1. Measuring the T m of DNA GC pairs connected by 3 H bonds AT pairs connected by 2 H bonds * Higher GC content  higher T m Absorbance of 260 nM light (UV) by DNA increases during strand separation Absorbance reaches plateau at maximum strand separation Midpoint of curve is the T m Question: diversity in GC content? From 21 to 79% Question: how/why did this occur?

2. Nucleic acid composition * *

3. Nucleic acid hybridization * Measure of sequence similarity * DNA heated above T m to form single stranded DNA * ssDNA incubated with radioactive ssDNA from other organism

4. Nucleic acid hybridization dsDNA heated to form ssDNA ssDNA bound to nitrocellulose membrane Membrane incubated with radioactive ssDNA from different organism Filter incubated at temp lower than Tm Filter washed and amount of bound DNA measured Percent DNA bound indicates relatedness of organisms DNA-rRNA hybridization can be used on more distantly related organisms dsDNA Heat ssDNA Cool Base pairing dsDNA

5. Nucleic acid sequencing Sequencing of nucleic acid only way to provide direct comparison of genes/genomes Sequence of 16 S rRNA gene often used to compare organisms 16 S rRNA gene amplified by PCR PCR product sequenced and sequence compared with that of known organism New development: comparative genomics

6. Why use rDNA for phylogeny? * Present in all organisms * Has highly conserved and weakly conserved regions * Risk of lateral gene transfer is low Sequences of other genes/proteins can also be used as molecular chronometers

7. Ribosomal RNA (rRNA) Some parts have changed very little over time and can serve as an indicator of evolutionary relatedness between distantly related organisms

8. Ribosomal RNA (rRNA) 16S rRNA often contains oligonucleotide signature sequences specific for members of a particular phylogenetic group This sequence is absent in other groups of organisms

9. rRNA analysis: Domains All organisms are divided into one of three domains based on rRNA studies conducted by Carl Woese and others Archaea Bacteria Eukaryotes

10. Phylogenetic trees Graphs that indicate phylogenetic (evolutionary) relationships Made up of nodes connected by branches Nodes represent taxonomical units e.g. species Rooted trees show the evolutionary path of the organisms Unrooted versus rooted tree

11. Domains Different theories exist regarding the evolution of the three domains The currently most widely used theory is (b)

12. Domains Widespread gene transfer between the different domains has occurred This creates difficulties in constructing phylogenetic trees Gene transfers were/are most likely virus-mediated; also: endosymbiosis

13. Kingdoms Some biologists prefer the kingdom classification system Simplest system includes the kingdoms; Monera (not phylogenetic!) Protista (not phylogenetic!) Fungi Plantae Animalia

14. Kingdoms

16. Bergey’s Manuals Bergey’s Manual of Determinative Bacteriology (in 9th edition) Classification of bacteria used for identification Bergey’s Manual of Systematic Bacteriology Contains detailed descriptions of each organism 2nd edition is in 5 volumes (currently being published)

17. Phylogeny of bacteria Bacteria divided into 23 phyla, including: Proteobacteria Low G+C gram +’s (Firmicutes) High G+C gram +’s (Actinobacteria) Cyanobacteria Bacteriodetes Spirochaetes

18. Phylogeny of archaea Archaea divided into 2 phyla Euryarchaeaota Crenarchaeaota

19. Major archaeal groups

20. Crenarchaeota Thought to resemble the ancestor of archaea Divided into 1 class 3 orders and 5 families

21. Crenarchaeota Most are thermophiles or hyperthermophiles Many grow chemolithoautotrophically by reducing sulfur to sulfate

22. Crenarchaeota Most are strict anaerobes Are often found in geothermally heated water and soils (e.g. Yellowstone National Park)

23. Euryarchaeota A very diverse phylum with many classes orders and families Will focus on the 5 major groups

24. Euryarchaeota Methanogens Anaerobes that obtain energy by converting compounds to methane (and CO 2 ) Halobacteria Growth is dependent on a high concentration of salt (at least 1 M)

25. Euryarchaeota Thermoplasms Thermoacidophiles that lack cell walls Thermophilic S 0 -reducers Anaerobes that can reduce sulfur to sulfide

26. Euryarchaeota Sulfate-reducing archaea Extract electrons from various molecules and reduces sulfate, sulfite or thiosulfate to sulfide Cannot use S 0 as an electron acceptor

27. Phylogeny of bacteria

28. Nonproteobacteria gram-negative bacteria Many gram-negative bacteria belong to diverse phyla which differ from the proteobacteria Some belong to the oldest branches of bacteria while others have arisen more recently

29. Aquificae and Thermotogae The two oldest branches of bacteria Both are hyperthermophilic

30. Deinococcus-Thermus Species belonging to the genus Deinococcus are best studied Very resistant to radiation and desiccation T. aquaticus  Taq polymerase

31. Deinococcus Often associate in pairs and tetrads Stain gram + although cell wall is similar to gram  cells

32. Photosynthetic nonproteobacteria

33. Phylum Chloroflexi Also contains nonphotosynthetic bacteria Are the green nonsulfur bacteria Can be isolated from neutral to alkaline hot springs

34. Photosynthetic nonproteobacteria Phylum Chlorobi Composed of 1 class, 1 order and 1 family Are the green sulfur bacteria Use sulfur and sulfur-containing compounds as electron sources

35. Photosynthetic nonproteobacteria Phylum Cyanobacteria Largest and most diverse group of photosynthetic bacteria Photosynthetic system resembles that of eukaryotes Employ a variety of reproductive mechanisms (e.g. binary fission, multiple fission, budding and fragmentation)

36. Photosynthetic nonproteobacteria Phylum Cyanobacteria Vary greatly in shape and appearance