bacterium Escherichia coli and measured its effect on adaptation at high and low mutation rates. Recombination increased the rate of adaptation ~3-fold more in the high mutation rate treatment, where beneficial mutations had to compete for fixation. Sequencing of candidate loci revealed the presence of a beneficial mutation in six high mutation rate lines. In the absence of recombination, this mutation took longer to fix and, over the course of its substitution, conferred a reduced competitive advantage, indicating interference between competing beneficial mutations. Together, these results provide experimental support for the Fisher–Muller model and demonstrate that plasmid-mediated gene transfer can accelerate bacterial adaptation.     

Clonal interference, multiple mutations and adaptation in large asexual populations

Two important problems affect the ability of asexual populations to accumulate beneficial mutations and hence to adapt. First, clonal interference causes some beneficial mutations to be outcompeted by more-fit mutations that occur in the same genetic background. Second, multiple mutations occur in some individuals, so even mutations of large effect can be outcompeted unless they occur in a good genetic background that contains other beneficial mutations. In this article, we use a Monte Carlo simulation to study how these two factors influence the adaptation of asexual populations. We find that the results depend qualitatively on the shape of the distribution of the fitness effects of possible beneficial mutations. When this distribution falls off slower than exponentially, clonal interference alone reasonably describes which mutations dominate the adaptation, although it gives a misleading picture of the evolutionary dynamics. When the distribution falls off faster than exponentially, an analysis based on multiple mutations is more appropriate. Using our simulations, we are able to explore the limits of validity of both of these approaches, and we explore the complex dynamics in the regimes where neither one is fully applicable.     

Clonal populations of thermotolerant Enterobacteriaceae in recreational water and their potential interference with fecal Escherichia coli counts

Bacterial strains were isolated from beach water samples using the original Environmental Protection Agency method for Escherichia coli enumeration and analyzed by pulsed-field gel electrophoresis (PFGE). Identical PFGE patterns were found for numerous isolates from 4 of the 9 days sampled, suggesting environmental replication. 16S rRNA gene sequencing, API 20E biochemical testing, and the absence of beta-glucuronidase activity revealed that these clonal isolates were Klebsiella, Citrobacter, and Enterobacter spp. In contrast, 82% of the nonclonal isolates from water samples were confirmed to be E. coli, and 16% were identified as other fecal coliforms. These nonclonal isolates produced a diverse range of PFGE patterns similar to those of isolates obtained directly from untreated sewage and gull droppings. beta-Glucuronidase activity was critical in distinguishing E. coli from other fecal coliforms, particularly for the clonal isolates. These findings demonstrate that E. coli is a better indicator of fecal pollution than fecal coliforms, which may replicate in the environment and falsely elevate indicator organism levels.     

A method for selection of mutations at the tdk locus in Escherichia coli.

Mutants of Escherichia coli which are resistant to 5-fluorodeoxyuridine all have mutations which map at a single locus at 27.5 min on the genetic map of E. coli. Extracts prepared from each mutant were deficient in thymidine kinase activity measured in vitro. Simple selective conditions which allowed detection of one mutant in the presence of 10(7) wild-type bacteria were found. These results show that loss of thymidine kinase activity is the usual mechanism for 5-fluorodeoxyuridine resistance and that all such mutations occur at the locus previously designated tdk.     

A new class of promoter mutations in the lactose operon of Escherichia coli

The isolation and genetic characterization of a number of mutations that are located in the promoter region of the lac2 operon are described. These mutations have reduced levels of lac operon expression in a wild, type (crp⁺cya⁺) genetic background. Three of the mutations also have lower levels of lac operon expression than lacP⁺ in a crp^?cya^? genetic background, that is in the absence of the catabolite activator protein and 3′,5′-adenosine cyclic monophosphate. These three mutations are located nearest to the lac operator. They define a second essential site in the promoter region.     

Suppression of mutations conferring penicillin tolerance by interference with the stringent control mechanism of Escherichia coli.

Mutations in Escherichia coli previously reported (R. E. Harkness and E. E. Ishiguro, J. Bacteriol. 155:15-21, 1983; L. C. Shimmin, D. Vanderwel, R. E. Harkness, B. R. Currie, A. Galloway, and E. E. Ishiguro, J. Gen. Microbiol. 130:1315-1323, 1984) as conferring a temperature-dependent tolerance to lysis induced by inhibitors of peptidoglycan synthesis were suppressed by treatment with inhibitors of the stringent response or by introduction of a relA mutation. The relA+ derivatives of the mutants exhibited a stringent response at the nonpermissive temperature. The consequent inhibition of the autolytic enzyme system (W. Kusser and E. E. Ishiguro, J. Bacteriol. 164:861-865, 1985) was apparently responsible for the lysis-tolerant phenotypes of these mutants.     

In vivo selection of conditional-lethal mutations in the gene encoding elongation factor G of Escherichia coli.

The ribosome translocation step that occurs during protein synthesis is a highly conserved, essential activity of all cells. The precise movement of one codon that occurs following peptide bond formation is regulated by elongation factor G (EF-G) in eubacteria or elongation factor 2 (EF-2) in eukaryotes. To begin to understand molecular interactions that regulate this process, a genetic selection was developed with the aim of obtaining conditional-lethal alleles of the gene (fusA) that encodes EF-G in Escherichia coli. The genetic selection depends on the observation that resistant strains arose spontaneously in the presence of sublethal concentrations of the antibiotic kanamycin. Replica plating was performed to obtain mutant isolates from this collection that were restrictive for growth at 42 degrees C. Two tightly temperature-sensitive strains were characterized in detail and shown to harbor single-site missense mutations within fusA. The fusA100 mutant encoded a glycine-to-aspartic acid change at codon 502. The fusA101 allele encoded a glutamine-to-proline alteration at position 495. Induction kinetics of beta-galactosidase activity suggested that both mutations resulted in slower elongation rates in vivo. These missense mutations were very near a small group of conserved amino acid residues (positions 483 to 493) that occur in EF-G and EF-2 but not EF-Tu. It is concluded that these sequences encode a specific domain that is essential for efficient translocase function.     

Estimation of the rate and effect of new beneficial mutations in asexual populations

The rate and effect of available beneficial mutations are key parameters in determining how a population adapts to a new environment. However, these parameters are poorly known, in large part because of the difficulty of designing and interpreting experiments to examine the rare and intrinsically stochastic process of mutation occurrence. We present a new approach to estimate the rate and selective advantage of beneficial mutations that underlie the adaptation of asexual populations. We base our approach on the analysis of experiments that track the effect of newly arising beneficial mutations on the dynamics of a neutral marker in evolving bacterial populations and develop efficient estimators of mutation rate and selective advantage. Using extensive simulations, we evaluate the accuracy of our estimators and conclude that they are quite robust to the use of relatively low experimental replication. To validate the predictions of our model, we compare theoretical and experimentally determined estimates of the selective advantage of the first beneficial mutation to fix in a series of ten replicate populations. We find that our theoretical predictions are not significantly different from experimentally determined selection coefficients. Application of our method to suitably designed experiments will allow estimation of how population evolvability depends on demographic and initial fitness parameters.     

Altered sugar selection and transport conferred by spontaneous point and deletion mutations in the lactose carrier of Escherichia coli

Spontaneous mutants harboring the lacY gene on an F'-factor were isolated. Those mutants that failed to grow on 5 mM lactose minimal media plates were chosen for further study. The mutants showed striking mutations in the lactose carrier as well as in sugar selection properties during transport assays. DNA sequencing of the lacY gene of the mutants revealed the following mutations: M-1-I, R-144-W, G-370-C and a deletion of residues 387-392, located in helix 12 of the carrier. Transport studies indicated that ONPG transport ranged between 8 and 25% of normal for the M-1-I, G-370-C and D387-392 mutants and 51% of normal for the R-144-W mutant. The downhill transport of lactose was 2-fold greater than for melibiose in cells harboring the M-1-I mutation and 3-fold higher for cells with the G-370-C mutation. On the other hand, cells with the D387-392-deletion mutation showed no lactose downhill transport, but 47% melibiose transport. Accumulation of TMG, a lactose analog, was 3-fold higher than the accumulation of melibiose in cells with the G-370-C mutation. On the other hand, in cells with the D387-392 mutation, TMG accumulation was completely defective, whereas melibiose accumulation was 50-fold higher than that of TMG, indicating that one or more of these residues in helix 12 of the carrier play a role in the active transport of b-galactoside, but not a-galactoside sugars. Initial lactose downhill transport rates were too unreliable to obtain trustworthy kinetic data. TMG and melibiose accumulation activities were present, but severely reduced in the mutant containing the R144W mutation, confirming that Arg-144 is important for active transport. All transport data were normalized for expression levels. The results indicate that the affected residues play a role in dictating sugar specificity and transport in the lactose carrier. The results here are novel in that they represent mutations in unique locations along the lactose carrier protein. For example, the M-1-I mutation was located at the N-terminal cytoplasmic tail of the carrier. Furthermore, G-370-C was located in the periplasmic loop between helices 11 and 12, suggesting a role for residues in this loop in mediating sugar selection.     

Oxidative and SOS-response in Escherichia coli and their interference]

Interference between the oxidative and SOS responses in Escherichia coli was studied. The oxidative response involves both reactive oxygen scavenging system and DNA repair systems which are distinct from either the SOS or adaptive response to alkylating agents. The oxyR gene is a positive regulatory gene for the oxidative response and controls at least 9 proteins which are induced by treatment with H2O2. This gene is not a portion of the SOS regulon that involves at least 17 different genes in E. coli and controls the SOS response--another inducible and nonspecific repair activity. The SOS response was measured in E. coli PQ37 by means of a sfiA: :lacZ operon fusion according to "SOS Chromotest" in a completely automated system "Bioscreen C" (Labsystems, Finland). Our data have shown that: 1) H2O2 was a potent inducer of sfiA gene--one of the SOS genes; 2) there was strong negative effect of the oxidative response on the subsequent induction of the SOS response. In common with our previous findings it should be concluded that there is an interference between the SOS response--on the one hand, and the adaptive and oxidative responses--on the other. The nonspecific heat shock response is proposed to be a main key in these interferences.     

Number of mutations required to evolve a new lactase function in Escherichia coli.

The frequency of mutation of the ebgAo allele to ebgA+ was compared with the frequency of mutation of strA+ to strA-. The observation that both spontaneous and ethyl methane sulfonate-induced mutations to ebgA+ occurred more frequently than mutations to strA- suggests that ebgA+ mutants arise as the result of single-point mutations.     

Preferential selection of deletion mutations of the outer membrane lipoprotein gene of Escherichia coli by globomycin.

Globomycin is an antibiotic which inhibits the processing of the prolipoprotein. Eighty globomycin-resistant mutants were independently isolated from Escherichia coli K-12 which had a deletion mutation in chromosomal lipoprotein gene (lpp), but contained a plasmid carrying the wild-type lpp gene. Twenty-six of the mutants did not have the lipoprotein in the membrane fractions. From the analysis of the plasmids of these mutants, all of the lipoprotein-deficient mutations were found to be due to deletion mutations around the lpp gene.     

Polymorphism and selection of rpoS in pathogenic Escherichia coli

Background  

Though RpoS is important for survival of pathogenic Escherichia coli in natural environments, polymorphism in the rpoS gene is common. However, the causes of this polymorphism and consequential physiological effects on gene expression in pathogenic strains are not fully understood.     

Direct selection for mutators in Escherichia coli

We have constructed strains that allow a direct selection for mutators of Escherichia coli on a single plate medium. The plate selection is based on using two different markers whose reversion is enhanced by a given mutator. Plates containing limiting amounts of each respective nutrient allow the growth of ghost colonies or microcolonies that give rise to full-size colonies only if a reversion event occurs. Because two successive mutational events are required, mutator cells are favored to generate full-size colonies. Reversion of a third marker allows direct visualization of the mutator phenotype by the large number of blue papillae in the full-size colonies. We also describe plate selections involving three successive nutrient markers followed by a fourth papillation step. Different frameshift or base substitution mutations are used to select for mismatch-repair-defective strains (mutHLS and uvrD). We can detect and monitor mutator cells arising spontaneously, at frequencies lower than 10(-5) in the population. Also, we can measure a mutator cascade, in which one type of mutator (mutT) generates a second mutator (mutHLS) that then allows stepwise frameshift mutations. We discuss the relevance of mutators arising on a single medium as a result of cells overcoming successive growth barriers to the development and progression of cancerous tumors, some of which are mutator cell lines.     

Up-promoter mutations in the lpp gene of Escherichia coli.

The promoter of the gene for the major outer membrane lipoprotein, the most abundant protein in Escherichia coli, is considered to be one of the strongest promoters in E. coli. The nucleotide sequences of the -10 and the -35 regions of the lpp promoter were altered in a step-wise manner to conform to their respective consensus sequences by synthetic oligonucleotide-directed site-specific mutagenesis. The mutated promoters were then fused to the lacZ gene to measure promoter activity. The beta-galactosidase activity increased approximately 1.9 and 2.4 fold when the -10 region (AATACT) was altered to TATACT(P1) and TATAAT (consensus sequence; P2), respectively. Similarly, it increased approximately 1.2 and 4.2 fold, when the -35 region (TTCTCA) was altered to TTCACA(R1) and TTGACA (consensus sequence; R2), respectively. When the mutations at the -10 and -35 regions were combined, the overall improvement of the promoter activity for R2-P1 was 4.0 fold over that of the wild-type promoter, while it was only 2.5 fold for R2-P2. These results indicate that substantial improvement of the promoter activity can be achieved by changing either of the two key regions to their respective consensus sequences. However, the complete conformity to consensus sequences at both regions does not necessarily result in the highest activity. With use of the improved lpp promoter in an expression cloning vehicle pIN-III-ompA, staphylococcal nuclease A was produced at a level of approximately 47% of the total cellular protein.     

Quinolone-resistant mutations of the gyrA gene of Escherichia coli

Summary DNA fragments of 8.5 kb containing the gyrA gene were cloned from Escherichia coli KL-16 and from four spontaneous gyrA mutants which showed various levels of resistance to quinolones. The gyrA gene was situated at about 4 kb in front of the nrdA gene and transcribed counterclockwise on the E. coli chromosome. It encoded a polypeptide of 875 amino acids with a molecular weight of about 97000. The four gyrA mutations were located strikingly close to one another within a small region near the N-terminus of the gyrA polypeptide, i.e., nucleotide changes from C to T, from C to G, from G to T and from G to T at nucleotides 248, 248, 318 and 199, respectively, resulting in amino acid changes from Ser to Leu, from Ser to Trp, from Gln to His and from Ala to Ser at amino acids 83, 83, 106 and 67, respectively. These mutations were situated in the relatively hydrophilic regions of the GyrA polypeptide and close to Tyr at amino acid 122 which has been shown to be the site covalently bound to DNA.