Download Microbial diversity

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 acid
hybridization
dsDNA
Heat
dsDNA heated to form ssDNA
ssDNA bound to nitrocellulose membrane
ssDNA
Membrane incubated with radioactive ssDNA
from different organism
Filter incubated at temp lower than Tm
Cool
Filter washed and amount of bound DNA
measured
Base
pairing
Percent DNA bound indicates relatedness of
organisms
DNA-rRNA hybridization can be used on
more distantly related organisms
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 and
fragmentation)
Photosynthetic nonproteobacteria
Phylum Cyanobacteria
Vary greatly in shape and appearance