Measuring the Tm of DNA

GC pairs connected by 3 H bonds

AT pairs connected by 2 H bonds

* Higher GC content higher Tm

Absorbance of 260 nM light (UV) by DNA increases during strand separation

Absorbance reaches plateau at maximum strand separation

Midpoint of curve is the Tm

Question: diversity in GC content?

From 21 to 79%

Question: how/why did this occur?

Nucleic acid composition

**

Nucleic acid hybridization* Measure of sequence similarity

* DNA heated above Tm to form single stranded DNA

* ssDNA incubated with radioactive ssDNA from other organism

Nucleic acidhybridization

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

Basepairing

dsDNA

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

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

Ribosomal RNA (rRNA)

Some parts have changed very little over time and can serve as an indicator of evolutionary relatedness between distantly related organisms

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

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

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

Domains

Different theories exist regarding the evolution of the three domains

The currently most widely used 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; also: endosymbiosis

Kingdoms

Some biologists prefer the kingdom classification system

Simplest system includes the kingdoms;

Monera (not phylogenetic!)

Protista (not phylogenetic!)

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

Phylogeny of bacteria

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

Aquificae and Thermotogae

The two oldest branches of bacteria

Both are hyperthermophilic

Deinococcus-Thermus

Species belonging to the genus Deinococcus are best studied

Very resistant to radiation and desiccation

T. aquaticus Taq polymerase

Deinococcus

Often associate in pairs and tetrads

Stain gram + although cell wall is similar to gram cells

Photosynthetic nonproteobacteria

Photosynthetic nonproteobacteria

Phylum Chloroflexi

Also contains nonphotosynthetic bacteria

Are the green nonsulfur bacteria

Can be isolated from neutral to alkaline hot springs

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

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 andfragmentation)

Photosynthetic nonproteobacteria

Phylum Cyanobacteria

Vary greatly in shape and appearance