1. Chapter 14 Microbial Evolution and Systematics

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

3. 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)

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

5. 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

6. Ancient and Modern Stromatolites Figure 14.2 3.5 billion yrs old Oldest (Western Australia) 1.6 billion yrs old (Northern Australia) Modern stromatolites (Western Australia) Modern stromatolites (Yellow Stone NP) Modern stromatolites (Western Australia)

7. More Recent Fossil Bacteria and Eukaryotes Figure 14.3 From 1 billion yrs old rocks in Central Australia Prokaryotes (bacteria) Eukaryotic cells

8. 14.2 Origin of Cellular Life  Early Earth was anoxic and much hotter than present day (over 100 o C)  First biochemical compounds were made by abiotic systems that set the stage for the origin of life

9.  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

10.  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., H 2 and H 2 S) may have been available at these sites

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

12.  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

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

14.  DNA, a more stable molecule, eventually became the genetic repository  Three-part systems (DNA, RNA, and protein) evolved and became universal among cells

15.  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 vesicles synthesized on the surfaces of montmorillonite clay

16. Lipid Vesicles Made in the Laboratory from Myristic Acid Figure 14.6 RNAs vesicle Vesicle synthesis is catalyzed by the surfaces of montmorillonite clay particles.

17.  Last universal common ancestor (LUCA)  Population of early cells from which cellular life may have diverged into ancestors of modern day Bacteria and Archaea

18.  As early Earth was anoxic, energy-generating metabolism of primitive cells was exclusively  Anaerobic and likely chemolithotrophic (autotrophic)  Obtained carbon from CO 2  Obtained energy from H 2 ; likely generated by H 2 S reacting with FeS or UV light

19. Major Landmarks in Biological Evolution Figure 14.7

20. A Possible Energy-Generating Scheme for Primitive Cells Figure 14.8

21.  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

22. 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

23.  ~ 2.7 billion years ago, cyanobacterial lineages 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 (i.e., FeS, FeS 2 ) in the oceans  Banded iron formations: iron oxides (e.g. Fe 2 O 3 ) in laminated sedimentary rocks; prominent feature in geological record

24. Banded Iron Formations Figure 14.9 Iron oxides

25.  Development of oxic atmosphere led to evolution of new metabolic pathways that yielded more energy than anaerobic metabolisms  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

26.  Consequence of O 2 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

27. 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

28.  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

29. 2) Eukaryotic cell arose from intracellular association between O 2 -consuming bacterium (the symbiont), which gave rise to mitochondria, and an archaean host

30.  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

31. Major Features Grouping Bacteria or Archaea with Eukarya Table 14.1

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

33. 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

34. 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

35.  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

36. Ribosomal RNA Figure 14.11 16S rRNA from E. coli

37.  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

38.  The ribosomal database project (RDP)  A large collection of rRNA sequences  Currently contains > 409,000 sequences  Provides a variety of analytical programs

39. 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

40. PCR-Amplification of the 16S rRNA Gene Figure 14.12

41. General PCR Protocol

42.  The first step in sequence analysis involves aligning the sequence of interest with sequences from homologous (orthologous) genes from other strains or species

43. Alignment of DNA Sequences Figure 14.13

44.  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

45.  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

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

47.  Evolutionary analysis uses character-state methods (cladistics) for tree reconstruction  The higher the proportion of characteristics that two organisms share, the more recently they diverged from a common ancestor  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)

48. Identification of Phylogenetically Informative Sites Figure 14.15 Dots: neutral sites. Arrows: phylogenetically informative sites, varying in at least two of the sequences.

49.  Common cladistic methods  Parsimony  Maximum likelihood  Bayesian analysis

50. 14.8 Microbial Phylogeny  The universal phylogenetic tree based on SSU rRNA genes is a genealogy of all life on Earth

51. Universal Phylogenetic Tree as Determined by rRNA Genes Figure 14.16

52.  Domain Bacteria  Contains at least 80 major evolutionary groups (phyla)  Many groups defined from environmental sequences (metagenome)alone  i.e., no cultured representatives  Many groups are phenotypically diverse  i.e., physiology and phylogeny not necessarily linked  Eukaryotic organelles originated within Bacteria  Mitochondria arose from Proteobacteria  Chloroplasts arose from the cyanobacterial phylum

53.  Domain Archaea consists of two major groups  Crenarchaeota  Euryarchaeota

54.  Each of the three domains of life can be characterized by various phenotypic properties

55. Major Features Distinguishing Prokaryotes from Eukarya

57. 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)

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

59.  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

60.  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

61. Ribotyping Figure 14.18

62. 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

63. 14.10 Phenotypic Analysis  Taxonomy  The science of identification, classification, and nomenclature  Systematics  The study of the diversity of organisms and their relationships  Links phylogeny with taxonomy

64.  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

65.  Phenotypic analysis examines the morphological, metabolic, physiological, and chemical characters of the cell

66. Some Phenotypic Characteristics of Taxonomic Value Table 14.3

67. Some Phenotypic Characteristics of Taxonomic Value Table 14.3

68.  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

69. Fatty Acid Methyl Ester (FAME) Analysis Figure 14.19a

70. Fatty Acid Methyl Ester (FAME) Analysis Figure 14.19b

71. 14.11 Genotypic Analysis  Several methods of genotypic analysis are available and used  DNA-DNA hybridization  DNA profiling  Multilocus Sequence Typing (MLST)  GC Ratio

72. Some Genotypic Methods Used in Bacterial Taxonomy

73.  DNA-DNA hybridization  Genomes of two organisms are hybridized to examine proportion of similarities in their gene sequences

74. Genomic Hybridization as a Taxonomic Tool Figure 14.20a

75. Figure 14.20b

76. Figure 14.20c

77.  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

78. Relationship Between SSU rRNA and DNA Hybridization 97 95 2570

79.  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 (SSU rRNA)  Repetitive extragenic palindromic PCR (rep-PCR): focused on highly conserved repetitive DNA elements  Amplified fragment length polymorphism (AFLP): focus on many genes located randomly throughout genome - digestion of genomic DNA with one or two restriction enzymes and selective PCR of resulting fragments

80. DNA Fingerprinting with rep-PCR Figure 14.22

81.  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

82. Multilocus Sequence Typing

83.  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

84. 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%

85.  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

86.  Whole-genome sequence analyses are becoming more common  Provide many traits for comparative genotypic analysis  Genome structure - size and number of chromosomes, GC ratio, linear or circular, etc.  Gene content  Gene order

87. 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

88. Taxonomic Hierarchy for Allochromatium warmingii

89.  Biological species concept: not meaningful for prokaryotes as they are haploid and do not undergo sexual reproduction  Genealogical species concept: 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

90. Multi-Gene Phylogenetic Analysis Figure 14.24 16S rRNA genes gyrB genes luxABFE genes 50 nucleotide changes

91.  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

92. A Model for Bacterial Speciation Figure 14.25

93.  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

94.  No firm estimate on the number of prokaryotic species  Nearly 7,000 species of Bacteria and Archaea are presently known

95. 14.14 Classification and Nomenclature  Classification  Organization of organisms into progressively more inclusive groups on the basis of either phenotypic similarity or evolutionary relationship

96.  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

97.  Major references in bacterial diversity  Bergey’s Manual of Systematic Bacteriology (Springer)  The Prokaryotes (Springer)

98.  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

99. Some National Microbial Culture Collections Table 14.6 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