1. Taxonomy of Bacteria and Archaea Modern taxonomy comprises the following features: –Nomenclature: giving names of appropriate taxonomic rank to the classified organisms. –Classification: the theory and process of ordering the organisms, on the basis of shared properties, into groups. –Identification: obtaining data on the properties of the organism (characterization) and determination which species it belongs to. This is based on direct comparison to known taxonomic groups.

2. Nomenclature of Bacteria and Archaea There are a, quite complicated, set of rules for the naming Bacteria and Archaea. They must have two names: the first refers to the genus (= slekt) and the second refers to the species (= art). The names can be derived from any language but they must be Latinized. Take for example Staphylococcus aureus. The genus name is capitalized and the species name is lower case. The name is italized to indicate that is Latinized. Staphyl is derived from the Greek staphyle meaning ”a bunch of grapes” and coccus from the Greek meaning ”a berry”. Aurous is from Latin and means ”gold”. A yellow bunch of berries. The higher taxonomic orders are family, order, class, phylum and domain but except for domain these are rarely used.

4. Classification of Bacteria and Archaea Prokaryotes can be classified using artificial or natural (phylogenetic) systems. Historically, prokaryotes were classified on the basis of their phenotype (morphology, staining reactions, biochemistry, substrates/products, antigens etc). In other words a phenotypic characterization is based on the information carried in the products of the genes. These classification systems were artificial. Modern characterization is based on the information carried in the genes i.e. the genome. This is genetic information and can also tell us something about the evolution of the organism. In other words phylogenetics.

5. Numerical Taxonomy Numerical taxonomy is a methods which is used to differentiate a large number of similar bacteria, i.e. species. A large number of tests (~100) are carried out and the results are scored as positive or negative. Several control species are included in the analysis. All characteristics are given equal weight and a computer based analysis is carried out to group the bacteria according to shared properties.

6. Homologous genes are used in the construction of phylogenetic trees Homologous means that genes have a common anscestor Orthologs are homologous genes that belong to different species but still retain their original function Paralogs are homologous genes that have arrisen by gene duplication and are found in the same organism Only orthologes can be used in the construction of phylogenetic trees. The classical example is the 16S ribosomal RNA gene.

7. 16S RNA Secondary structure of the 16S rRNA molecule from the small ribosomal subunit of the bacterium Escherichia coli. The bases are numbered from 1 at the 5' end to 1,542 at the 3' end. Every tenth nucleotide is marked with a tick mark, and every fiftieth nucleotide is numbered. Tertiary interactions with strong comparative data are connected by solid lines. From the Comparative RNA Web Site, www.rna.icmb.utexas.edu; courtesy of Robin Gutell.

8. Conservation and variation in small subunit rRNA This diagram shows conserved and variable regions of the small subunit rRNA (16S in prokaryotes or 18S in eukaryotes). Each dot and triangle represents a position that holds a nucleotide in 95% of all organisms sequenced, though the actual nucleotide present (A, U, C, or G) varies among species. Figure by Jamie Cannone, courtesy of Robin Gutell; data from the Comparative RNA Web Site: www.rna.icmb.utexas.edu

9. Conservation and variation in small subunit rRNA The starred region from part A as it appears in a bacterium (Escherichia coli), an archaean (Methanococcus vannielii), and a eukaryote (Saccharomyces cerevisiae). This region includes important signature sequences for the Bacteria and Archaea. Figure by Jamie Cannone, courtesy of Robin Gutell; data from the Comparative RNA Web Site: www.rna.icmb.utexas.edu

10. Phylogenetic trees Two different formats of phylogenetic trees used to show relatedness among species.

11. Unrooted and rooted trees Representations of the possible relatedness between three species, A, B, and C. (A) A single unrooted tree (shown in both formats; see Figure 17.4). (B) Three possible rooted trees (in one format).

12. (Part 1) Phylogenetic analysis

13. (Part 2) Phylogenetic analysis

14. (Part 3) Phylogenetic analysis

15. (Part 4) Phylogenetic analysis Phylogenetic analysis of four different strains, a, b, c, and d, showing a hypothetical region of their 16S rRNA that contains nine bases. (B) The maximum parsimony method (see text for details).

16. Universal phylogenetic tree as determined from comparative ribosomal RNA sequencing.

17. Detailed phylogenetic tree of the major lineages (phyla) of Bacteria based on 16S ribosomal RNA sequence comparisons

18. Detailed phylogenetic tree of the Archaea based on 16S ribosomal RNA sequence comparisons.

19. Novel phyla discovered by molecular analysis of natural habitats A phylogenetic tree of 16S rDNA sequences of Bacteria, based on pure cultures and clonal libraries from natural samples. Note the existence of many phyla (shown in outline rather than as solid black lines) that have not yet been cultivated. Courtesy of Phil Hugenholz and ASM Publications (Hugenholz, P., B. M. Goebel and N. R. Pace. 1998. J. Bacteriol. 180:4765- 4774).

20. Ribosomal Database project The database contains over 78,000 bacterial 16S rDNA sequences Approximately 7000 Type strains (the bacteria are in pure culture) Approximately 70000 Environmental samples (bacteria and archaea samples have been collected from the environment and characterized by molecular methods.) http://rdp.cme.msu.edu/html/index.html

21. Horizontal gene transfer

23. Species concept The species concept applied to eukaryotes cannot be applied to bacteria and archaea. In fact it is quite difficult to define prokaryote species. In order to be of the same species prokaryotes must share many more properties with each other than with other prokaryotes. They must have similar mol % G+C. Note that two species having the same mol % G+C are not necessary of the same species. The DNA from organisms of the same species must show a minimum of 70% reassociation.

24. DNA melting curve

25. Tm and DNA base composition

26. DNA base composition range

27. DNA/DNA reassociation In this example, which is a control experiment (the radiolabeled sample is reannealed with unlabeled DNA from the same strain), the degree of reassociation is highest and treated as 100%. If a different strain is reannealed with the radiolabeled DNA, it will show a lower degree of reannealing (compared with the 100% attributed to the control), indicative of the similarity between the two strains being tested. Strains with reannealing values of 70% or greater are considered to be the same species.

28. Mole percent guanine + cytosine (Mol% G+C)

29. Fatty acid analysis

30. Archaea Crenarchaeaota: most thermophilic archaea are found in this group. They use sulfur compounds as electron donors or as acceptors. Not all are thermophilic. Euryarcheota: methanogens, halophiles, thermophiles. Korarcheota; found in hot springs. None have been grown i pure culture.

31. Detailed phylogenetic tree of the Archaea based on 16S ribosomal RNA sequence comparisons.

32. Proteobacteria (2086) Purple phototrophic Bacteria The nitrifying Bacteria Sulphur and iron oxidizing Bacteria Hydrogen oxidizing Bacteria Methanotrophs and methylotrophs Pseudomonas and the Pseudomonads Acetic acid Bacteria Free living aerobic nitrogen fixing Bacteria Neisseria and Chromobacterium Enteric Bacteria Vibrio and photobacterium Rickettsia Spirilla Sheathed proteobacteria Budding and prosthecate/stalked Bacteria Gliding Myxobacteria Sulphate and sulphur reducing proteobacteria

33. Detailed phylogenetic tree of the major lineages (phyla) of Bacteria based on 16S ribosomal RNA sequence comparisons

34. Firmicutes (1421) and Actinobacteria(1626) Firmicutes Nonsporulating, low GC, Gram-positive bacteria: Lactic acid bacteria and relatives Endospore forming, low GC, Gram-positive bacteria: Bacillus (673), Clostridia (536) and relatives (212). Cell wall less, low GC, Gram-positive bacteria: the Mycoplasmas Actinobacteria High GC, Gram-positive bacteria: Coryneform and propionic acid bacteria High GC, Gram-positive bacteria: Mycobacteria Filamentous, high GC, Gram-positive bacteria: Streptomyces and other Actinomycetes

35. Detailed phylogenetic tree of the major lineages (phyla) of Bacteria based on 16S ribosomal RNA sequence comparisons

36. Other major groups of bacteria Chloroflexus (12) Chlorobium (13) Cyanobacteria and prochlorophytes (82) Aquifex (12) Thermatoga (23) Thermodesulphobacterium (4) Deinococcus / Thermus (23) Bacteriodes (288) Verrucomicrobium (6) and Prothecabacter Planctomyces Chlamydia Spirochetes (96) Fibrobacter Cytophaga

37. Detailed phylogenetic tree of the major lineages (phyla) of Bacteria based on 16S ribosomal RNA sequence comparisons

38. Fluorescent in situ hybridization (FISH)