MICROBIOL ENGNRD ENVRON SYSTM
MICROBIOL ENGNRD ENVRON SYSTM ESGN 586
Colorado School of Mines
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Date Created: 10/05/15
Invited article for Microbe 0807 in press The Tree of Life Grows Implications for Understanding and Teaching Microbial Diversity Norman R Pace Department of Molecular Cellular and Developmental Biology University of Colorado Boulder CO 803090347 Summary Comparisons of gene sequences provide an objective view of evolutionary relationships and the course of evolution in the context of a molecular tree of life Cultureindependent sequencebased identi cations of microbes in the environment have dramatically expanded the known extent of microbial diversity The results of environmental surveys af rm the threedomain model for phylogenetic organization and the course of evolution The experimental results represented by the molecular tree are not consistent with the textbook paradigm of procaryote and eucaryote procaryote is obsolete and a distraction for students Recent decades have seen significant advances in the ways that we can perceive the diversity and evolution of microbial life However revisions oftextbooks to incorporate new information generally have not kept pace with either the discoveries or the concepts that result from the discoveries Consequently there are two intents of this article One is to provide an outline of the molecular view of biological diversity in general and how recent discoveries in environmental microbiology have considerably expanded the known scope of microbial diversity The second intent is to explain how this molecular view of life s diversity shows that the textbook explanations of life s organization and evolutionary history in terms of procaryote and eucaryote are wrong and conceptually misleading Procaryote is obsolete Toward a Tree of Life Biologists have long been intrigued by the possibility of relating all organisms As early as the mid19th Century Charles Darwin Ernst Haeckel and others compiled elaborate evolutionary trees based on life such as they knew it The relationships depicted were based on morphological and developmental properties of organisms however so could not include microbial life in any meaningful way With those criteria how can a plant be compared realistically to an animal or to a bacterium In general microbes did not t into early thought on biological diversity Little was known about such organisms and there was no objective way to relate them to one another let alone to complex eucaryotes The few physiological properties that have been and still are used to identify microbes provided little and often misleading information with which to deduce actual relationships That all changed in the 1960 s and 70 s with the development of technology for determination of gene sequences In contrast to physiological properties gene sequences provide an objective metric for evolutionary diversity the extent of sequence difference between orthologous genes in different organisms is a measure of evolutionary distance Consequently comparisons of sequences can be used to extract maps of evolutionary relatedness socalled phylogenetic trees Carl Woese in the early 1970s presciently chose to use comparisons of small subunit rRNA sequences to relate the most diverse organisms Ribosomal RNA sequences are ubiquitous and highly conserved and have become the gold standard for many phylogenetic studies Moreover comparisons of rRNA sequences were amenable to the technology available to Woese namely catalogs of oligonucleotides released by nuclease digestion of 32Plabeled rRNAs Early results provided the rst outlines of relationships among bacteria and led in 1977 to the discovery of archaebacteria which in 1990 were renamed archaea as it had become clear that they were fundamentally different from bacteria Thus Woese s results showed that all known organisms fall into three distinct relatedness groups archaea bacteria and eucarya eucaryotes The molecular description of diversity is based on gene sequences not properties of particular organisms Not all gene sequences have tracked with the rRNA sequences in overall evolution lateral transfer of genes from one genetic environment to another is well known and likely has been a significant driver of evolution The rRNA genes however have not to our knowledge undergone lateral transfer Moreover other widely conserved genes of the nucleic acidsbased information processing system also track with the rRNA tree Consequently the rRNAbased tree represents the evolutionary flow of the genetic machinery the essence of cells Largescale molecular trees should not be viewed as organismic trees rather as the more abstract lines of descent of the genetic machinery Thus we may think of dinosaurs as extinct but the line of descent from which dinosaurs emerged is alive today in the form of birds Into the Natural Microbial World It has long been recognized that most microbes in the environment gtgt99 do not thrive in culture and so remained unknown V th the use of gene sequences for identi cation of microbes molecular cloning and sequencing technology could be used to bypass the traditional but often impossible requirement for pure culture Microbiologists could now begin to explore the makeup of the natural microbial world and the results have expanded dramatically the known extent of microbial diversity With molecular technology rRNA genes are cloned from environmental DNA and sequenced sequences are used to relate the environmental organisms to others with known sequences Some properties oforganisms identified solely through rRNA sequence analysis can be predicted depending on how closely they are related to organisms with characterized properties The presumption here is that the representatives of a phylogenetic group are expected to share many common traits Even if the environmental organisms are not closely related to known ones however the rRNA sequences are specific identifiers and they provide a basis for the design of tools such as PCR primers and hybridization probes with which to study organisms of interest The number of environmental rRNA sequences in the databases now substantially exceeds the number from cultured organisms and encompasses far more diversity The basic foundation of the threedomains tree comprised of archaea bacteria and eucarya has remained solid but each of the domains has expanded significantly in known phylogenetic diversity An overview ofthe universal phylogenetic tree based on known rRNA sequences is diagrammed in Figure 1 The Molecular Tree of Life A profound lesson to emerge from molecular phylogenetics is that all known cellular life is related and had a common origin The location ofthe root of the universal tree the last universal common ancestral state cannot be inferred solely from rRNA data However other phylogenetic results as well as biochemical correlates put the root of the molecular tree on the bacterial line indicated as origin in Figure 1 This would mean that the eucaryal and archaeal lines separated subsequent to the separation from the bacterial line Eucaryotes and archaea thus are more closely related than either is to bacteria and they are expected to share fundamental properties not found in bacteria There are many biochemical correlates that support the phylogenetic interpretation For instance whereas bacteria use sigma factors to control transcription initiation both archaea and eucarya use a different mechanism TATAbinding proteins As another example bacteria wrap their DNA in a variety of basic proteins while eucaryotes and archaea both use histones The details ofthe major radiations in the domains cannot be resolved with current data and are interpreted as unresolved star radiations polytomies in Figure 1 The overall extent of diversity represented in the universal tree recently has been influenced heavily by molecular analyses of environmental organisms Among the bacteria for example in 1987 12 phyla sometimes called divisions the main relatedness groups of bacteria could be identi ed All these phyla were represented by cultured organisms Now gt70 phylumlevel lines are known but only 20 have any cultured representation Only seven of the cultured phyla those that contain human pathogens have significant representation through culture studies Archaeal phylogeny also has proven much more complex than originally thought as environmental sequences have swamped those from the few cultured versions of these organisms The molecular observations contradict some previous notions For instance based on limited culture studies the crenarchaeota Fig 1 commonly are portrayed both popularly and in textbooks as extreme thermophiles Yet in the light of environmental sequences the cultured thermophilic crenarchaeotes turned out to be only one line of a radiation of lowtemperature organisms that occur globally Although these environmental crenarchaeota are among the most abundant kinds of organisms on Earth their roles in the global ecosystem remain obscure The largescale structure of the eucaryotic tree based on rRNA sequences is still unclear The molecular results confirm that the major organelles mitochondria and chloroplasts were bacterial in origin However most eucaryal rRNA sequences available for comparison derive from a relatively limited diversity oforganisms metazoa metaphyta fungi and the detail of phylogenetic trees can be distorted by uneven representation of sequences used in tree calculations The rst broadrange eucaryal rRNA sequence comparisons by Mitchell Sogin and colleagues outlined the topology diagrammed in Figure 1 Environmental sequences have bolstered that result by discovery of additional deeply divergent kingdom level groups which increases the resolution of phylogenetic calculations based on the sequences Regardless ofthe detail it is evident from the rRNA perspective that the nuclear line of descent is as old as the archaeal line eucaryotes have been around since the beginning Whether or not the earliest representatives ofthe eucaryotic line of descent had a nuclear membrane becomes moot in the face of the genetic relationships established by DNA sequences From the rRNA perspective the general path of eucaryotic evolution seems to have been a basal radiation one line of which resulted in subsequent radiations one line of which gave rise to the crown radiation of lineages most familiar to us animals plants fungi stamenopiles alveolates etc Many thousands of new species of microbial life from all three domains have now been identified in the environment by sequencing rRNA and other genes It is clear only that we are barely scratching the surface of an enormous extent of biological diversity The emerging eld of metagenomics which involves determination and interpretation of natural community DNA sequences will play a central role as we learn more about how the microbial world in uences the biosphere Impact of the Molecular Tree on Biological Concepts The molecular tree of life provides an objective measurement of evolutionary relationships While scientists continue to re ne the molecular tree the essence ofthe three domain model for phylogenetic organization and evolution is established and is summarized in the simpli ed diagram of Figure 2A The tree shows that fundamentally there are three phylogenetic kinds oforganisms representative ofthe three primary domains The tree also shows that none of the primary domains is derived from another These experimental observations represented by the molecular tree are at sharp odds with the notion of procaryote and eucaryote that currently dominates education in biology The molecular tree shows that the procaryoteeucaryote model for biological organization and evolution is wrong and perhaps more importantly it is distracting In the following paragraphs l summarize what I mean by the procaryoteeucaryote model then weigh the precepts ofthat model against the experimental results An Unfortunate name Procaryote The name procaryote was popularized in the 1960s by Roger Stanier and colleagues Michael Douderoffand Julius Adelberg in the 2nd edition of their widely used text The Microbial World Originally posed as a taxonomic distinction without specific evolutionary implication choice of the language pro before and eu true nucleus inevitably invoked an evolutionary model I think that to most biologists procaryote was never much more than a name change from the monera at the base of Haeckel s 19th Century trees and a dumpingground taxon for organisms that were little understood Indeed some texts use monera and procaryotes interchangeably However even the textbook definition of procaryote as an organism without a nucleus more properly nuclear membrane is negative and therefore not a useful basis for classification No one can say what is a procaryote only what it is not Consequently procaryote has attracted different and confusing usages In one context it is extant bacteria and lately archaea organisms without nuclear membranes In another context procaryote is some longgone ancestor of eucaryotes Merger ofthese two notions results in the intellectual muddle ofthe procaryote eucaryote model for phylogenetic organization and evolution that confronts our students The newest editions of many textbooks have incorporated the threedomains molecular tree to some extent although generally only in the context of comparison to other systems of classi cation vekingdoms sixkingdoms etc Nonetheless the fundamental lesson of the molecular tree that none of the three rRNA sequencedefined domains emerged from another is not acknowledged in textbook presentations of cellular evolution which inevitably show procaryote giving rise to eucaryote The textbooks are slippery in how they portray procaryotes in evolution and the literature is rife with speculation about the formation of eucaryotes by various fusions of bacteria and archaea Based on review of main text books and discussions with educators and students I venture that the typical student of biology emerges from the university education system with the following basic notions of biological relationships and evolution These I would posit are the main precepts of the procaryoteeucaryote model as diagrammed in Figure 2B 1 All eucaryotes are speci cally related 2 All procaryotes are speci cally related 3 Eucaryotes evolved from procaryotes Each of these precepts can now be weighed scienti cally as follows in the light ofthe molecular tree Fig 2A an experimental result not simply a hypothesis 1 Relationships ofeucaryotes True All eucaryotes fall into the eucaryal domain the relatedness group determined by the sequence comparisons 2 Relationships of procaryotes False The molecular tree shows there are two fundamentally distinct kinds of noneucaryotes bacteria and archaea Moreover one of those archaea is more closely related to eucaryotes than is the other bacteria 3 Evolutionary origin of eucaryotes False The molecular tree Fig 2A shows clearly that the eucaryotic nuclear line did not derive from either bacteria or archaea The procaryoteeucaryote model for the origin of eucaryotes Fig 2B is wrong It s About More Than Terminology Procaryote ls Misinformation Biologists embraced the procaryoteeucaryote model for the large scale of phylogenetic organization and evolution and that model has been proven incorrect With a halfcentury of usage procaryote has come to permeate our journals and texts at all levels and even our language The language through which science expresses itself is a critical matter because science is conceptualized through language We must teach and understand with accurate terminology Continued usage of procaryote perpetuates incorrect information and concepts about phylogenetic organization and evolution the very foundations of biological thought Procaryote has no place in modern scienti c discourse What to Do about It It will take time for the terminology of procaryote to disappear from our textbooks and the lexicon of biology Nonetheless the process needs to start and it needs to be catalyzed Microbiologists will need to take the lead in rectifying the misconceptions because they are closest to the problem of how to understand and describe disparate organisms An early challenge to microbiologists is to stop using the term procaryote This is hard to do because of long conditioning However those who are tempted still to use it saddle their students with misconception and muddy their thinking about important biological problems What else to use For microorganisms in general I usually use microbe which as well brings in the poorly acknowledged microbial eucaryotes But beyond that it is necessary to be more speci c For example it is far more illuminating to distinguish bacterial and archaeal transcription ratherthan to lump them into prokaryotic transcription How to teach this issue in the context ofthe pervasive use of procaryote in textbooks and journals In fact the discordance between currently emerging data and traditional thought on deep evolution and relationships is a wonderful example of how science biology in this case is an ongoing living process Bringing the subject to students shows them how new ideas based on justemerging experimental evidence can change the ways in which we understand natural processes Dealing with the procaryoteeucaryote issue is a good example of weighing specific models hypotheses for testing in the face of experimental data Phylogenetic trees maps of evolutionary relationships are not hard to understand in essence eg Fig 2 They are abstract to be sure but provide graphic information that is readily interpreted by students The three domains concept of course poses many questions but it also provides a solid framework for progress toward answering such questions Acknowledgements A number of colleagues contributed significantly to this text particularly Jim Darnell Corrie Detweiler Mike Klymkowsky Carl Woese and members of the Pace lab Thanks to Kirk Harris for help with artwork This article is based on the 2007 AbbottASM Lifetime Achievement Award lecture presented at the 2007 Annual Meeting in Toronto Canada May 22 2007 Suggested Reading Rapp MS and SJ Giovannoni 2003 The uncultured microbial majority Annu Rev Microbiol 57369394 Sapp J 2005 The prokaryoteeukaryote dichotomy meanings and mythology Microbiol Mol Biol Rev 69 292305 Sogin ML JH Gunderson HJ Elwood RA Alonso and DA Peattie 1989 Phylogenetic meaning of the kingdom concept an unusual ribosomal RNA from Giardia lamblia Science 2437577 Woese CR 1994 There must be a prokaryote somewhere microbiology s search for itself Microbiol Mol Biol Rev 5819 Woese CR 2004 A new biology for a new century Microbiol Mol Biol Rev 68173186 Figure Legends Figure 1 Molecular tree of life based on rRNA sequence comparisons The diagram compiles the results of many rRNA sequence comparisons Only a few known lines of descent are shown The lineages TM7 OP11WS6 BRC1 BOL3 and BAC1 and many not shown are known only from environmental sequences Shaded zones represent areas in the diagram in which branching orders are not resolved Figure 2 Two models for phylogenetic organization and the course of evolution The wedges represent relatedness groups of organisms Figure 1 Molecular tree of life Bacteria m 7 11 17 gt70 mam phyla g 43 D Q 2 g 0 lt3 o a 9 E quot to 0 2 a 6 E 2 E 0 s 9 s Q 6 O Chlom eXi Origin Crenarchaeota Archaea two major groups severalImany deep subgroups Euryarchaeota SDEUOLUOLIOLQ speuowmdlq xo g Eucarya 30 main groups known Figure 2 Models of phylogenetic organization and evolution A ThreeDomain Model Bacteria Origin Eucarya Sequence change Archaea B ProcaryoteEucaryote Model Origin Procaryote Eucaryote
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