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The effect of mobile element IS10 on experimental regulatory evolution in Escherichia coli.

ABSTRACT: 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.

SUBMITTER: Stoebel DM

PROVIDER: S-EPMC2922620 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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The effect of mobile element IS10 on experimental regulatory evolution in Escherichia coli.

Stoebel Daniel M DM Dorman Charles J CJ

Molecular biology and evolution 20100416 9

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 e ...[more]

PMID: 20400481

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Project description:Background and Objectives:Metallic antimicrobial materials are of growing interest due to their potential to control pathogenic and multidrug-resistant bacteria. Yet we do not know if utilizing these materials can lead to genetic adaptations that produce even more dangerous bacterial varieties. Methodology:Here we utilize experimental evolution to produce strains of Escherichia coli K-12 MG1655 resistant to, the iron analog, gallium nitrate (Ga(NO3)3). Whole genome sequencing was utilized to determine genomic changes associated with gallium resistance. Computational modeling was utilized to propose potential molecular mechanisms of resistance. Results:By day 10 of evolution, increased gallium resistance was evident in populations cultured in medium containing a sublethal concentration of gallium. Furthermore, these populations showed increased resistance to ionic silver and iron (III), but not iron (II) and no increase in traditional antibiotic resistance compared with controls and the ancestral strain. In contrast, the control populations showed increased resistance to rifampicin relative to the gallium-resistant and ancestral population. Genomic analysis identified hard selective sweeps of mutations in several genes in the gallium (III)-resistant lines including: fecA (iron citrate outer membrane transporter), insl1 (IS30 tranposase) one intergenic mutations arsC ?/? yhiS; (arsenate reductase/pseudogene) and in one pseudogene yedN ?; (iapH/yopM family). Two additional significant intergenic polymorphisms were found at frequencies > 0.500 in fepD ?/? entS (iron-enterobactin transporter subunit/enterobactin exporter, iron-regulated) and yfgF ?/? yfgG (cyclic-di-GMP phosphodiesterase, anaerobic/uncharacterized protein). The control populations displayed mutations in the rpoB gene, a gene associated with rifampicin resistance. Conclusions:This study corroborates recent results observed in experiments utilizing pathogenic Pseudomonas strains that also showed that Gram-negative bacteria can rapidly evolve resistance to an atom that mimics an essential micronutrient and shows the pleiotropic consequences associated with this adaptation. Lay summary:We utilize experimental evolution to produce strains of Escherichia coli K-12 MG1655 resistant to, the iron analog, gallium nitrate (Ga(NO3)3). Whole genome sequencing was utilized to determine genomic changes associated with gallium resistance. Computational modeling was utilized to propose potential molecular mechanisms of resistance.

Project description:Experimental evolution of Escherichia coli K-12 W3110 by serial dilutions for 2,200 generations at high pH extended the range of sustained growth from pH 9.0 to pH 9.3. pH 9.3-adapted isolates showed mutations in DNA-binding regulators and envelope proteins. One population showed an IS1 knockout of phoB (encoding the positive regulator of the phosphate regulon). A phoB::kanR knockout increased growth at high pH. phoB mutants are known to increase production of fermentation acids, which could enhance fitness at high pH. Mutations in pcnB [poly(A) polymerase] also increased growth at high pH. Three out of four populations showed deletions of torI, an inhibitor of TorR, which activates expression of torCAD (trimethylamine N-oxide respiration) at high pH. All populations showed point mutations affecting the stationary-phase sigma factor RpoS, either in the coding gene or in genes for regulators of RpoS expression. RpoS is required for survival at extremely high pH. In our microplate assay, rpoS deletion slightly decreased growth at pH 9.1. RpoS protein accumulated faster at pH 9 than at pH 7. The RpoS accumulation at high pH required the presence of one or more antiadaptors that block degradation (IraM, IraD, and IraP). Other genes with mutations after high-pH evolution encode regulators, such as those encoded by yobG (mgrB) (PhoPQ regulator), rpoN (nitrogen starvation sigma factor), malI, and purR, as well as envelope proteins, such as those encoded by ompT and yahO Overall, E. coli evolution at high pH selects for mutations in key transcriptional regulators, including phoB and the stationary-phase sigma factor RpoS.IMPORTANCEEscherichia coli in its native habitat encounters high-pH stress such as that of pancreatic secretions. Experimental evolution over 2,000 generations showed selection for mutations in regulatory factors, such as deletion of the phosphate regulator PhoB and mutations that alter the function of the global stress regulator RpoS. RpoS is induced at high pH via multiple mechanisms.

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Project description:In previous work (D. R. Harris et al., J Bacteriol 191:5240-5252, 2009, https://doi.org/10.1128/JB.00502-09; B. T. Byrne et al., Elife 3:e01322, 2014, https://doi.org/10.7554/eLife.01322), we demonstrated that Escherichia coli could acquire substantial levels of resistance to ionizing radiation (IR) via directed evolution. Major phenotypic contributions involved adaptation of organic systems for DNA repair. We have now undertaken an extended effort to generate E. coli populations that are as resistant to IR as Deinococcus radiodurans After an initial 50 cycles of selection using high-energy electron beam IR, four replicate populations exhibit major increases in IR resistance but have not yet reached IR resistance equivalent to D. radiodurans Regular deep sequencing reveals complex evolutionary patterns with abundant clonal interference. Prominent IR resistance mechanisms involve novel adaptations to DNA repair systems and alterations in RNA polymerase. Adaptation is highly specialized to resist IR exposure, since isolates from the evolved populations exhibit highly variable patterns of resistance to other forms of DNA damage. Sequenced isolates from the populations possess between 184 and 280 mutations. IR resistance in one isolate, IR9-50-1, is derived largely from four novel mutations affecting DNA and RNA metabolism: RecD A90E, RecN K429Q, and RpoB S72N/RpoC K1172I. Additional mechanisms of IR resistance are evident.IMPORTANCE Some bacterial species exhibit astonishing resistance to ionizing radiation, with Deinococcus radiodurans being the archetype. As natural IR sources rarely exceed mGy levels, the capacity of Deinococcus to survive 5,000?Gy has been attributed to desiccation resistance. To understand the molecular basis of true extreme IR resistance, we are using experimental evolution to generate strains of Escherichia coli with IR resistance levels comparable to Deinococcus Experimental evolution has previously generated moderate radioresistance for multiple bacterial species. However, these efforts could not take advantage of modern genomic sequencing technologies. In this report, we examine four replicate bacterial populations after 50 selection cycles. Genomic sequencing allows us to follow the genesis of mutations in populations throughout selection. Novel mutations affecting genes encoding DNA repair proteins and RNA polymerase enhance radioresistance. However, more contributors are apparent.

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The effect of mobile element IS10 on experimental regulatory evolution in Escherichia coli.

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The effect of mobile element IS10 on experimental regulatory evolution in Escherichia coli.

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