Major characteristics used in taxonomy Classical characteristics Molecular characteristics

Classical characteristics Morphological characteristics Physiological and metabolic characteristics Ecological characteristics Genetic analysis

Molecular characteristics Comparison of proteins Nucleic acid composition Nucleic acid hybridization Nucleic acid sequencing

Comparison of proteins Protein amino acid sequence reflects gene sequence DNA  mRNA  protein Comparison of proteins from different organisms can be used for taxonomical classification

Comparison of proteins Amino acid sequencing Comparison of electrophoretic mobility Immunological techniques Comparison of enzymatic properties

Nucleic acid composition Usually expressed as the G + C content (% G + C) G + C = (G + C / G + C + A + T) x 100 Can be determined in a number of ways Hydrolysis of DNA and analysis of of bases using HPLC Measurement of melting point (Tm)

Measuring the Tm of DNA GC pairs connected by 3 H bonds AT pairs connected by 2 H bonds Higher GC content  higher Tm

Measuring the Tm of DNA Absorbance of 260 nM light (UV) by DNA increases during strand separation Absorbance reaches plateau at maximum strand separation Midpoint of rising curve is the Tm

Nucleic acid composition

Nucleic acid hybridization Measure of sequence homology DNA heated above Tm to form single stranded DNA ssDNA incubated with radioactive ssDNA from other organism

Nucleic acid hybridization dsDNA heated to form ssDNA ssDNA bound to nitrocellulose membrane Membrane incubated with radioactive ssDNA from different organism

Nucleic acid hybridization Filter incubated at temp lower than Tm Filter washed and amount of bound DNA measured

Nucleic acid hybridization Percent DNA bound indicates relatedness of organisms DNA-rRNA hybridization can be used on more distantly related organisms

Nucleic acid hybridization

Nucleic acid sequencing Sequencing of nucleic acid only way to provide direct comparison of 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

Phylogenetic trees Graphs that indicate phylogenetic (evolutionary) relationships Made up of nodes connected by branches Nodes represent taxonomical units e.g. species

Phylogenetic trees Trees can be rooted or unrooted Rooted trees show the evolutionary path of the organisms

Indicators of phylogeny Different cell constituents can be used as indicators of phylogeny Sometimes referred to as molecular chronometers Include: Ribosomal RNA (rRNA) DNA Proteins

Ribosomal RNA (rRNA) Has changed very little over time and can serve as an indicator of evolutionary relatedness

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

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

Domains

Domains

Domains Different theories exist regarding the evolution of the three domains The currently accepted theory is (b)

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

Kingdoms Some biologists prefer the kingdom classification system Simplest system includes the kingdoms; Monera Protista Fungi Plantae Animalia

Kingdoms

Kingdoms

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)

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

Phylogeny of archaea Archaea divided into 2 phyla Euryarchaeaota Crenarchaeaota

Major archaeal groups

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

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

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

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

Euryarchaeota Methanogens Anaerobes that obtain energy by converting compounds to methane (and CO2) Halobacteria Growth is dependent on a high concentration of salt (at least 1 M)

Euryarchaeota Thermoplasms Thermoacidophiles that lack cell walls Thermophilic S0-reducers Anaerobes that can reduce sulfur to sulfide

Euryarchaeota Sulfate-reducing archaea Extract electrons from various molecules and reduces sulfate, sulfite or thiosulfate to sulfide Cannot use S0 as an electron acceptor