PowerPoint ® Lecture Presentations prepared by John Zamora Middle Tennessee State University C H A P T E R © 2015 Pearson Education, Inc. Microbial Evolution and Systematics 12

© 2015 Pearson Education, Inc Formation and Early History of Earth The Earth is ~4.5 billion years old First evidence for microbial life can be found in rocks ~3.86 billion years old Figure 12.2 SEM of microfossil bacteria in rocks dated 3.45 billon years Figure 12.1

© 2015 Pearson Education, Inc. Science: The intellectual and practical activity encompassing the systematic study of the structure and behavior of the physical and natural world through observation and experiment. Supernatural: (of a manifestation or event) attributed to some force beyond scientific understanding or the laws of nature Generator Of Diversity

© 2015 Pearson Education, Inc Formation and Early History of Earth Early Earth was anoxic and much hotter than present day First biochemical compounds were made by abiotic systems that set the stage for the origin of life Chemical Evolution

© 2015 Pearson Education, Inc. Figure Formation and Early History of Earth Subsurface origin hypothesis Life originated at hydrothermal springs on ocean floor Conditions would have been more stable Steady and abundant supply of energy (e.g., H 2 and H 2 S) may have been available at these sites

© 2015 Pearson Education, Inc Formation and Early History of Earth Prebiotic chemistry of early Earth set stage for self- replicating systems First self-replicating systems may have been RNA-based RNA can bind small molecules (e.g., ATP) RNA has catalytic activity; may have catalyzed its own synthesis Figure 12.4

© 2015 Pearson Education, Inc Formation and Early History of Earth DNA, a more stable molecule, eventually became the genetic repository Three-part systems (DNA, RNA, and protein) evolved and became universal among cells Figure 12.4

© 2015 Pearson Education, Inc Formation and Early History of Earth Other important steps in emergence of cellular life Buildup of lipids Synthesis of phospholipid membrane vesicles that enclosed the cell's biochemical and replication machinery Figure 12.4

© 2015 Pearson Education, Inc Formation and Early History of Earth Last universal common ancestor (LUCA) Population of early cells from which cellular life may have diverged into ancestors of modern-day Bacteria and Archaea Figure 12.4

© 2015 Pearson Education, Inc Formation and Early History of Earth Because early Earth was anoxic, energy-generating metabolism of primitive cells was exclusively anaerobic and likely chemolithotrophic Obtained carbon from CO 2 Obtained energy from H 2

© 2015 Pearson Education, Inc. Figure Formation and Early History of Earth FeS + H 2 S  FeS 2 + H 2 ∆G 0 =-42 kJ Formation of pyrite leads to H 2 production and S 0 reduction H 2 S plays a catalytic role

© 2015 Pearson Education, Inc Formation and Early History of Earth Early forms of chemolithotrophic metabolism would have supported production of large amounts of organic compounds Organic material provided an abundant, diverse, and continually renewed source of reduced organic carbon, stimulating evolution of various chemoorganotrophic metabolisms Development of oxygenic photosynthesis dramatically changed course of evolution

© 2015 Pearson Education, Inc Photosynthesis and the Oxidation of Earth Stromatolites – Earth’s oldest fossils Fossilized microbial mats of filamentous prokaryotes and trapped sediment Found in rocks 3.5 billion years old or younger Figure 12.6 Oldest known stomatolite 3.5 billion years Conical stomatolites From Northern Australia

© 2015 Pearson Education, Inc. Figure 12.6 Stromatolites Comparisons of ancient and modern stromatolites Anoxygenic phototrophic filamentous bacteria formed ancient stromatolites Oxygenic phototrophic cyanobacteria dominate modern stromatolites 12.2 Photosynthesis and the Oxidation of Earth 1mm Darby Island, Bahamas Northern Australia Shark Bay, Australia 2mm 1m

© 2015 Pearson Education, Inc Photosynthesis and the Oxidation of Earth ~2.7 billion years ago, cyanobacteria developed a photosystem that could use H 2 O instead of H 2 S, generating O 2 By 2.4 billion years ago, O 2 concentrations raised to 1 part per million; initiation of the Great Oxidation Event O 2 could not accumulate until it reacted with abundant reduced materials in the oceans (e.g., FeS, FeS 2 ) Figure 12.7a Microfossils of eukaryotic cells Similar to ancient cyanobacteria 1 billion years old

© 2015 Pearson Education, Inc. Figure Photosynthesis and the Oxidation of Earth Banded iron formations: laminated sedimentary rocks; prominent feature in geological record Layers of iron oxides O 2 released from cyanobacterial photosynthesis results in: Fe 2+  Fe 3+

© 2015 Pearson Education, Inc Photosynthesis and the Oxidation of Earth Development of oxic atmosphere led to evolution of new metabolic pathways that yielded more energy than anaerobic metabolisms O 2 essential for the formation of ozone layer that provides a barrier against UV radiation Without ozone shield, life would be possible only in protected terrestrial environments

© 2015 Pearson Education, Inc Endosymbiotic Origin of Eukaryotes Oxygen also spurred evolution of organelle- containing eukaryotic microorganisms Oldest eukaryotic microfossils ~2 billion years old Endosymbiosis Well-supported hypothesis for origin of eukaryotic cells Contends that mitochondria (genome related to Alphaproteobacteria) and chloroplasts (genome related to Cyanobacteria) arose from symbiotic association of prokaryotes within another type of cell

© 2015 Pearson Education, Inc Endosymbiotic Origin of Eukaryotes Two hypotheses exist to explain the formation of the eukaryotic cell: 1.Eukaryotes began as nucleus-bearing lineage that later acquired mitochondria and chloroplasts by endosymbiosis (Figure 12.9a) 2.Eukaryotic cell arose from intracellular association between O 2 -consuming bacterium (the symbiont), which gave rise to mitochondria, and an archaeal host (Figure 12.9b)

© 2015 Pearson Education, Inc. Figure 12.9a 12.3 Endosymbiotic Origin of Eukaryotes Theory 1 Doesn’t explain the similar lipid profiles in Bacteria and Eukarya

© 2015 Pearson Education, Inc. Figure 12.9b 12.3 Endosymbiotic Origin of Eukaryotes Theory 2 “Hydrogen Hypothesis” Lipid biosythenthesis genes were transferred from symbiont to the host

© 2015 Pearson Education, Inc Endosymbiotic Origin of Eukaryotes Both hypotheses suggest eukaryotic cell is chimeric This is supported by several features: Eukaryotes have lipids and energy metabolisms similar to those of Bacteria Eukaryotes have transcription and translational machinery most similar to those of Archaea

© 2015 Pearson Education, Inc Molecular Phylogeny and the Tree of Life Figure Phylogenetics: is the study of the evolutionary history and relationships among individuals or groups of organisms Historically based on phenotypes

© 2015 Pearson Education, Inc Molecular Phylogeny and the Tree of Life Carl Woese Pioneered the use of rRNA for phylogenetic studies in the 1970s Established the presence of three domains of life: Bacteria, Archaea, and Eukarya Provided a unified phylogenetic framework for Bacteria

© 2015 Pearson Education, Inc Molecular Phylogeny and the Tree of Life The most widely used rRNAs are small subunit ribosomal RNA (SSU rRNA) genes Found in all domains of life 16S rRNA in prokaryotes and 18S rRNA in eukaryotes Functionally constant Sufficiently conserved (change slowly) Sufficient length

© 2015 Pearson Education, Inc. Figure Molecular Phylogeny and the Tree of Life 16S rRNA – E. coli Constant areas (white) Variable regions (colour) Variable regions can be used to determine relationships

© 2015 Pearson Education, Inc Molecular Phylogeny and the Tree of Life Phylogeny Evolutionary history of a group of organisms Inferred indirectly from nucleotide sequence data The universal phylogenetic tree based on SSU rRNA genes is a genealogy of all life on Earth

© 2015 Pearson Education, Inc. Figure BACTERIA ARCHAEA Tenericutes Fusobacteria Gemmatimonadetes Lentisphaerae Fibrobacteres Verrucomicrobia Chlamydiae Planctomycetes Cyanobacteria Plastids Chlorobi Spirochaetes Thermodesulfobacteria Chloroflexi Deinococcus– Thermus Aquificae Thermotogae Epsilonproteobacteria Deltaproteobacteria Mitochondria Alphaproteobacteria Gammaproteobacteria Nitrospira Acidobacteria Bacteroidetes Actinobacteria Firmicutes Betaproteobacteria Crenarchaeota Thaumarchaeota Euryarchaeota Korarchaeota Nanoarchaeota Plants Cercozoans Stramenopiles Alveolates Parabasalids Diplomonads Euglenozoa Amoebozoa Fungi Animals Origin of life LUCA EUKARYA 12.4 Molecular Phylogeny and the Tree of Life

© 2015 Pearson Education, Inc Molecular Phylogeny and the Tree of Life Domain Bacteria Contains at least 80 major evolutionary groups (phyla) Many groups are defined from environmental sequences alone = not cultured Many groups are phenotypically diverse—i.e., physiology and phylogeny are not necessarily linked Domain Archaea consists of 7 major phyla: e.g. Crenarchaeota, Euryarchaeota, Nanoarchaeoota

© 2015 Pearson Education, Inc Molecular Phylogeny and the Tree of Life Domain Eukarya Eukaryotic organelles originated within Bacteria Mitochondria arose from Proteobacteria Chloroplasts arose from Cyanobacteria Each of the three domains of life can be characterized by various phenotypic properties

© 2015 Pearson Education, Inc. Comparative rRNA sequencing is a routine procedure that involves the following: Amplification of the gene encoding SSU rRNA Sequencing of the amplified gene Analysis of sequence in reference to other sequences 12.5 Molecular Phylogeny: Making Sense of Molecular Sequences

© 2015 Pearson Education, Inc Molecular Phylogeny: Making Sense of Molecular Sequences PCR can be used to amplify SSU rRNA genes from members of a microbial community Genes can be sorted out, sequenced, and analyzed Such approaches have revealed key features of microbial community structure and microbial interactions Figure 12.14

© 2015 Pearson Education, Inc. The first step in sequence analysis involves aligning the sequence of interest with sequences from homologous (orthologous) genes 12.5 Molecular Phylogeny: Making Sense of Molecular Sequences Figure 12.15

© 2015 Pearson Education, Inc Molecular Phylogeny: Making Sense of Molecular Sequences Phylogenetic tree Graphic illustration of the relationships among sequences Composed of nodes and branches Nodes represent ancestral sequence from which two branches derived Branches define the order of descent and ancestry of the nodes. Branch length represents the number of changes that have occurred along that branch

© 2015 Pearson Education, Inc. Figure Molecular Phylogeny: Making Sense of Molecular Sequences

© 2015 Pearson Education, Inc Molecular Phylogeny: Making Sense of Molecular Sequences Evolutionary analysis uses character-state methods (cladistics) for tree reconstruction Cladistic methods Define phylogenetic relationships by examining changes in nucleotides at individual positions in the sequence Use those characters that are phylogenetically informative and define monophyletic groups

© 2015 Pearson Education, Inc Molecular Phylogeny: Making Sense of Molecular Sequences Common cladistic methods: Algorithms: programmed series of steps (Figure 12.17) Unweighted pair group method with arithmetic mean Neighbor joining methods Optimality criteria: pick the best of many possible trees Parsimony Maximum-likelihood Bayesian analysis

© 2015 Pearson Education, Inc. Figure Molecular Phylogeny: Making Sense of Molecular Sequences

© 2015 Pearson Education, Inc. Ancestral Sequence Reconstruction Phylogenetic trees are a prediction of how sequences have evolved Nodes represent theoretical ancestral sequences (1-4) Question: is it possible to “calculate” these ancestral sequences? If so….Can those sequences be synthesized and characterized?

© 2015 Pearson Education, Inc. Ancestral Sequence Reconstruction

© 2015 Pearson Education, Inc. ** *, ** * * * EndolyticExolytic Ancestral Sequence Reconstruction McLean et al (2015) JBC 290:21231 ? ? ? ? ?

© 2015 Pearson Education, Inc. Ancestral Sequence Reconstruction

© 2015 Pearson Education, Inc. AAGAGGTGG ++ McLean et al (2015) JBC 290:21231 Ancestral Sequence Reconstruction

© 2015 Pearson Education, Inc The Evolutionary Process Mutations Changes in the nucleotide sequence of an organism's genome Occur because of errors in replication, UV radiation, and other factors Adaptative mutations improve fitness of an organism, increasing its survival Other genetic changes include gene duplication, horizontal gene transfer, and gene loss

© 2015 Pearson Education, Inc The Evolutionary Process Recombination Physical exchange of DNA between genetic elements Selection Based on fitness The ability to produce progeny and contribute to genetic makeup of future generations Genetic drift Random process that can cause gene frequencies to change over time New traits can evolve quickly

© 2015 Pearson Education, Inc. Figure The Evolutionary Process Genetic drift

© 2015 Pearson Education, Inc The Evolutionary Process Loss of Function In the absence of light, bacteria that lose bacteriochlorophyll grow and are selected Figure 12.21

© 2015 Pearson Education, Inc The Evolutionary Process Figure Been running since ,000 divisions Grow in glucose minimal media = nutrient poor Both strains engineered to provide a simple color diagnostic to differentiate between ancestor and evolved strains Gain of Function E. coli Long-term Evolution Experiment (LTEE)

© 2015 Pearson Education, Inc The Evolutionary Process Figure Gain of Function E. coli Long-term Evolution Experiment (LTEE) Mutations lead to rapid positive changes in fitness – then taper off One strain adapted to use citrate as an energy source – now grows to much higher density

© 2015 Pearson Education, Inc The Evolutionary Process Speciation of microorganisms Species can posses a variety of individuals with different traits Molecular clocks (chronometers) Certain genes and proteins that are measures of evolutionary change Major assumptions of this approach are that nucleotide changes occur at a constant rate, are generally neutral, and are random

© 2015 Pearson Education, Inc The Evolution of Microbial Genomes Core genes: Genes shared by all members of a species Pan genes: Core genes plus genes not shared by other members of species Deletions play an important role in microbial genome dynamics Figure 12.23

© 2015 Pearson Education, Inc The Species Concept in Microbiology Biological species concept not meaningful, because prokaryotes are haploid and do not undergo sexual reproduction Phylogenetic species concept is an alternative Prokaryotic species is a group of strains that, based on DNA sequences of multiple genes, cluster closely with others phylogenetically and are distinct from other groups of strains

© 2015 Pearson Education, Inc The Species Concept in Microbiology No universally accepted concept of species for prokaryotes Current definition of prokaryotic species Collection of strains sharing a high degree of similarity in several independent traits Most important traits include 97% or greater 16S rRNA gene sequence identity and 70% or greater DNA–DNA hybridization

© 2015 Pearson Education, Inc The Species Concept in Microbiology DNA–DNA hybridization Genomes of two organisms are hybridized to examine proportion of similarities in their gene sequences Figure 12.24

© 2015 Pearson Education, Inc The Species Concept in Microbiology DNA–DNA hybridization Genomes of two organisms are hybridized to examine proportion of similarities in their gene sequences Figure 12.24

© 2015 Pearson Education, Inc The Species Concept in Microbiology DNA–DNA hybridization Genomes of two organisms are hybridized to examine proportion of similarities in their gene sequences Figure 12.24

© 2015 Pearson Education, Inc Taxonomic Methods Used in Systematics Taxonomy The science of identification, classification, and nomenclature Systematics The study of the diversity of organisms and their relationships Links phylogeny with taxonomy

© 2015 Pearson Education, Inc Taxonomic Methods Used in Systematics Bacterial taxonomy incorporates multiple methods for identifying and describing new species The polyphasic approach to taxonomy uses three methods: 1. Phylogenetic analysis 2.Genotypic analysis 3.Phenotypic analysis

© 2015 Pearson Education, Inc Taxonomic Methods Used in Systematics Phylogenetic analysis The lack of divergence of the 16S rRNA gene limits its effectiveness in discriminating between bacteria at the species level; thus, a multigene approach can be used Protein encoding genes accumulate mutations faster than 16s rRNA Examples: recA and gyrB

© 2015 Pearson Education, Inc. Figure Multigene sequence analysis uses complete sequences, and comparisons are made using cladistic methods 12.9 Taxonomic Methods Used in Systematics 16S gyrB luxABFE

© 2015 Pearson Education, Inc Taxonomic Methods Used in Systematics Multilocus sequence typing (MLST) Method in which several different "housekeeping genes" from an organism are sequenced and collectively analyzed Has sufficient resolving power to distinguish between very closely related strains Figure 12.27

© 2015 Pearson Education, Inc Taxonomic Methods Used in Systematics Ribotyping: DNA fragments generated from restriction enzyme digestion are probed with labeled probes for SSU rRNA Highly specific and rapid Used in bacterial identification in clinical diagnostics and microbial analyses of food, water, and beverages Figure 12.28

© 2015 Pearson Education, Inc Taxonomic Methods Used in Systematics Repetitive extragenic palindromic PCR (rep- PCR) Primers designed against diverse genes randomly throughout genome Products separated on agarose gel to generate “DNA fingerprint” Figure different strains of the same species

© 2015 Pearson Education, Inc Taxonomic Methods Used in Systematics Whole genome sequence analyses are becoming more common Enabled by advanced sequencing technologies now available and rapid drop in sequencing costs Directly compare size and number of chromosomes, GC ratio, gene content, gene order with nucleotide resolution Synteny: conservation of blocks of order within two sets of chromosomes that are being compared with each other

© 2015 Pearson Education, Inc. Table 12.2

© 2015 Pearson Education, Inc Taxonomic Methods Used in Systematics Fatty acid analysis (FAME: fatty acid methyl ester) Relies on variation in type and proportion of fatty acids present in membrane lipids for specific prokaryotic groups More than 200 known fatty acids in bacteria Requires rigid standardization because FAME profiles can vary as a function of temperature, growth phase, and growth medium

© 2015 Pearson Education, Inc. Figure 12.30a 12.9 Taxonomic Methods Used in Systematics

© 2015 Pearson Education, Inc. Figure 12.30b 12.9 Taxonomic Methods Used in Systematics

© 2015 Pearson Education, Inc Classification and Nomenclature Organization of organisms into progressively more inclusive groups on the basis of either phenotypic similarity or evolutionary relationship Prokaryotes are given descriptive genus names and species epithets following the binomial system of nomenclature used throughout biology Assignment of names for species and higher groups of prokaryotes is regulated by the International Code of Nomenclature of Bacteria