Microbial Evolution and Systematics Chapter 14 Microbial Evolution and Systematics Lectures by Buchan & LeCleir

I. Early Earth and the Origin and Diversification of Life 14.1 Formation and Early History of Earth 14.2 Origin of Cellular Life 14.3 Microbial Diversification: Consequences for Earth’s Biosphere 14.4 Endosymbiotic Origin of Eukaryotes

14.1 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 (southwestern Green land)

3.45 billion-year-old rocks, South Africa Ancient Microbial Life 3.45 billion-year-old rocks, South Africa Figure 14.1

14.1 Formation and Early History of Earth Stromatolites Fossilized microbial mats consisting of layers of filamentous prokaryotes and trapped sediment Found in rocks 3.5 billion years old or younger Comparisons of ancient and modern stromatolites provide evidence that Anoxygenic phototrophic filamentous bacteria formed ancient stromatolites (relatives of the green nonsulfur bacterium Chloroflexus) Oxygenic phototrophic cyanobacteria dominate modern stromatolites

Ancient and Modern Stromatolites 3.5 billion yrs old 1.6 billion yrs old Figure 14.2

More Recent Fossil Bacteria and Eukaryotes 1 billion yrs old rocks prokaryotes eukaryotic cells Figure 14.3

14.2 Origin of Cellular Life Early Earth was anoxic and much hotter than present day (over 100 oC) First biochemical compounds were made by abiotic systems that set the stage for the origin of life

14.2 Origin of Cellular Life Surface Origin Hypothesis Contends that the first membrane-enclosed, self-replicating cells arose out of primordial soup rich in organic and inorganic compounds in ponds on Earth’s surface Dramatic temperature fluctuations and mixing from meteor impacts, dust clouds, and storms argue against this hypothesis

14.2 Origin of Cellular Life Subsurface Origin Hypothesis States that life originated at hydrothermal springs on ocean floor Conditions would have been more stable Steady and abundant supply of energy (e.g., H2 and H2S) may have been available at these sites

Submarine Mound Formed at Ocean Hydrothermal Spring Cooler, more oxidized, more acidic ocean water Hot, reduced, alkaline hydrothermal fluid Figure 14.4

14.2 Origin of Cellular Life Prebiotic chemistry of early Earth set stage for self-replicating systems First self-replicating systems may have been RNA-based (RNA world theory) RNA can bind small molecules (e.g., ATP, other nucleotides) RNA has catalytic activity; may have catalyzed its own synthesis

Last Universal Common Ancestor A Model for the Origin of Cellular Life Last Universal Common Ancestor Figure 14.5

14.2 Origin of Cellular Life DNA, a more stable molecule, eventually became the genetic repository Three-part systems (DNA, RNA, and protein) evolved and became universal among cells

14.2 Origin of Cellular Life Other Important Steps in Emergence of Cellular Life Build up of lipids Synthesis of phospholipid membrane vesicles that enclosed the cell’s biochemical and replication machinery May have been similar to montmorillonite clay vesicles

Vesicles formed on Montmorillonite clay particles Lipid Vesicles Made in the Laboratory from Myristic Acid vesicle RNAs Vesicles formed on Montmorillonite clay particles Figure 14.6

14.2 Origin of Cellular Life Last Universal Common Ancestor (LUCA) Population of early cells from which cellular life may have diverged into ancestors of modern day Bacteria and Archaea

14.2 Origin of Cellular Life As early Earth was anoxic, energy-generating metabolism of primitive cells was exclusively Anaerobic and likely chemolithotrophic (autotrophic) Obtained carbon from CO2 Obtained energy from H2; likely generated by H2S reacting with FeS or UV light

Major Landmarks in Biological Evolution Figure 14.7

A Possible Energy-Generating Scheme for Primitive Cells Figure 14.8

14.2 Origin of Cellular Life Early forms of chemolithotrophic metabolism would have supported production of large amounts of organic compounds Organic material provided abundant, diverse, and continually renewed source of reduced organic carbon, stimulating evolution of various chemoorganotrophic metabolisms

14.3 Microbial Diversification Molecular evidence suggests ancestors of Bacteria and Archaea diverged ~ 4 billion years ago As lineages diverged, distinct metabolisms developed Development of oxygenic photosynthesis dramatically changed course of evolution

14.3 Microbial Diversification ~ 2.7 billion years ago, cyanobacterial lineages developed a photosystem that could use H2O instead of H2S, generating O2 By 2.4 billion years ago, O2 concentrations raised to 1 part per million; initiation of the Great Oxidation Event O2 could not accumulate until it reacted with abundant reduced materials in the oceans (i.e., FeS, FeS2) Banded iron formations: laminated sedimentary rocks; prominent feature in geological record

Banded Iron Formations Iron oxides Figure 14.9

14.3 Microbial Diversification Development of oxic atmosphere led to evolution of new metabolic pathways that yielded more energy than anaerobic metabolisms

14.3 Microbial Diversification Oxygen also spurred evolution of organelle-containing eukaryotic microorganisms Oldest eukaryotic microfossils ~ 2 billion years old Fossils of multicellular and more complex eukaryotes are found in rocks 1.9 to 1.4 billion years old

14.3 Microbial Diversification Consequence of O2 for the evolution of life Formation of ozone layer that provides a barrier against UV radiation Without this ozone shield, life would only have continued beneath ocean surface and in protected terrestrial environments

14.4 Endosymbiotic Origin of Eukaryotes Endosymbiosis Well-supported hypothesis for origin of eukaryotic cells Contends that mitochondria and chloroplasts arose from symbiotic association of prokaryotes within another type of cell

14.4 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

Models for the Origin of the Eukaryotic Cell Figure 14.10a

14.4 Endosymbiotic Origin of Eukaryotes Two hypotheses exist to explain the formation of the eukaryotic cell (cont’d) 2) Eukaryotic cell arose from intracellular association between O2-consuming bacterium (the symbiont), which gave rise to mitochondria and an archaean host

Models for the Origin of the Eukaryotic Cell Figure 14.10b

14.4 Endosymbiotic Origin of Eukaryotes Both hypotheses suggest eukaryotic cell is chimeric This is supported by several features Eukaryotes have similar lipids and energy metabolisms to Bacteria Eukaryotes have transcription and translational machinery most similar to Archaea

Major Features Grouping Bacteria or Archaea with Eukarya Table 14.1

II. Microbial Evolution 14.5 The Evolutionary Process 14.6 Evolutionary Analysis: Theoretical Aspects 14.7 Evolutionary Analysis: Analytical Methods 14.8 Microbial Phylogeny 14.9 Applications of SSU rRNA Phylogenetic Methods

14.5 The Evolutionary Process Mutations Changes in the nucleotide sequence of an organism’s genome Occur because of errors in the fidelity of 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

14.6 Evolutionary Analysis: Theoretical Aspects Phylogeny Evolutionary history of a group of organisms Inferred indirectly from nucleotide sequence data 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 random

14.6 Evolutionary Analysis: Theoretical Aspects The most widely used molecular clocks 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

Ribosomal RNA 16S rRNA from E. coli Figure 14.11

14.6 Evolutionary Analysis: Theoretical Aspects Carl Woese Pioneered the use of SSU rRNA for phylogenetic studies in 1970s Established the presence of three domains of life: Bacteria, Archaea, and Eukarya Provided a unified phylogenetic framework for Bacteria

14.6 Evolutionary Analysis: Theoretical Aspects The Ribosomal Database Project (RDP) A large collection of rRNA sequences Currently contains > 409,000 sequences Provides a variety of analytical programs

14.7 Evolutionary Analysis: Analytical Methods Comparative rRNA sequencing is a routine procedure that involves Amplification of the gene encoding SSU rRNA Sequencing of the amplified gene Analysis of sequence in reference to other sequences

PCR-Amplification of the 16S rRNA Gene Figure 14.12

General PCR Protocol

14.7 Evolutionary Analysis: Analytical Methods The first step in sequence analysis involves aligning the sequence of interest with sequences from homologous (orthologous) genes from other strains or species

Alignment of DNA Sequences Figure 14.13

14.7 Evolutionary Analysis: Analytical Methods BLAST (Basic Local Alignment Search Tool) Web-based tool of the National Institutes of Health Aligns query sequences with those in GenBank database Helpful in identifying gene sequences

14.7 Evolutionary Analysis: Analytical Methods Phylogenetic Tree Graphic illustration of the relationships among sequences Composed of nodes and branches Branches define the order of descent and ancestry of the nodes Branch length represents the number of changes that have occurred along that branch

Phylogenetic Trees: Unrooted (a) and Rooted (b-d) Forms Figure 14.14

14.7 Evolutionary Analysis: Analytical Methods 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 (a group which contains all the descendants of a common ancestor; a clade)

Identification of Phylogenetically Informative Sites Dots: neutral sites. Arrows: phylogenetically informative sites. Figure 14.15

14.7 Evolutionary Analysis: Analytical Methods Common cladistic methods Parsimony Maximum likelihood Bayesian analysis

14.8 Microbial Phylogeny The universal phylogenetic tree based on SSU rRNA genes is a genealogy of all life on Earth Animation: Generating Phylogenetic Trees

Universal Phylogenetic Tree as Determined by rRNA Genes Figure 14.16

14.8 Microbial Phylogeny Domain Bacteria Contains at least 80 major evolutionary groups (phyla) Many groups defined from environmental sequences alone i.e., no cultured representatives Many groups are phenotypically diverse i.e., physiology and phylogeny not necessarily linked

14.8 Microbial Phylogeny Eukaryotic organelles originated within Bacteria Mitochondria arose from Proteobacteria Chloroplasts arose from the cyanobacterial phylum

14.8 Microbial Phylogeny Domain Archaea consists of two major groups Crenarchaeota Euryarchaeota

14.8 Microbial Phylogeny Each of the three domains of life can be characterized by various phenotypic properties

Major Features Distinguishing Prokaryotes from Eukarya

Major Features Distinguishing Prokaryotes from Eukarya

14.9 Applications of SSU rRNA Phylogenetic Methods Signature Sequences Short oligonucleotides unique to certain groups of organisms Often used to design specific nucleic acid probes Probes Can be general or specific Can be labeled with fluorescent tags and hybridized to rRNA in ribosomes within cells FISH: fluorescent in situ hybridization Circumvent need to cultivate organism(s)

Fluorescently Labeled rRNA Probes: Phylogenetic Stains Stained with universal rRNA probe Stained with a eukaryotic rRNA probe Figure 14.17

14.9 Applications of SSU rRNA Phylogenetic Methods 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

14.9 Applications of SSU rRNA Phylogenetic Methods Ribotyping Method of identifying microbes from analysis of DNA fragments generated from restriction enzyme digestion of genes encoding SSU rRNA Highly specific and rapid Used in bacterial identification in clinical diagnostics and microbial analyses of food, water, and beverage

Ribotyping Figure 14.18

III. Microbial Systematics 14.10 Phenotypic Analysis 14.11 Genotypic Analysis 14.12 Phylogenetic Analysis 14.13 The Species Concept in Microbiology 14.14 Classification and Nomenclature

14.10 Phenotypic Analysis Taxonomy Systematics The science of identification, classification, and nomenclature Systematics The study of the diversity of organisms and their relationships Links phylogeny with taxonomy

14.10 Phenotypic Analysis Bacterial taxonomy incorporates multiple methods for identification and description of new species The polyphasic approach to taxonomy uses three methods 1) Phenotypic analysis 2) Genotypic analysis 3) Phylogenetic analysis

14.10 Phenotypic Analysis Phenotypic analysis examines the morphological, metabolic, physiological, and chemical characters of the cell

Some Phenotypic Characteristics of Taxonomic Value Table 14.3

Some Phenotypic Characteristics of Taxonomic Value Table 14.3

14.10 Phenotypic Analysis Fatty Acid Analyses (FAME: fatty acid methyl ester) Relies on variation in type and proportion of fatty acids present in membrane lipids for specific prokaryotic groups Requires rigid standardization because FAME profiles can vary as a function of temperature, growth phase, and growth medium

Fatty Acid Methyl Ester (FAME) Analysis Figure 14.19a

Fatty Acid Methyl Ester (FAME) Analysis Figure 14.19b

14.11 Genotypic Analysis Several methods of genotypic analysis are available and used DNA-DNA hybridization DNA profiling Multilocus Sequence Typing (MLST) GC Ratio

Some Genotypic Methods Used in Bacterial Taxonomy

14.11 Genotypic Analysis DNA-DNA hybridization Genomes of two organisms are hybridized to examine proportion of similarities in their gene sequences

Genomic Hybridization as a Taxonomic Tool Figure 14.20a

Genomic Hybridization as a Taxonomic Tool Figure 14.20b

Genomic Hybridization as a Taxonomic Tool Figure 14.20c

14.11 Genotypic Analysis DNA-DNA hybridization Provides rough index of similarity between two organisms Useful complement to SSU rRNA gene sequencing Useful for differentiating very similar organisms Hybridization values 70% or higher suggest strains belong to the same species Values of at least 25% suggest same genus

Relationship Between SSU rRNA and DNA Hybridization 97 95 25 Figure 14.21

14.11 Genotypic Analysis DNA Profiling Several methods can be used to generate DNA fragment patterns for analysis of genotypic similarity among strains, including Ribotyping: focuses on a single gene Repetitive extragenic palindromic PCR (rep-PCR) and Amplified fragment length polymorphism (AFLP): focus on many genes located randomly throughout genome

DNA Fingerprinting with rep-PCR Figure 14.22

14.11 Genotypic Analysis Multilocus Sequence Typing (MLST) Method in which several different “housekeeping genes” from an organism are sequenced (~450-bp) Has sufficient resolving power to distinguish between very closely related strains

Multilocus Sequence Typing

14.11 Genotypic Analysis GC Ratios Percentage of guanine plus cytosine in an organism’s genomic DNA Vary between 20 and 80% among Bacteria and Archaea Generally accepted that if GC ratios of two strains differ by ~ 5% they are unlikely to be closely related

14.12 Phylogenetic Analysis 16S rRNA gene sequences are useful in taxonomy; serve as “gold standard” for the identification and description of new species Proposed that a bacterium should be considered a new species if its 16S rRNA gene sequence differs by more than 3% from any named strain, and a new genus if it differs by more than 5%

14.12 Phylogenetic Analysis The lack of divergence of the 16S rRNA gene limits its effectiveness in discriminating between bacteria at the species level, thus, a multi-gene approach can be used Multi-gene sequence analysis is similar to MLST, but uses complete sequences and comparisons are made using cladistic methods

14.12 Phylogenetic Analysis Whole-genome sequence analyses are becoming more common Genome structure; size and number of chromosomes, GC ratio, etc. Gene content Gene order

14.13 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 70% or greater DNA-DNA hybridization and 97% or greater 16S rRNA gene sequence identity

Taxonomic Hierarchy for Allochromatium warmingii

14.13 The Species Concept in Microbiology Biological species concept not meaningful for prokaryotes as they are haploid and do not undergo sexual reproduction Genealogical 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

Multi-Gene Phylogenetic Analysis 16S rRNA genes gyrB genes luxABFE genes Figure 14.24

14.13 The Species Concept in Microbiology Ecotype Population of cells that share a particular resource Different ecotypes can coexist in a habitat Bacterial speciation may occur from a combination of repeated periodic selection for a favorable trait within an ecotype and lateral gene flow

A Model for Bacterial Speciation Figure 14.25

14.13 The Species Concept in Microbiology This model is based solely on the assumption of vertical gene flow New genetic capabilities can also arise by horizontal gene transfer; the extent among bacteria is variable

14.13 The Species Concept in Microbiology No firm estimate on the number of prokaryotic species Nearly 7,000 species of Bacteria and Archaea are presently known

14.14 Classification and Nomenclature Organization of organisms into progressively more inclusive groups on the basis of either phenotypic similarity or evolutionary relationship

14.14 Classification and Nomenclature 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 Bacteriological Code - The International Code of Nomenclature of Bacteria

14.14 Classification and Nomenclature Major references in bacterial diversity Bergey’s Manual of Systematic Bacteriology (Springer) The Prokaryotes (Springer)

14.14 Classification and Nomenclature Formal recognition of a new prokaryotic species requires Deposition of a sample of the organism in two culture collections Official publication of the new species name and description in the International Journal of Systematic and Evolutionary Microbiology (IJSEM) The International Committee on Systematics of Prokaryotes (ICSP) is responsible for overseeing nomenclature and taxonomy of Bacteria and Archaea

Some National Microbial Culture Collections KCCM Korean Culture Center of Microorganisms Seoul, Korea http://www.kccm.or.kr KACC Korean Agricultural Culture Collection Suwon, Korea http://kacc.rda.go.kr Table 14.6