16.1 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 (Figure 16.1) © 2012 Pearson Education, Inc.

16.1 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 Anoxygenic phototrophic filamentous bacteria formed ancient stromatolites Oxygenic phototrophic cyanobacteria dominate modern stromatolites © 2012 Pearson Education, Inc.

Figure 16.1 © 2012 Pearson Education, Inc.

16.2 Origin of Cellular Life 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 © 2012 Pearson Education, Inc.

16.2 Origin of Cellular Life Surface origin hypothesis –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 © 2012 Pearson Education, Inc.

16.2 Origin of Cellular Life Subsurface origin hypothesis (Figure 16.4) –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 © 2012 Pearson Education, Inc.

Figure 16.4 Evolutionary events Early Bacteria Early Archaea Dispersal to other habitats Diversification of molecular biology, lipids, and cell wall structure LUCA DNA RNA and proteins RNA life Prebiotic chemistry Mound: precipitates of clay, metal sulfides, silica, and carbonates Ocean water (  20°C, containing metals, CO 2 and PO 4 2  ) Flow of substances up through mound Ocean crust Nutrients in hot hydrothermal water Time (  0.3 to 0.5 billion years) Amino acids Sugars Nitrogen bases © 2012 Pearson Education, Inc.

16.2 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 © 2012 Pearson Education, Inc.

16.2 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 © 2012 Pearson Education, Inc.

16.2 Origin of Cellular Life 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 May have been similar to montmorillonite clay vesicles © 2012 Pearson Education, Inc.

Figure 16.5 © 2012 Pearson Education, Inc.

16.2 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 © 2012 Pearson Education, Inc.

16.2 Origin of Cellular Life As early Earth was anoxic, energy-generating metabolism of primitive cells was exclusively anaerobic and likely chemolithotrophic (autotrophic; Figure 16.6) –Obtained carbon from CO 2 –Obtained energy from H 2 ; likely generated by H 2 S reacting with H 2 S or UV light (Figure 16.7) © 2012 Pearson Education, Inc.

Figure 16.7 Alternative source of H 2 In Out Primitive hydrogenase Primitive ATPase Cytoplasmic membrane S 0 reductase © 2012 Pearson Education, Inc.

16.3 Microbial Diversification ~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 ) –Banded iron formations: laminated sedimentary rocks; prominent feature in geological record (Figure 16.8) © 2012 Pearson Education, Inc.

16.3 Microbial Diversification 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 © 2012 Pearson Education, Inc.

16.3 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 © 2012 Pearson Education, Inc.

16.4 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 © 2012 Pearson Education, Inc.

16.5 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 © 2012 Pearson Education, Inc.

16.6 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 are random © 2012 Pearson Education, Inc.

16.6 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 © 2012 Pearson Education, Inc.

Figure © 2012 Pearson Education, Inc.

16.6 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 © 2012 Pearson Education, Inc.

16.6 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 © 2012 Pearson Education, Inc.

16.7 Evolutionary Analysis: Analytical Methods Comparative rRNA sequencing is a routine procedure that involves the following (Figure 16.12): –Amplification of the gene encoding SSU rRNA –Sequencing of the amplified gene –Analysis of sequence in reference to other sequences © 2012 Pearson Education, Inc.

Figure Kilo- bases – 2.0– 1.5– 1.0– 0.5– 16 S gene Ancestral cell Distinct species Align sequences; generate tree Sequence Run on agarose gel; check for correct size Amplify 16S gene by PCR Isolate DNA A C G G T © 2012 Pearson Education, Inc.

Figure Bacteria Archaea Eukarya PROKARYOTES EUKARYOTES LUCA Flavobacteria Thermotoga Thermodesulfobacterium Aquifex Cyanobacteria Chloroplast Proteobacteria Mitochondrion Gram- positive bacteria Green nonsulfur bacteria Crenarchaeota Euryarchaeota Thermoproteus Pyrodictium Thermococcus Marine Crenarchaeota Methano- bacterium Methano- coccus Pyrolobus Methanosarcina Thermoplasma Methanopyrus Extreme halophiles Entamoebae Slime molds Animals Fungi Plants Ciliates Flagellates Trichomonads Microsporidia Diplomonads (Giardia) © 2012 Pearson Education, Inc.

16.8 Microbial Phylogeny Domain Bacteria –Contains at least 80 major evolutionary groups (phyla) –Many groups defined from environmental sequences alone—i.e., there are no cultured representatives –Many groups are phenotypically diverse—i.e., physiology and phylogeny not necessarily linked © 2012 Pearson Education, Inc.

16.8 Microbial Phylogeny Eukaryotic organelles originated within Bacteria –Mitochondria arose from Proteobacteria –Chloroplasts arose from the cyanobacteria Domain Archaea consists of two major groups: –Crenarchaeota –Euryarchaeota Each of the three domains of life can be characterized by various phenotypic properties © 2012 Pearson Education, Inc.

16.10 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 © 2012 Pearson Education, Inc.

16.10 Phenotypic Analysis Phenotypic analysis examines the morphological, metabolic, physiological, and chemical characters of the cell © 2012 Pearson Education, Inc.

16.11 Genotypic Analysis Several methods of genotypic analysis are available: –DNA–DNA hybridization –DNA profiling –Multilocus sequence typing (MLST) –GC ratio © 2012 Pearson Education, Inc.

16.11 Genotypic Analysis DNA–DNA hybridization –Genomes of two organisms are hybridized to examine proportion of similarities in their gene sequences (Figure 16.20) © 2012 Pearson Education, Inc.

Figure Organisms to be compared: Hybridization experiment: Results and interpretation: DNA preparation Genomic DNA Shear and label (– P ) Genomic DNA Shear DNA Organism 1 Organism 2 Mix DNA, adding unlabeled DNA in excess: Heat to form single strands 1  11  1 1  21  2 Hybridized DNA 1  11  1 1  21  2 100%  25% Same strain (control) 1 and 2 are likely different genera Percent hybridization Same species Same genus, but different species Different genera © 2012 Pearson Education, Inc.

16.11 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 of 70% or higher suggest strains belong to the same species Values of at least 25% suggest same genus © 2012 Pearson Education, Inc.

16.11 Genotypic Analysis Multilocus sequence typing (MLST) –Method in which several different “housekeeping genes” from an organism are sequenced (Figure 16.22) –Has sufficient resolving power to distinguish between very closely related strains © 2012 Pearson Education, Inc.

Figure New isolate or clinical sample Strains 1–5 New strain Strain 6 Strain Isolate DNA Amplify 6–7 target genes Sequence Allele analysis Compare with other strains and generate tree Linkage Distance © 2012 Pearson Education, Inc.

16.11 Genotypic Analysis GC ratios –Percentage of guanine plus cytosine in an organism’s genomic DNA –Vary from 20 to 80% among Bacteria and Archaea –Generally accepted that if GC ratios of two strains differ by ~5% they are unlikely to be closely related © 2012 Pearson Education, Inc.

16.12 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 © 2012 Pearson Education, Inc.

16.12 The Species Concept in Microbiology Biological species concept not meaningful as prokayotes 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 © 2012 Pearson Education, Inc.

16.12 The Species Concept in Microbiology 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% © 2012 Pearson Education, Inc.

16.12 The Species Concept in Microbiology Phylogenetic analysis (cont’d) –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 –Multigene sequence analysis is similar to MLST, but uses complete sequences and comparisons are made using cladistic methods (Figure 16.24) © 2012 Pearson Education, Inc.

Figure S rRNA Gene Tree Multigene Tree ATCC T ATCC T BAA-1194 T 50 changes Photobacterium damselae Photobacterium leiognathi Photobacterium mandapamensis Photobacterium angustum Photobacterium phosphoreum Photobacterium iliopiscarium Photobacterium kishitanii Photobacterium phosphoreum Photobacterium iliopiscarium Photobacterium kishitanii FS-2.1 FS-4.2 FS-3.1 FS-5.1 FS-2.2 FS-5.2 ATCC NCIMB NCIMB NCIMB chubb.1.1 ckamo.3.1 canat.1.2 hstri.1.1 calba.1.1 apros.2.1 ckamo.1.1 vlong.3.1 © 2012 Pearson Education, Inc.

16.12 The Species Concept in Microbiology Phylogenetic analysis (cont’d) Whole-genome sequence analyses are becoming more common –Genome structure: size and number of chromosomes, GC ratio, etc. –Gene content –Gene order © 2012 Pearson Education, Inc.

16.12 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 © 2012 Pearson Education, Inc.

16.12 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 © 2012 Pearson Education, Inc.

16.13 Classification and Nomenclature Classification –Organization of organisms into progressively more inclusive groups on the basis of either phenotypic similarity or evolutionary relationship © 2012 Pearson Education, Inc.

16.13 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 International Code of Nomenclature of Bacteria © 2012 Pearson Education, Inc.