The Effect of Mobile Element IS10 on Experimental Regulatory Evolution in Escherichia coli

Mobile genetic elements are widespread in bacteria, where they cause several kinds of mutations. Although their effects are on the whole negative, rare beneficial mutations caused by insertion sequence elements are frequently selected in some experimental evolution systems. For example, in earlier work, we found that strains of Escherichia coli that lack the sigma factor RpoS adapt to a high-osmolarity environment by the insertion of element IS10 into the promoter of the otsBA operon, rewiring expression from RpoS dependent to RpoS independent. We wished to determine how the presence of IS10 in the genome of this strain shaped the evolutionary outcome. IS10 could influence the outcome by causing mutations that confer adaptive phenotypes that cannot be achieved by strains without the element. Alternatively, IS10 could influence evolution by increasing the rate of appearance of certain classes of beneficial mutations even if they are no better than those that could be achieved by a strain without the element. We found that populations evolved from an IS10-free strain did not upregulate otsBA. An otsBA-lacZY fusion facilitated the recovery of a number of mutations that upregulate otsB without involving IS10 and found that two caused greater fitness increases than IS10 insertion, implying that evolution could have upregulated otsBA in the IS10-free strain. Finally, we demonstrate that there is epistasis between the IS10 insertion into the otsBA promoter and the other adaptive mutations, implying that introduction of IS10 into the otsBA promoter may alter the trajectory of adaptive evolution. We conclude that IS10 exerts its effect not by creating adaptive phenotypes that could not otherwise occur but by increasing the rate of appearance of certain adaptive mutations

Adaptive Evolution of the Lactose Utilization Network in Experimentally Evolved Populations of Escherichia coli

Adaptation to novel environments is often associated with changes in gene regulation. Nevertheless, few studies have been able both to identify the genetic basis of changes in regulation and to demonstrate why these changes are beneficial. To this end, we have focused on understanding both how and why the lactose utilization network has evolved in replicate populations of Escherichia coli. We found that lac operon regulation became strikingly variable, including changes in the mode of environmental response (bimodal, graded, and constitutive), sensitivity to inducer concentration, and maximum expression level. In addition, some classes of regulatory change were enriched in specific selective environments. Sequencing of evolved clones, combined with reconstruction of individual mutations in the ancestral background, identified mutations within the lac operon that recapitulate many of the evolved regulatory changes. These mutations conferred fitness benefits in environments containing lactose, indicating that the regulatory changes are adaptive. The same mutations conferred different fitness effects when present in an evolved clone, indicating that interactions between the lac operon and other evolved mutations also contribute to fitness. Similarly, changes in lac regulation not explained by lac operon mutations also point to important interactions with other evolved mutations. Together these results underline how dynamic regulatory interactions can be, in this case evolving through mutations both within and external to the canonical lactose utilization network

Unique Cost Dynamics Elucidate the Role of Frameshifting Errors in Promoting Translational Robustness

There is now considerable evidence supporting the view that codon usage is frequently under selection for translational accuracy. There are, however, multiple forms of inaccuracy (missense, premature termination, and frameshifting errors) and pinpointing a particular error process behind apparently adaptive mRNA anatomy is rarely straightforward. Understanding differences in the fitness costs associated with different types of translational error can help us devise critical tests that can implicate one error process to the exclusion of others. To this end, we present a model that captures distinct features of frameshifting cost and apply this to 641 prokaryotic genomes. We demonstrate that, although it is commonly assumed that the ribosome encounters an off-frame stop codon soon after the frameshift and costs of mis-elongation are therefore limited, genomes with high GC content typically incur much larger per-error costs. We go on to derive the prediction, unique to frameshifting errors, that differences in translational robustness between the 5′ and 3′ ends of genes should be less pronounced in genomes with higher GC content. This prediction we show to be correct. Surprisingly, this does not mean that GC-rich organisms necessarily carry a greater fitness burden as a consequence of accidental frameshifting. Indeed, increased per-error costs are often more than counterbalanced by lower predicted error rates owing to more diverse anticodon repertoires in GC-rich genomes. We therefore propose that selection on tRNA repertoires may operate to reduce frameshifting errors

Chloramphenicol Selection of IS10 Transposition in the cat Promoter Region of Widely Used Cloning Vectors

The widely used pSU8 family of cloning vectors is based on a p15A replicon and a chloramphenicol acetyltransferase (cat) gene conferring chloramphenicol resistance. We frequently observed an increase in the size of plasmids derived from these vectors. Analysis of the bigger molecular species shows that they have an IS10 copy inserted at a specific site between the promoter and the cat open reading frame. Promoter activity from both ends of IS10 has been reported, suggesting that the insertion events could lead to higher CAT production. Insertions were observed in certain constructions containing inserts that could lead to plasmid instability. To test the possibility that IS10 insertions were selected as a response to chloramphenicol selection, we have grown these constructs in the presence of different amounts of antibiotic and we observed that insertions arise promptly under higher chloramphenicol selective pressure. IS10 is present in many E. coli laboratory strains, so the possibility of insertion in constructions involving cat-containing vectors should be taken into account. Using lower chloramphenicol concentrations could solve this problem

Nonlinear Fitness Landscape of a Molecular Pathway

Genes are regulated because their expression involves a fitness cost to the organism. The production of proteins by transcription and translation is a well-known cost factor, but the enzymatic activity of the proteins produced can also reduce fitness, depending on the internal state and the environment of the cell. Here, we map the fitness costs of a key metabolic network, the lactose utilization pathway in Escherichia coli. We measure the growth of several regulatory lac operon mutants in different environments inducing expression of the lac genes. We find a strikingly nonlinear fitness landscape, which depends on the production rate and on the activity rate of the lac proteins. A simple fitness model of the lac pathway, based on elementary biophysical processes, predicts the growth rate of all observed strains. The nonlinearity of fitness is explained by a feedback loop: production and activity of the lac proteins reduce growth, but growth also affects the density of these molecules. This nonlinearity has important consequences for molecular function and evolution. It generates a cliff in the fitness landscape, beyond which populations cannot maintain growth. In viable populations, there is an expression barrier of the lac genes, which cannot be exceeded in any stationary growth process. Furthermore, the nonlinearity determines how the fitness of operon mutants depends on the inducer environment. We argue that fitness nonlinearities, expression barriers, and gene–environment interactions are generic features of fitness landscapes for metabolic pathways, and we discuss their implications for the evolution of regulation

Molecular Characterization of Transcriptional Regulation of rovA by PhoP and RovA in Yersinia pestis

BACKGROUND: Yersinia pestis is the causative agent of plague. The two transcriptional regulators, PhoP and RovA, are required for the virulence of Y. pestis through the regulation of various virulence-associated loci. They are the global regulators controlling two distinct large complexes of cellular pathways. METHODOLOGY/PRINCIPAL FINDINGS: Based on the LacZ fusion, primer extension, gel mobility shift, and DNase I footprinting assays, RovA is shown to recognize both of the two promoters of its gene in Y. pestis. The autoregulation of RovA appears to be a conserved mechanism shared by Y. pestis and its closely related progenitor, Y. pseudotuberculosis. In Y. pestis, the PhoP regulator responds to low magnesium signals and then negatively controls only one of the two promoters of rovA through PhoP-promoter DNA association. CONCLUSIONS/SIGNIFICANCE: RovA is a direct transcriptional activator for its own gene in Y. pestis, while PhoP recognizes the promoter region of rovA to repress its transcription. The direct regulatory association between PhoP and RovA bridges the PhoP and RovA regulons in Y. pestis

The Transcriptional Regulator Rok Binds A+T-Rich DNA and Is Involved in Repression of a Mobile Genetic Element in Bacillus subtilis

The rok gene of Bacillus subtilis was identified as a negative regulator of competence development. It also controls expression of several genes not related to competence. We found that Rok binds to extended regions of the B. subtilis genome. These regions are characterized by a high A+T content and are known or believed to have been acquired by horizontal gene transfer. Some of the Rok binding regions are in known mobile genetic elements. A deletion of rok resulted in higher excision of one such element, ICEBs1, a conjugative transposon found integrated in the B. subtilis genome. When expressed in the Gram negative E. coli, Rok also associated with A+T-rich DNA and a conserved C-terminal region of Rok contributed to this association. Together with previous work, our findings indicate that Rok is a nucleoid associated protein that serves to help repress expression of A+T-rich genes, many of which appear to have been acquired by horizontal gene transfer. In these ways, Rok appears to be functionally analogous to H-NS, a nucleoid associated protein found in Gram negative bacteria and Lsr2 of high G+C Mycobacteria

Compensatory Evolution of Gene Regulation in Response to Stress by Escherichia coli Lacking RpoS

The RpoS sigma factor protein of Escherichia coli RNA polymerase is the master transcriptional regulator of physiological responses to a variety of stresses. This stress response comes at the expense of scavenging for scarce resources, causing a trade-off between stress tolerance and nutrient acquisition. This trade-off favors non-functional rpoS alleles in nutrient-poor environments. We used experimental evolution to explore how natural selection modifies the regulatory network of strains lacking RpoS when they evolve in an osmotically stressful environment. We found that strains lacking RpoS adapt less variably, in terms of both fitness increase and changes in patterns of transcription, than strains with functional RpoS. This phenotypic uniformity was caused by the same adaptive mutation in every independent population: the insertion of IS10 into the promoter of the otsBA operon. OtsA and OtsB are required to synthesize the osmoprotectant trehalose, and transcription of otsBA requires RpoS in the wild-type genetic background. The evolved IS10 insertion rewires expression of otsBA from RpoS-dependent to RpoS-independent, allowing for partial restoration of wild-type response to osmotic stress. Our results show that the regulatory networks of bacteria can evolve new structures in ways that are both rapid and repeatable