Johann Peter Gogarten, PhD - US grants

Transport ATPases play a central role in the bioenergetics of all living cells. In the past we have shown that the F-(eubacterial) and V-type (eukaryotic and archaebacterial) ATPases are very useful molecular markers for early organismal evolution. The proposed research aims to extend the use of the nuclear encoded ATPAse subunits to the early radiation of eukaryotes (especially flagellates and algae) and to the evolution of land plants. Parts of the genes encoding the catalytic subunits of V-type ATPASes will be amplified directly from the genomic or cDNA of various organisms using the polymerase chain reaction (PCR). The obtained sequences will be used to determine the phylogenetic relations between these organisms, including evaluations of the significance of the derived branching patterns.

Horizontal or lateral gene transfer (HGT) denotes the exchange of genetic information between organisms as opposed to vertical inheritance, i.e. the passing of genes from parent to daughter cells. This project will study the relative contributions of HGT and vertical inheritance to microbial evolution. First, the extent of intra-gene mosaicism will be explored. Individual genes can be mosaic as a result of recombination. Preliminary data indicate that in the case of ribosomal RNA intra-gene mosaicism might not be a rare event. Statistical tests will be developed to assess how frequently intra-gene mosaicism occurred during microbial evolution. These tests will utilize simulated sequence evolution incorporating different models with varying amounts of HGT and recombination. Second, genome evolution will be simulated to address the following question: Are the confidently detected instances of HGT the tip of an iceberg, the bulk of which remains below the detection limit? Or are many of the alleged transfer events artifacts due to compositional bias, convergent evolution and unrecognized gene duplications? The simulations will include different frequencies of recombination and will utilize different models to simulate sequence evolution. Simulations that take the detection levels for horizontal gene transfer and intra-gene mosaicism into consideration will move the study of horizontal gene transfer beyond a compilation of anecdotal evidence. Third, a relaxed molecular clock model will be used to analyze the early evolution of prokaryotic life. Time estimates and their credibility intervals will be obtained for individual gene families. Dates for key evolutionary events estimated from compatible datasets will be combined to achieve more accurate divergence time estimates

A tree has been the organizing image to explain the relationships among living creatures since before Darwin's explanations for natural descent. The modern practice of comparing DNA and protein sequences to discern these relationships, however, has revealed complexities in the process of descent never before imagined. Organisms, particularly microbes, evolve by horizontal as well as vertical inheritance of traits with different groups of organisms exchanging genes, in addition to passing them down to the next generation. This research project addresses the question: Can the large scale structure of the tree/web of life be discerned? While the current data may be interpreted to mean that the depiction of life's history as a single bifurcating tree is incomplete, this should not be seen as justification to give up on reconstructing life's history. The working hypothesis underlying this research is that a reconstruction of life's early history is indeed possible, provided care is taken to guard against artifacts and that new, informative molecular characters are added to the analyses. Four complementary approaches will be used to address the question through analyses of microbial genome sequences. (1) Genome sequence data will be examined to find ancient horizontal gene transfer events that created useful taxonomic characters. An automated genome analysis pipeline will be developed to find these events, and these characters will be incorporated into new examinations of evolutionary relationships. (2) Complete genome sequences allow comparisons of all the genes of an organism with those of others to measure their evolutionary relatedness. However, the impact of horizontal acquisition of genes on these analyses is unknown. Computer simulations and genome sequences from two unrelated groups of microbes that have undergone extensive horizontal gene transfer, the Haloarchaea and the Thermotogales, will be used to gauge the extent to which different phylogenomic approaches are impacted by highways of gene sharing between different groups of organisms. (3) A public database will be constructed to store information on all putatively identified gene transfer events. This database will be a valuable resource for the wider scientific community to trace the role of gene transfer in the assembly of metabolic pathways and to examine the adaptation of organisms to new ecological niches. (4) The nature of the last common ancestor of all living creatures, the trunk of the tree of life, has been difficult to discern given all the changes in nucleotide composition that have occurred during evolutionary time. A compositional analysis will be used to detect the remaining signal of the genetic code's expansion that occurred early in life's history in order to "root" the tree of life. These in-depth analyses of several prokaryotic and eukaryotic genomes will offer insights into the impacts of horizontal gene transfer on genome evolution and phylogenetic reconstructions, as well as identify natural groups in the tree of life and help to resolve its deep structure. Unraveling the structure of the tree of life will have percussions in all fields of biology.

This research program and its associated education activities will provide an ideal training ground in which high school, undergraduate, and graduate students along with postdoctoral fellows can learn about genome and organismal evolution. Plans are for three postdoctoral fellows, nine graduate students, and about twenty undergraduate and high school students to be trained through computational and laboratory research experiences. The goal is to train computer scientists and biologists to become experts in their respective disciplines and to become effective in trans-disciplinary communication; thereby enabling successful interdisciplinary collaborations. Results from the proposed research will be introduced into related courses on three campuses and will be accessible through an integrated database, which also includes a forum for public discussions on topics related to gene transfer and organismal evolution.

One of the most important, but poorly understood, drivers of microbial evolution is horizontal gene transfer (HGT), when whole genes or large gene fragments are passed along between two bacterial cells, sometimes representing different species. Understanding how HGT occurs may shed light on the adaptation of microbes to new ecological niches, evolution and spread of antibiotic resistance genes, the rise of emerging pathogens, and competition between microbes. The outcomes of the proposed research will include new computational methods and techniques for analyzing HGT events, open-source software tools for use by biologists, and valuable new insights into different types of HGT events in oceanic bacteria. The project will train three PhD students and at least three undergraduate students in interdisciplinary research skills. The project will also introduce several high-school students to bioinformatics research and provide training to several high-school science teachers on computational molecular evolution. Research results will also be integrated into graduate and undergraduate level classroom teaching.

The project focuses on two fundamental properties of HGTs. The first property deals with the 'mode' of an HGT event, which defines whether the transferred gene is 'additive' or 'replacing', i.e., whether it adds itself as a new gene to the recipient genome or replaces an existing homologous gene. The second property concerns the units of HGT events, i.e., whether the HGT event involved a gene fragment, entire gene, several genes, or entire operons. The project entails development of new, broadly applicable, computational methods to infer the modes and units of HGT events, application of these methods to microbial genome datasets to investigate the frequencies and roles of the different modes and units of HGT events in various evolutionary scenarios, and testing of specific hypotheses relating these properties of HGT events to evolutionary divergence, HGT integration mechanism, and gene function.

The research will address the following questions: (1) How do microorganisms (bacteria and archaea) diverge and become adapted to new environments? (2) What is the role of rare genes in the adaptation process? Evolution and adaptation of bacterial and archaeal populations, the spread of "selfish" or parasitic genes in those populations, and especially the maintenance of within-lineage variation, in which different microbial cells contain slightly different complements of genes, impacts plant, animal and human life in many ways. This interdisciplinary project combines theoretical explorations with the study of environmental samples and laboratory experimentation on archaea that inhabit saline environments. The project will train its participants in theoretical and computational biology and genetic research that involves genome sequencing, genetic engineering and laboratory tests. The research will involve high-school students and teachers, undergraduate and graduate students, and postdoctoral fellows in an international collaboration to develop and test hypotheses on the long-term survival of rare genes.

The research will execute the following tasks: (1) Through theory, modeling, and in silico experiments determine which population dynamics can lead to persistence of alleles in the non-core portion of the pan-genome. Find conditions that allow for the emergence of a within population division of labor, and for the within population survival of molecular parasites. In particular, explore the impact of population bottlenecks, gene flow and recombination within and between populations and species on the persistence of rare genes over time. (2) Determine which community dynamics can lead to the coexistence of alleles and to species diversification. Research will focus on the effect of low or uneven rates of within population gene transfer, possibly due to incompatibility of restriction modification (RM) or targeting by clustered regularly spaced short palindromic repeats (CRISPR) systems and we will explore when a division of labor that arose within a population is expected to lead to diversification. (3) Identify genes having limited distribution in Haloferax spp. and Halorubrum spp. populations and species from genome and meta-genome sequences, characterize these genes through bioinformatics based approaches, and select suitable candidates for genetic experimentation. (4) Perform experiments to determine the effect of selfish genetic elements, RM and CRISPR-Cas systems on recombination and gene frequencies in the off-spring. (5) Perform growth competition experiment in Hfx. volcanii (plus versus minus a gene of interest) to measure fitness cost of genes contributing to the production of a common shared good and for selfish genetic elements.

This collaborative US/Israel project is supported by the Division of Molecular and Cellular Biosciences and the Office of International Science and Engineering at the US National Science Foundation and the Israeli Binational Science Foundation.