Source: MICHIGAN STATE UNIV submitted to
GENOME EVOLUTION AND COMPETITIVENESS IN BACTERIA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1013880
Grant No.
(N/A)
Project No.
MICL02520
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Nov 1, 2017
Project End Date
Oct 31, 2022
Grant Year
(N/A)
Project Director
Lenski, R.
Recipient Organization
MICHIGAN STATE UNIV
(N/A)
EAST LANSING,MI 48824
Performing Department
Plant, Soil and Microbial Science
Non Technical Summary
Bacteria are critically important components of agricultural and natural ecosystems. Their activities may be either beneficial (e.g., decomposition of wastes) or harmful (e.g., pathogens of crops) to human interests, depending on the bacterial species and context. Many bacteria can evolve rapidly owing to their short generations and large populations. Such evolution often impedes efforts to control undesirable species, as exemplified by the emergence and spread of pathogenic bacteria that are resistant to antibiotics and other efforts to control them. The potential for rapid evolution also offers opportunities to develop microbes with improved properties for environmental and other applications. The specific objectives of this project are to characterize changes in the DNA, competitive fitness, and antibiotic-resistance of E. coli bacteria that are evolving under well-defined conditions. The knowledge that is gained may ultimately improve applications that seek to control those bacteria with undesirable properties and enhance those species that perform valuable functions.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2124010110035%
3114010110035%
7124010110030%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
Bacteria are essential components of both agricultural and natural ecosystems. They perform functions that may be either beneficial or harmful, depending on the species and ecological context. Many bacteria, including plant pathogens and food-borne pathogens such as Escherichia coli, have short generation times and large population sizes, enablingthem to rapidly evolve. Such rapid evolution often limits efforts to control harmful species, as exemplified by the emergence of pathogenic bacteria that are resistant to antibiotics. The overarching goals of this project are to quantify and characterize changes in the genome sequences, competitive fitness, and antibiotic-resistance profiles in bacteria that have evolved under defined and controlled conditions. The project will address the following specific objectives: (i) We will analyze metagenomic samples obtained at multiple time points from 12 replicate E. coli populations that have been propagated under the same conditions for over 60,000 generations, providing insights into bacterial genome evolution including rates of change, types of mutations, and extent of genetic diversity within and between populations. (ii) We will quantify the dynamics of competitive fitness over time from one population and compare the resulting trajectory with the metagenomic dynamics obtained above, providing insights into the coupling of genome evolution and competitive fitness. (iii) We will examine the effects of horizontal gene transfer on genome structure by analyzing E. coli isolates from an experiment in which donor strains carrying plasmids were mixed with recipients, providing insights into the effects of gene transfer and its interplay with selection. (iv) We will examine how the antibiotic-resistance profiles of bacteria have changed over the course of long periods without exposure to antibiotics, providing insights into the tension between antibiotic resistance and bacterial competitiveness.
Project Methods
Samples: This project will use samples of E. coli taken during experiments in which cell populations have been serially propagated over long periods under defined conditions. These samples are stored at -80C, and the bacteria remain viable and available for study. Metagenomic analysis: DNA is harvested from whole-population samples from the long-term experiment, sequenced using the Illumina platform, and analyzed using the breseq pipeline and other bioinformatics tools, as appropriate. Mutations are identified by comparing sequence data to the ancestral strain. In the Competitive fitness assays: A selectively neutral genetic marker allows competitors to be distinguished. Before each assay, the competitors are removed from the freezer and acclimated to the competition environment. An evolved population sample and the marked reference strain are then mixed and allowed to compete, and their relative growth rates are measured by dilution-plating on an appropriate indicator-agar medium at multiple time-points. Analysis of horizontal gene transfer: A similar approach will be used to the metagenomic analysis, except that sequencing will be performed on clonal isolates rather than population samples. Gene transfer events will be identified by comparing the genomes of recombinant clones with sequenced donor and recipient strains. Antibiotic-resistance profiles: Changes in resistance profiles are determined by comparing growth inhibition of evolved bacterial clones from several long-term experimental populations with their common ancestor as a reference. Assays are performed on a nutrient-rich agar-based medium supplemented with a range of concentrations of three antibiotics with different modes of action. Costs of resistance are assessed using competition assays described above; mutations are identified based on sequencing clones also as described above. The methods are evaluated via discussions with various colleagues (at Michigan State University and elsewhere) and based on success in the peer-review process.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The primary target audience for this research includes scientists in the fields of microbiology, ecology, genomics, and evolutionary biology. The PI also sometimes communicates findings to the general public via occasional lectures and other media. Changes/Problems:Work has proceeded largely as planned. Owing to the continuing pandemic, however, the lab has been mostly closed for several months, and the collection of new data has therefore been limited. We have adjusted and compensated by directing our efforts to analyzing already-collected data and preparing the results for publication. What opportunities for training and professional development has the project provided?This project does not provide direct financial support for training or professional development. However, a number of postdoctoral researchers, graduate students, and undergraduate students were engaged in research related to the objectives of this project, including several individuals from groups underrepresented in science. Two individuals completed their Ph.D. programs during this period. How have the results been disseminated to communities of interest?Results were shared with the scientific community via the publications reported above. For the general public, this long-term project was the subject of a feature article in Discover Magazine (November 2019) titled "How a 30-year experiment has fundamentally changed our view of how evolution works." What do you plan to do during the next reporting period to accomplish the goals?We have made substantial progress on the overarching goals and all four specific objectives for this project, with several major papers already published or in press that address them (Good et al., 2017, Nature; Maddamsetti and Lenski, 2018, PLOS Genetics; Lamrabet et al., 2019, mBio; Card et al., 2019; Blount et al., 2020; Grant et al, in press). We have obtained considerable data relevant to the coupling of genome evolution and competitive fitness in bacteria (objective ii in the Major Goals above), which we are currently analyzing. We also have two additional papers in preparation on the evolution of antibiotic resistance (objective iv in the Major Goals above). We will continue to identify new questions and pursue further analyses as our findings dictate and opportunities arise.

Impacts
What was accomplished under these goals? The PI and colleagues published two refereed journal articles during the reporting period that advanced the goals of this AgBio project. A third paper is in press, and other papers are in various stages of preparation. (1) Our previous work documented changes in the antibiotic-resistance profiles of E. coli during 50,000 generations without exposure to antibiotics, finding a clear trend towards increased susceptibility. The new paper by Card et al. (2019, PLOS Biology) examined the evolution of resistance when the ancestral and derived strains were challenged with four different antibiotics. We found that resistance was idiosyncratic with respect to the initial genotype; that is, the derived strains did not generally compensate for their greater susceptibility by "catching up" to the resistance level of the ancestor. Instead, the capacity to evolve resistance was constrained in some genetic backgrounds, implying that evolvability depended on previously fixed mutations in a contingent fashion. On-going work involves sequencing and analyzing the resistant genomes to better understand the constraints on resistance. (2) The paper by Blount et al. (2020, eLife) studied the genomic and phenotypic evolution of E. coli adapting to growth on citrate, a novel resource for this species. It was previously demonstrated that the capacity to use citrate evolved in one of12 experimental populations. In this new paper, we demonstrate ongoing refinement of this new ability, while also finding a persistent mismatch between the physiology of E. coli and growth on citrate. (3) The paper by Grant et al. (in press, American Naturalist) examines the effect of long-term adaptation to strictly oxic conditions on anaerobic metabolism in E. coli. During 60,000 generations in the presence of oxygen, anaerobic-specific genes accumulated excess mutations relative to aerobic-specific genes, which indicates relaxed selection. However, competitions between evolved and ancestral bacteria in anoxic conditions showed that anaerobic fitness had not decayed. These discordant results may be reconciled by recognizing the highly interconnected genetic architecture of these two metabolic networks.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Card, K. J., T. LaBar, J. B. Gomez, and R. E. Lenski. 2019. Historical contingency in the evolution of antibiotic resistance after decades of relaxed selection. PLOS Biology 17: e3000397.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Blount, Z. D., R. Maddamsetti, N. A. Grant, S. T. Ahmed, T. Jagdish, J. A. Baxter, B. A. Sommerfeld, A. Tillman, J. Moore, J. L. Slonczewski, J. E. Barrick, and R. E. Lenski. 2020. Genomic and phenotypic evolution of Escherichia coli in a novel citrate-only resource environment. eLife 9: e55414.
  • Type: Journal Articles Status: Awaiting Publication Year Published: 2021 Citation: Grant, N. A., R. Maddamsetti, and R. E. Lenski. In press. Maintenance of metabolic plasticity despite relaxed selection in a long-term evolution experiment with Escherichia coli. American Naturalist, awaiting publication.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The primary target audience for this research includes scientists in the fields of microbiology, ecology, genomics, and evolutionary biology. The PI also sometimes communicates findings to the general public via occasional lectures and other media. Changes/Problems:Work is proceeding as planned. There have been no substantive problems, and no major changes are anticipated. What opportunities for training and professional development has the project provided?This project does not provide direct financial support for training or professional development. However, a number of postdoctoral researchers, graduate students, and undergraduates were engaged in research related to the objectives of this project, including several individuals from groups that are underrepresented in science. How have the results been disseminated to communities of interest?Results were shared with the scientific community via the publications reported above as well as in seminars at universities and talks at conferences including, among others, a symposium of the American Society for Biochemistry and Molecular Biology on "Evolution and Core Processes in Gene Expression." What do you plan to do during the next reporting period to accomplish the goals?We have made substantial progress on the four specific objectives for this project, with major empirical papers already published for three of them (Good et al., 2017, Nature; Maddamsetti and Lenski, 2018, PLOS Genetics; Lamrabet et al., 2019, mBio). Additional work is currently underway on the evolution of antibiotic resistance (objective iv in Major Goals above), and we have gathered considerable data relevant to the temporal coupling of genome evolution and competitive fitness in bacteria (objective ii in Major Goals above). We will continue to identify new questions and pursue further analyses as our findings dictate and opportunities arise.

Impacts
What was accomplished under these goals? The PI and colleagues published three refereed journal articles during the reporting period that advanced the goals of this AgBio project. Additional papers are in various stages of preparation. One paper by Lamrabet et al. (2019, mBio) documented changes in the antibiotic-resistance profiles of E. coli cells over the course of 50,000 generations without exposure to antibiotics, finding a trend towards increased susceptibility. Related on-going work examines how the changes in susceptibility relate to the potential to evolve increased resistance when exposed to antibiotics. The second paper by Lamrabet et al. (2019, Molecular Biology and Evolution) showed that E. coli could evolve to compensate for the loss of a global regulatory gene, one that affects the expression of many other genes. They showed that the compensatory mutations had occurred in the promoter region of a gene involved in glucose uptake, and that this compensation was specific to that resource environment. The third paper by Blount et al. (2019, Science) was a review article that broadly examined the repeatability of evolution, and the roles of chance and historical contingencies in evolution. While not specifically focused on microorganisms, evolution experiments with bacteria featured prominently in that review.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Blount, Z. D., R. E. Lenski, and J. B. Losos. 2018. Contingency and determinism in evolution: replaying lifes tape. Science 362: eaam5979.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Lamrabet, O., M. Martin, R. E. Lenski, and D. Schneider. 2019. Changes in intrinsic antibiotic susceptibility during a long-term evolution experiment with Escherichia coli. mBio 10: e00189-19.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Lamrabet, O., J. Plumbridge, M. Martin, R. E. Lenski, D. Schneider, and T. Hindr�. 2019. Plasticity of promoter-core sequences allows bacteria to compensate for the loss of a key global regulatory gene. Molecular Biology and Evolution 36: 1121-1133.


Progress 11/01/17 to 09/30/18

Outputs
Target Audience:The primary target audience for this research includes scientists in the fields of microbiology, ecology, genomics, and evolutionary biology. In addition, the PI sometimes communicates to the general public via public lectures and various media. Changes/Problems:Work is proceeding as planned. There have been no substantive problems, and no major changes are anticipated. What opportunities for training and professional development has the project provided?This project does not provide direct financial support for training or professional development. However, multiple postdoctoral researchers, graduate students, and undergraduates were engaged in research related to the objectives of this project, including several individuals from groups that are underrepresented in science. How have the results been disseminated to communities of interest?Results were shared with the scientific community via the publications reported above as well as in seminars at universities and talks at conferences including, among others, the International Boehringer Ingelheim Conference on "From pathogen evolution to microbiome dynamics." What do you plan to do during the next reporting period to accomplish the goals?We have already made substantial progress on the four specific objectives for this project, with major papers already published for two of them (Good et al., 2017; Maddamsetti and Lenski, 2018). During the next reporting period, we will continue to work on the other two objectives, while also exploring new questions and hypotheses as they arise.

Impacts
What was accomplished under these goals? The PI and colleagues published five journal articles that directly or indirectly advanced the goals of this AgBio project (including during the period after the final progress report for a previous, related project). One paper analyzed more than a thousand metagenomes (i.e., whole-population samples) of the E. coli sampled from 12 experimental populations over the course of 60,000 generations, providing insights into bacterial genome evolution including rates of change, types of mutations, and genetic diversity within and divergence between populations (Good et al., 2017). Another paper examined the effects of evolved hypermutability on genome structure, using previously published sequences from clonal isolates from these same populations (Couce et al., 2017). A third paper examined the effects of horizontal gene transfer on genome structure by analyzing E. coli isolates from another experiment in which donor strains carrying plasmids were mixed with recipients, revealing high levels of gene transfer and illuminating its interplay with selection (Maddamsetti and Lenski, 2018). Two review articles placed the results of the long-term evolution experiment in the broader contexts of microbial ecology (Lenski, 2017) and economics (Lenski and Burnham, 2018). We also gathered data on changes in competitive fitness in relation to the metagenomic dynamics, and other data on changes in the antibiotic-resistance profiles of the evolving bacteria.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Maddamsetti, R., and R. E. Lenski. 2018. Analysis of bacterial genomes from an evolution experiment with horizontal gene transfer shows that recombination can sometimes overwhelm selection. PLOS Genetics 14: e1007199.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Lenski, R. E., and T. C. Burnham. 2018. Experimental evolution of bacteria across 60,000 generations, and what it might mean for economics and human decision-making. Journal of Bioeconomics 20: 107-124.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Lenski, R. E. 2017. Experimental evolution and the dynamics of adaptation and genome evolution in microbial populations. ISME Journal 11: 2181-2194.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Couce, A., L. V. Caudwell, C. Feinauer, T. Hindr�, J. P. Feugeas, M. Weigt, R. E. Lenski, D. Schneider, and O. Tenaillon. 2017. Mutator genomes decay, despite sustained fitness gains, in a long-term experiment with bacteria. Proceedings of the National Academy of Sciences, USA 114: E9026-E9035.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Good, B. H., M. J. McDonald, J. E. Barrick, R. E. Lenski, and M. M. Desai. 2017. The dynamics of molecular evolution over 60,000 generations. Nature 551: 45-50.