Conference abstracts from the Colloqium Microbial : Concepts and Controversies

organised by The Canada Research Chair in the history of

at the Université du Québec à Montréal, from October 17 to 19 2002

Beyond neo-Darwinism: The Origins of Microbial Jan Sapp Department fo History, Université du Québec à Montréal, CIRST Chairholder of the Canada Research Chair in the

The neo-Darwinian evolutionary synthesis of the 1930s and 1940s dealt with the evolution of plants and animals over the last 560 million years. It did not address the evolution of and the previous 3000 million years of evolutionary change on earth. During the last two decades of the twentieth century, biologists developed new comparative molecular techniques and concepts to trace life back thousands of millions of years to investigate early microbial evolution with the aim to create a universal phylogeny. Studies of microbial phylogeny have brought about a conceptual revolution in the way in which evolutionary change occurs in microbes with the evidence for the fundamental importance of symbiotic mergers, fusions, and various other mechanisms for horizontal transfer. The scope and significance of these mechanisms remain subjects of controversy.

The Origin of Intermediate Metabolism Harold Morowitz Krasnow Institute, George Mason University, Fairfax, VA 2030, USA

The case is made for autotrophs preceding heterotrophs, chemoautorophs preceding photoautotrophs, and the reductive tricarboxylic acid cycle preceding the Calvin-Benson cycle. The acetyl Co-A pathway is less certain. A group of universal features of the primary chart of autotrophic metabolism is discussed. This includes the universal nitrogen entry point and the universal sulfur entry point. The ecological significance of universal features of primary metabolism is discussed. The existence of biological laws at various hierarchical levels is outlined. The Pauli Exclusion Principle is presented as an example of hierarchical laws. The anomalous features of Vitamin B12 are discussed.

Molecular phylogeny of based on comparative sequence analysis of conserved

1 Karl Heinz Schleifer and Wolfgang Ludwig Department of , Technical University Munich, Am Hochanger 4, D-85350 Freising; Germany

Carl Woese and his coworkers achieved a breakthrough regarding the reconstruction of the phylogeny of by introducing rapid methods for comparative sequence analysis of small subunit rRNAs. Based on their data a phylogenetic tree of prokaryotes could be reconstructed for the first time. Currently more than 30000 small subunit rRNA sequences from pro- and eukaryotes are available in public databases. Due to the lack of comprehensive sequence data bases for other potential phylogenetic marker molecules, our view of the phylogeny of bacteria is mainly based on data derived from only one class of molecule, the 16S rRNA. Now in the era of genomics the rapidly increasing number of accessible full genome and further gene sequence data allows a sound evaluation of small subunit rRNA based phylogenetic conclusions. The comparative analysis of the available genome data showed that only a rather limited number of genes or gene products fulfils the criteria for phylogentic markers such as universal distribution as well as sufficient sequence conservation and complexity. The large subunit rRNA, elongation and initiation factors, subunits of proton translocation ATPase, RNA polymerase, DNA gyrase, recA, heat shock , and amino acyl tRNA synthetases are among the most informative marker molecules. Comparative phylogenetic analyses of these markers are in good agreement with that of the small subunit rRNA, at least with respect to the major groups. However, local tree topologies often differ depending on the molecule analysed. This was to be expected. In a few cases there are also more drastic differences between phylogenetic trees derived from rRNA and genes. This, however, can mostly be explained by the presence of multiple genes which may originate from gene duplication or lateral gene transfer. Since functional consistency of multiple genes cannot be assumed for all variants, there may be the risk of comparing paralogous markers. The latter can only be recognized as such if there are genomes containing all variants of the multiple genes. This may complicate comparative phylogenetic analysis. However, with the availability of more full genome sequences it should be possible to handle such problems more properly.Despite the problems with multiple genes, the phylogenetic analyses based upon different alternative markers showed that small subunit rRNA derived trees globally reflect the phylogeny of the corresponding organisms, however, locally more their own history.

Interactions between and the Environment. James Lake and R. Jain, M. Rivera, J. Moore Molecular Biology Institure, University of California, Los Angeles

Horizontal gene transfer (HGT) is a process through which disparate prokaryotic groups can obtain foreign genetic material in response to a changing environment. It is an ancient process that has altered genomes since, at least, the last common ancestor of life and is influenced by abiotic factors such as temperature and pH. HGT is selective and preferentially favors the exchange of some gene types. Operational genes (mostly housekeeping genes) are readily incorporated through HGT, but informational genes (translation, transcription, and other genes) are less readily incorporated through this mechanism. HGT is also influenced by abiotic factors

2 such as temperature and pH. It has been suggested that the collection of organisms that exchange genes can be thought of as a globalorganism, having an immense population size and a correspondingly great potential for evolving novel genes. Here we ask whether the organisms which share genes truly share them on a global scale, or whether environmental factors can affect its scale. In other words, can geography and environment, or abiotic and biotic parameters, influence genetic exchange by HGT? Our analyses of some 20,000 genes in eight complete genomes show that environmental factors can significantly alter horizontal transfer among prokaryotes.

Mountains and molehills what is at stake in the lateral gene transfer debate? W. Ford Doolittle Program in Evolutionary Biology, Canadian Institute for Advanced Research and Department of Biochemistry and Molecular Biology ,

During the 1980s and 90s, microbiologists constructed, and came to believe in, a universal species tree based on sequences of a single gene (small subunit rRNA) even though they had known since the mid 1960s that some genes can be readily transferred between species. There were sound and unsound reasons for being optimistic about this enterprise, and many still endorse it, although complete prokaryotic genome sequences, ever more numerous and accessible, show that we had drastically underestimated the extent of transfer, over short and long evolutionary time scales. I will survey the current state of this debate over gene transfer the evidence, the arguments and the underlying reasons for the heat.

CONTEMPORARY ISSUES IN MITOCHONDRIAL ORIGINS AND EVOLUTION Michael W. Gray Program in Evolutionary Biology, Canadian Institute for Advanced Research and Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS B3H 4H7

Questions about the origin of the mitochondrion and its subsequent evolution are intimately tied to the mitochondrial genome, for it is the latter that has provided the clearest indications of a bacterial (specifically a-Proteobacterial) origin for at least those functions (coupled oxidative phosphorylation, protein synthesis) that this genome partly encodes. One of the most remarkable features of the mitochondrial genome is its extreme structural and organizational diversity, which underpins a basically uniform genetic function. Mitochondrial (mtDNAs) in animals, plants, fungi and look very different from one another, and they evolve in radically different ways. Comparative sequence analysis, based on the determination of complete mtDNA sequences, has proven to be a powerful method for assessing the pathways and processes of mitochondrial genome evolution, and for making inferences about the origin of the mitochondrion. This approach has defined three general organizational patterns (designated ancestral, expanded ancestral and derived) into which mitochondrial genomes can be roughly classified, and has also identified the most gene-rich and eubacteria-like mitochondrial genomes yet known, providing our clearest picture to date of the ancestral mtDNA progenitor. Various types of data, including mitochondrial gene content, gene order and phylogenetic trees based on

3 concatenated mitochondrial protein-coding sequences, argue strongly in favour of a single origin of the mitochondrial genome.

Although the mitochondrial genome is essential to the process of mitochondrial biogenesis, it encodes a relatively small number of the protein components of the mitochondrion: genes for the vast majority of mitochondrial proteins are carried by the nuclear genome. The availability of complete nuclear genome sequences (e.g., yeast) has provided an opportunity to assess the phylogenetic affiliations of nucleus-encoded mitochondrial proteins. A small proportion of these proteins can be traced unambiguously to the a-Proteobacterial lineage of eubacteria; genes for these proteins likely resided originally in the proto-mitochondrial genome but were transferred to the nucleus in the course of evolution. A large proportion of nucleus- encoded mitochondrial proteins appear to be unique within the eukaryotic lineage, suggesting that at least some of these proteins may have emerged specifically within this lineage. A still- contentious question is whether any extant amitochondriate eukaryotes derive from ancestors that primitively lacked mitochondria, or whether the amitochondriate condition is in all cases a derived trait, due to secondary loss of the organelle in a primitive ancestor. The finding in some amitochondriate eukaryotes of nuclear genes encoding typically “mitochondrial” proteins has strengthened the case for secondary loss of mitochondria. Nevertheless, at this time we cannot confidently discount the existence of primitively amitochondriate eukaryotes.

On the Origins of Cells -- Prokaryotic and Eukaryotic William Martin Institut für Botanik, Universität Düsseldorf, D-40225 Düsseldorf, Germany Tel ++49-211-811-3011; Fax ++49-211-811-3554 e-mail [email protected]

The discrepancy between the number of genes that organelle genomes encode (<100) and the number of eubacterial proteins that they contain (>1000) is generally explained by something known as 'endosymbiotic gene transfer'- during evolution, organelles export their genes to the nucleus, but re-import the gene products, so that proteins are retained in organelles, but most of the genes are not. Traditional views posit that the host of mitochondrial symbiosis was a heterotrophic, primitively amitochondriate eukaryote. The hydrogen hypothesis posits that the heterotrophic lifestyle ancestral to all eukaryotes is an acquisition via gene transfer from a facultatively anaerobic, heterotrophic purple non-sulphur eubacterium - the common ancestor of mitochondria and hydrogenosomes - that became an endosymbiont in an H2-dependent, autotrophic host. The hydrogen hypothesis provides a simple rationale to explain the findings of i) common ancestry of mitochondria and hydrogenosomes, ii) that all eukaryotes studied to date possessed a mitochondrial symbiont in their evolutionary past, and iii) that eukaryotes, generally speaking, possess an archaebacterial genetic apparatus that proliferates with the help of eubacterial energy metabolism. If time permits, some thought will be given to the origins of cells in general.

4 Evolutionary Relationships Among Prokaryotes and the Origin of the Eukaryotic Cell Radhey S. Gupta Department of Biochemistry, McMaster University, Hamilton, Canada L8N 3Z5

To understand the origin of the eukaryotic cell, it is essential at first to clarify the evolutionary relationships among prokaryotic organisms which predated the eukaryotes. Our work employing conserved inserts and deletions (indels or signature sequences) in highly conserved proteins as phylogenetic tools is providing valuable information in this regard. Based on the identified signatures, it is now possible to define in clear molecular terms all the main groups within Bacteria and logically deduce how they are related to each other and in what order have they branched off from a common ancestor. These issues, which are central to understanding bacterial phylogeny, were not resolved in the past. The branching order of different groups is indicated as follows: Low G+C gram-positive Y High G+C gram-positive Y Clostridium- Fusobacteria- Thermotoga Y Deinococcus-Thermus- Green nonsulfur bacteria Y Y Spirochetes Y Chlamydia-Cytophaga -Bacteroides- Green sulfur bacteria Y Aquifex Y Proteobacteria-1 (, and *) Y Proteobacteria-2 (") Y Proteobacteria-3 ($) and Y Proteobacteria -4 ((). The reliability and predictive power of this model was objectively tested using sequence data for bacterial genomes. The model correctly predicted the presence or absence of various indels in all 67 bacterial genomes with only a single exception in 1322 observations (>99.9 % reliability). These results also provide strong evidence that the genes/proteins containing these indels have not been affected by factors such as lateral gene transfer, although such events play an important role in evolution.

Signature sequences and phylogenetic analysis based on many proteins also point to a specific relationship between Archaebacteria and Gram-positive bacteria, a relationship which is supported by the similarity in their cell structures. The genes/proteins which indicate to be distinct from Bacteria are primarily those involved in the information transfer processes, and they provide the main targets for antibiotics produced by Gram-positive bacteria. We have suggested that both Archaebacteria and Diderm bacteria have evolved from Gram-positive bacteria as different strategies to cope with the antibiotic selection pressure.

Unlike the evolutionary relationships among prokaryotes, the eukaryotic cells are indicated to be of chimeric origin having received major gene contributions from both archaebacteria and Gram-negative bacteria ("-proteobacteria). We have suggested that the formation of the ancestral eukaryotic cell involved a unique fusion event between these two groups of prokaryotes, which resulted in the formation of nucleus as well as ER. Our analyses also indicate that this primary fusion event was distinct from the latter endosymbiotic event that gave rise to mitochondria.

The missing piece: the microtubule cytoskeleton and the origin of eukaryotes Michael F. Dolan Department of Geosciences, University of Massachusetts, Amherst

5

Eukaryotes are characterized by a membrane-bounded nucleus and a microtubule cytoskeleton that is used to separate the chromosomes in mitosis. The recent, molecular- and biochemical- based hypotheses on the origin of eukaryotes fail to adequately address the evolutionary origin of microtubules. This stems in part from the replacement of morphological- and organism- based approaches to cell evolution with molecular- and biochemical-based ones. Morphological, natural historical approaches stress the biology of contemporary organisms. Such an approach looks for tubules or tubule-organizing centers in bacteria, or for simplified microtubule structures in protists, and then considers the biochemical components involved. Proponents of such approaches usually insist that the clues to the earliest lineages can be found among extant taxa. Molecular and biochemical approaches emphasize gene or sequences and protein chemistry, and consider the organismic biology secondarily, as in the case of FtsZ, the putative prokaryotic tubulin homolog. Proponents of these approaches are willing to write-off higher-level taxa, such as the primordial Eukarya or pre-mitochondrial eukaryotes, or the pre-microtubule-containing eukaryotes, that are purported to be evolutionary intermediates, but have left no descendants. An hypothesis on the origin of microtubules that synthesizes both approaches is lacking.

"Thiodendron"-like consortia to chimeric archaeprotists” Department of Geosciences, University of Massachussetts, Amherst

Since all nucleated cells (eukaryotes) evolved via symbiogenesis from at least two kinds of ancestors (as did no bacterial cells, prokaryotes), the three classification system (Archeae, Bacteria, Eukarya) is inadequate and misleading. To those who seek logical, descriptive and useful classification systems all living organisms can be placed unambiguoulsy into one or another of the two highest taxa (most inclusive groups): Prokaryotae and Eukaryotae. All members of the four mutually exclusive unambiguous taxa of eukaryotes, by hypothesis, evolved from anaerobic prokaryotic consortia similar to the extant Thiodendron association. From an analogous prokaryotic consortium of a sulfur-reducing archaebacterium (like Thermoplasma acidophila) and a motile sulfide-oxidizing eubacterium (like the Spirochaeta of Thiodendron) evolved the chimeric ancestor (such as that suggested by Gupta). The descendants of the chimera include mitotic anaerobes (archaeprotists) such as the amitochondriate amoebae, the diplomonads and the parabasalids The former attachment apparatus of the consortium became the karyomastigont (organellar system of nucleus, kinetosome and their connector). Karyomastigont then became mitotic spindle, the single distinguishing aspect of all nucleated organisms. The nucleus evolved by release in several lineages, both without and with mitochondria.

The role of symbiotic bacteria in eukaryotic speciation John H. Werren Biology Department, University of Rochester, Rochester, N.Y. 14627

6 Symbiotic bacteria are widespread in nature and could play important roles in the rates and patterns of speciation in their eukaryotic hosts. Here the empirical and recent theoretical investigations of the role potential role of symbionts in eukaryotic speciation is explored. Special attention is paid the endosymbiont Wolbachia, which is widespread in arthropods and causes various reproductive manipulations in its hosts that may promote reproductive isolation and speciation. However, more general principles that relate to the diverse array of endosymbiont-host evolutionary interactions are also considered. Future direction of research to evaluate the role of endosymbionts in eukaryotic speciaton are discussed.

7