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

2. 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

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

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
3.5 billion yrs old
1.6 billion yrs old
Figure 14.2

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

8. 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

9. 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

10. 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

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

12. 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

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

14. 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

15. 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

16. 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

17. 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

18. 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

19. Major Landmarks in Biological Evolution
Figure 14.7

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

21. 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

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. 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

24. Banded Iron Formations
Iron oxides
Figure 14.9

25. 14.3 Microbial Diversification
Development of oxic atmosphere led to evolution of new metabolic pathways that yielded more energy than anaerobic metabolisms

26. 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

27. 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

28. 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

29. 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

30. Models for the Origin of the Eukaryotic Cell
Figure 14.10a

31. 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

32. Models for the Origin of the Eukaryotic Cell
Figure 14.10b

33. 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

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

35. 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

36. 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

37. 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

38. 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

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

40. 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

41. 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

42. 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

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

44. General PCR Protocol

45. 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

46. Alignment of DNA Sequences
Figure 14.13

47. 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

48. 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

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

50. 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)

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

52. 14.7 Evolutionary Analysis: Analytical Methods
Common cladistic methods
Parsimony
Maximum likelihood
Bayesian analysis

53. 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

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

55. 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

56. 14.8 Microbial Phylogeny
Eukaryotic organelles originated within Bacteria
Mitochondria arose from Proteobacteria
Chloroplasts arose from the cyanobacterial phylum

57. 14.8 Microbial Phylogeny Domain Archaea consists of two major groups
Crenarchaeota
Euryarchaeota

58. 14.8 Microbial Phylogeny
Each of the three domains of life can be characterized by various phenotypic properties

59. Major Features Distinguishing Prokaryotes from Eukarya

60. Major Features Distinguishing Prokaryotes from Eukarya

61. 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)

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

63. 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

64. 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

65. Ribotyping
Figure 14.18

66. III. Microbial Systematics
Phenotypic Analysis
Genotypic Analysis
Phylogenetic Analysis
The Species Concept in Microbiology
Classification and Nomenclature

67. 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

68. 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

69. 14.10 Phenotypic Analysis
Phenotypic analysis examines the morphological, metabolic, physiological, and chemical characters of the cell

70. Some Phenotypic Characteristics of Taxonomic Value
Table 14.3

71. Some Phenotypic Characteristics of Taxonomic Value
Table 14.3

72. 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

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

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

75. 14.11 Genotypic Analysis
Several methods of genotypic analysis are available and used
DNA-DNA hybridization
DNA profiling
Multilocus Sequence Typing (MLST)
GC Ratio

76. Some Genotypic Methods Used in Bacterial Taxonomy

77. 14.11 Genotypic Analysis DNA-DNA hybridization
Genomes of two organisms are hybridized to examine proportion of similarities in their gene sequences

78. Genomic Hybridization as a Taxonomic Tool
Figure 14.20a

79. Genomic Hybridization as a Taxonomic Tool
Figure 14.20b

80. Genomic Hybridization as a Taxonomic Tool
Figure 14.20c

81. 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

82. Relationship Between SSU rRNA and DNA Hybridization97 95 25
Figure 14.21

83. 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

84. DNA Fingerprinting with rep-PCR
Figure 14.22

85. 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

86. Multilocus Sequence Typing

87. 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

88. 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%

89. 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

90. 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

91. 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

92. Taxonomic Hierarchy for Allochromatium warmingii

93. 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

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

95. 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

96. A Model for Bacterial Speciation
Figure 14.25

97. 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

98. 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

99. 14.14 Classification and Nomenclature
Organization of organisms into progressively more inclusive groups on the basis of either phenotypic similarity or evolutionary relationship

100. 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

101. 14.14 Classification and Nomenclature
Major references in bacterial diversity
Bergey’s Manual of Systematic Bacteriology (Springer)
The Prokaryotes (Springer)

102. 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

103. Some National Microbial Culture Collections
KCCM Korean Culture Center of Microorganisms Seoul, Korea
KACC Korean Agricultural Culture Collection Suwon, Korea
Table 14.6