Bacterial Genetics 1
• Genetic information
• Genetic elements
• Replication
• Genetic information transfer
• Regulation of gene expression
• Mutation and recombination
Ch. 7, 8, 10
Sugar backbone (ribose or deoxiribose)
Antiparallel – complementary
(phosphate diester bond)
purine – pyrimidine
A
G
T
C
Deoxi-nucleotide triphosphate units:
dATP, dTTP, dGTP, dCTP
The three key processes of macromolecular
synthesis are:
• DNA replication;
• transcription (the synthesis of RNA from a
DNA template); and
• translation (the synthesis of proteins using
messenger RNA as template).
DNA
Deoxyribose
Thymine
T-A G-C
Double stranded
vs.
RNA
Ribose
Uracil
U-A G-C
Single but
Loop-forming
Types of RNA
mRNA – messenger
tRNA – transfer
rRNA – ribosomal
-------------------------------------------------Roles: Genetic (informational)
Functional: structure (ribosomes)
function (ribozymes)
• Bacteria: promoters are recognized by the sigma
subunit of RNA polymerase. These promoters have
very similar sequences.
• Eukarya: the major classes of RNA are
transcribed by three different RNA polymerases,
with RNA polymerase II producing most mRNA.
• Archaea: have a single RNA that resembles in
structure and function the RNA polymerase II.
Although the basic processes are the same in
both prokaryotes and eukaryotes, but more
complex in eukaryotes.
1. Eukaryotic genes have both:
• Exons (coding regions and
• Introns (noncoding regions).
2. Both are transcribed to primary mRNA
3. Introns are excised Exons ligated
4. A cap is added at 3’ end
5. A poly-A tail added at 5’ end is clipped and a
6. Mature mRNA leaves the nucleus
7. Translation takes place in cytoplasm
Transformation:
Free DNA transfer;
Requires competence factor
Transduction:
Virus mediated transfer
Generalized: Accidental DNA fragments
packed in the virion (at random; lytic cycle)
Specialized: Genes next to prophage are
transducted (lysogenic cycle)
Conjugation:
FF+
Hfr
F’
Plasmid-stimulated transfer
Recipient w/o plasmid
Plasmid only is transferred
Plasmid is integrated in the
chromosome both transferred
Plasmid w. chromosomal genes
Bacterial Genetics 2
• Mutation
• Recombination
• In vivo – techniques, transformation
transduction, conjugation
• Plasmids, Transposalbe elements
• In vitro – techniques
• Bacterial genomics
• Genetic engineering
Ch. 10, 15, 31
Different types of mutations can occur at
different frequencies. For a typical bacterium,
mutation rates of 10–7–10–11 per base pair are
generally seen. Although RNA and DNA
polymerases make errors at about the same
rate, RNA genomes typically accumulate
mutations at much higher frequencies than
DNA genomes.
Genetic
Recombination
General:
Site-specific:
RecA
Transposase
Eukaryotes:
mating + crossing over
Prokaryotes:
transformation
transduction
conjungation
Transformation:
Free DNA transfer;
Requires competence factor
Transduction:
Virus mediated transfer
Generalized: Accidental DNA fragments
packed in the virion (at random; lytic cycle)
Specialized: Genes next to prophage are
transducted (lysogenic cycle)
Conjugation:
FF+
Hfr
F’
Plasmid-stimulated transfer
Recipient w/o plasmid
Plasmid only is transferred
Plasmid is integrated in the
chromosome both transferred
Plasmid w. chromosomal genes
Recombination
General:
homologous: same sequence-different source --- complement
partial heteroduplex (prokaryotes). --- crossing-over (eukaryotes)
Plasmids: mobilize external receptors & pilus
Fno plasmid (competent vs. incompetent)
F+ complete transfer regular--- separate circular
Hfr complete transfer rare --- integrated
F'
plasmid + chromosomal genes
Interrupted mating
Site-specific:
Transposable elements:
transposase, inverted sequences & repeats
Conservative
Replicative
Integrons
Inversions
PCR – Polymerase Chain Reaction
1.
2.
3.
4.
5.
6.
7.
8.
Target DNA (organismal)
Oligonucleotide primers flanking the target sequence,
DNA polymerase of a hyperthermophile (Taq)
Heat denaturation of the target dsDNA
Cooling – annealing of the Primers
Primer extension by polymerase in both directions
Repeat of the cycle
Accumulated sequence 10-6 – 10-9
(Taq = Thermus aquaticus)
Microbial Evolution and
Systematics
EARLY EARTH, THE ORIGIN OF LIFE, AND
MICROBIAL DIVERSIFICATION
Origin of Earth, Evidence for Microbial Life on
Early Earth, Conditions on Early Earth: Hot
and Anoxic.
Origin of Life
Catalysis and the Importance of
Montmorillonite
Primitive Life: The RNA World and Molecular
Coding
RNA Life
The Modern Cell: DNA —> RNA —> Protein
Cambrian stromatolite, ca. 500 My. Old,
South Australia
Scale is in inches, Courtesy of Stanley M. Awramik
Great Slave Lake, Canada
Ancient
Stromatolites
dominated 5/6 of the
Recent
etire history of Earth
Kalkowsky, V.H. 1908:
Cryptozoon
Oolith und Stromatolith im
Norddeutschen Buntsandstein
Hamelin Pool,
Shark Bay,
W. Australia
ca. 2 • 109 y.
Early Proterozoic stromatolite
ca. 3000 My. old,
South Africa
Scale bar is 10 cm long
Modern subtidal stromatolites
Shark Bay, Australia
Modern Conophyton equivalent
Old Faithful Geyser
Microbially guided
silica deposition
Yellowstone National Park
Microbial reefs
with silica deposition
Yellowstone National Park
Tom Brock
in Yellowstone
1975
Modern subtidal stromatolites,
Lee Stocking Island, Bahamas
Sediment accumulation
Microbial mat (red) – Stromatolite (yellow)
Fossil 2000 My old stromatolite
Modern marine stromatolite
Belcher Island Formation, Canada
Shark bay, Western Australia
Eoentophysalis belcherensis
Entophysalis major
Hofmann
Ercegovic
Entophysalis major
Baja California, Mexico
Eoentophysalis belcherensis
Mesoproterozoic, ca. 1400 Ma.
Gauyuzhuang Formation, China
Planet Earth is approximately 4.6 billion years old.
The first evidence for microbial life can be found in
rocks about 3.86 billion years old. Early Earth was
anoxic and much hotter than the present. The first
biochemical compounds were made by abiotic
syntheses that set the stage for the origin of life.
Condition on Early Earth:
Hot and Anoxic
Organic synthesis and stability
Catalysis on clay and pyrite surfaces
RNA and molecular coding
The Modern Cell:
DNA —> RNA —> Protein
The first life forms may have been selfreplicating RNAs. These were both catalytic
and informational. Eventually, DNA became
the genetic repository of cells and the three
part system, DNA, RNA, and protein, became
universal among cells.
Primitive Life: Energy and
Carbon Metabolism
Molecular Oxygen:
Banded Iron Formations
Oxygenation of the Atmosphere:
New Metabolisms and the Ozone
Shield
• Primitive metabolism was anaerobic and
likely chemolithotrophic, exploiting the
abundant sources of FeS and H2S present.
• Carbon metabolism may have included
autotrophy.
• Oxygenic photosynthesis led to
development of banded iron formations, an
oxic environment, and great bursts of
biological evolution.
Phanerozoic
0-0.54
Neoproterozoic
0.54-1.0
Mesoproterozoic
1.0-1.6
Paleoproterozoic
1.6-2.5
Late Archaean
2.5-3.0
Early Archaean
3.0-3.5
Hadean
3.5-4.6
• Origin
of the Nucleus
• Origin of organelles as organisms
• Endosymbiosis
• Lateral flow of genetic information
• Reduction of redundancies
Evoluntionary history of endosymbiosis:
Mitochondria and Chloroplasts
Evoluntionary history of chloroplast endosymbiosis
2
3
1. Cyanobacteria 6. Euglenophyta
2. Glaucophyta
7. Chlorachniophyta
3. Cryptomonads 8. Dinoflagellata (green) .
4. Rhodophyta
9. Dinoflagellata (brown)
5. Chlorophyta
10. Chrysophyta, Heterocontae, Diatoms
Evoluntionary
history of
chloroplast
endosymbiosis:
The Hosts
Evidence of Endosymbiosis
•
•
•
•
•
Size of ribosomes 80S vs. 70S
Organellar DNA present
Organellar DNA is circular
Multiple membranes
Sensitivity to antibiotics
• Models of Symbioses
• The eukaryotic nucleus and mitotic apparatus
probably arose as a necessity for ensuring the
orderly partitioning of DNA in large-genome
organisms.
• Mitochondria and chloroplasts, the principal
energy-producing organelles of eukaryotes, arose
from symbiotic association of prokaryotes of the
domain Bacteria within eukaryotic cells,
• The process is called endosymbiosis.
• Assuming that an RNA world existed, selfreplicating entities have populated Earth for over 4
billion years.
Microbial evolution, phylogeny
and classification
Fossil record vs. Molecular view of evolution
DNA composition GC to AT ratio
DNA-DNA-hybridization: melting – reanealing
DNA-sequence similarity and interrelatedness
DNA-sequencing: in vivo vs. in vitro
Synthetic DNA – PCR – Molecular cloning
Fossil record, and endosymbiotic events
Phylogenetic classification
Bacterial Classification by phenotypes
Criteria for Molecular
Chronometers
• Universal distribution
•
•
•
•
Functional homology
Conserved sequences for alignment
Slow rates of evolutionary change
Lack of functional constraint
• Ribosomal database Project (RDP)
>100,000 sequences.
• Ribosomal RNAs as Evolutionary
Chronometers
• Ribosomal RNA Sequences as a Tool of
Molecular Evolution
• Sequencing Methodology
• Generating Phylogenetic Trees from RNA
Sequences
• Comparisons of sequences of ribosomal
RNA can be used to determine the
evolutionary relationships between organisms.
• Phylogenetic trees based on ribosomal RNA
have now been prepared for all the major
prokaryotic and eukaryotic groups.
• Comparative ribosomal RNA sequencing is
now a routine procedure involving:
• Amplification of the gene encoding 16S
rRNA,
• Sequencing it, and
• Analyzing the sequence in reference to
other sequences.
• Two major treeing algorithms are:
distance and parsimony.
Signature sequences, short oligonucleotides
found within a ribosomal RNA molecule, can
be highly diagnostic of a particular organism
or group of related organisms. Signature
sequences can be used to generate specific
phylogenetic probes, useful for FISH or
microbial community analyses.
Archaea
Crenarchaeota
Euryarchaeota
Life on Earth evolved along three major lines,
called domains, all derived from a common
ancestor. Each domain contains several
phyla. Two of the domains, Bacteria and
Archaea, remained prokaryotic, whereas the
third line, Eukarya, evolved into the modern
eukaryotic cell.
• Although the three domains of living
organisms were originally defined by
ribosomal RNA sequencing, subsequent
studies have shown that they differ in many
other ways.
• In particular, the Bacteria and Archaea differ
extensively in cell wall and lipid chemistry and
in features of transcription and protein
synthesis.
Conventional bacterial taxonomy places
heavy emphasis on analyses of
phenotypic properties of the organism.
Determining the guanine plus cytosine
base ratio of the DNA of the organism
can be part of this process.
Chemotaxonomy
DNA-DNA hybridization
Ribotyping
MultiLocus Sequence Typing
(MLST)
Lipid profiling
The species concept applies to
prokaryotes as well as eukaryotes, and a
similar taxonomic hierarchy exists, with
the domain as the highest level taxon.
Bacterial speciation may occur from a
combination of repeated periodic
selection for a favorable trait within an
ecotype and lateral gene flow.
Domain
Bacteria
Phylum
Gamma Proteobacteria
Class
Zymobacteria
Order
Chromatiales
Family
Chromatiaceae
Genus
Allochromatium
Species
warmingii
Prokaryotes are given descriptive genus
names and species epithets. Formal
recognition of a new prokaryotic species
requires depositing a sample of the organism
in a culture collection and official publication of
the new species name and description.
Bergey’s Manual of Systematic Bacteriology is
a major taxonomic compilation of Bacteria and
Archaea.
International Journal of Systematic and
Evolutionary Microbiology
IJSEM
Methods in Microbial Ecology
The enrichment culture technique is a
means of obtaining microorganisms from
natural samples. Specific reactions can
be investigated by enrichment methods
by choosing media and incubation
conditions selective for particular
organisms.
Once a successful enrichment culture
has been established, a pure culture can
be obtained by conventional
microbiological procedures, including
streak plates, agar shakes, and dilution
methods. Laser tweezers allow one to
“pick” a cell from a microscope field and
literally move it away from contaminants.
DAPI is a general stain for identifying
microorganisms in natural samples. Some
stains can differentiate live versus dead cells,
and fluorescent antibodies that are specific for
one or a small group of related cells can be
prepared. The green fluorescent protein
makes cells autofluorescent and is a means
for tracking cells introduced into the
environment. Unlike pure cultures, in natural
samples, morphologically similar cells may
actually be quite different genetically.
The polymerase chain reaction (PCR) can be
used to amplify specific target genes such as
small subunit ribosomal RNA genes or key
metabolic genes. Denaturing gradient gel
electrophoresis (DGGE) can be used to
resolve slightly to greatly different versions of
these genes present in the different species
inhabiting a natural sample.
The activity of microorganisms in natural
samples can be assessed very sensitively
using radioisotopes and/or microelectrodes. In
most cases, measurements are of the net
activity of a microbial community rather than
of a population of a single species.
Isotopic fractionation can reveal the biological
origin of various substances. Fractionation is a
result of the activity of enzymes that
discriminate against the heavier form of an
element when binding their substrates.
1. Microbial impact on global nutrient cycling
Carbon - Nitrogen - Sulfur - Phosphorus – Iron
2. Aquatic environments, nutrients & microbes
Benthos – Plankton. Biofilms – DeeP Sea – Hydrotermal
vents – Coastal environments – Microbial endoliths –
bioerosion – Freshwater eutrophication and pollution –
Thermal springs.
3. Aquatic environments
Soil microbiology – Rhizobium symbiosis - Mycorhzae
• Microbial communities consist of
guilds of metabolically related
organisms.
• Microorganisms play major roles in
energy transformations and
biogeochemical processes, thus in
• The recycling of elements essential to
living systems.
• Microorganisms are very small and their
habitats are likewise small.
• The microenvironment is the place in which
the microorganism actually lives.
• Microorganisms in nature often live a feastor-famine existence such that only the bestadapted species thrive in a given niche.
• Cooperation among microorganisms is also
important in many microbial
interrelationships.
• The soil is a complex habitat with numerous
microenvironments and niches.
• Microorganisms are present in the soil
primarily attached to soil particles.
• The most important factor influencing
microbial activity in surface soil is the
availability of water, whereas in deep soil
(the subsurface environment) nutrient
availability plays a major role.
• In aquatic ecosystems phototrophic
microorganisms are usually the main
primary producers.
• Most of the organic matter produced is
consumed by bacteria, which can lead to
depletion of oxygen in the environment.
• BOD is a measure of the oxygenconsuming properties of a water sample.
• Marine waters are more nutrient deficient
than many freshwaters, yet substantial
numbers of microorganisms exist there.
• Many of these use light to drive ATP
synthesis.
• Among the prokaryotes, species of the
domain Bacteria tend to predominate in
oceanic surface waters whereas Archaea
are more prevalent in deeper waters.
Energetic Base for Chemolithotrophy
at the
Deep Ocean Hydrothermal Vents
S - reduciers
Methanogens
H - oxidizers
Nitrifyiers
S - oxidizers
Methylotrophs
Fe - Mn oxidizers
Donors
Acceptors
Products
H2
H2
H2
NH4+, NO2
HS, S°, S2O3
CH4, CO
Fe 2+, Mn 2+
S°, S2O3
CO
O2 , NO3
O2
O2, NO3
O2
O2
H2S
CH4
H2O, NO2
NO2, NO3
S°, SO4
CO2
Fe3+, Mn4+
• Iron exists in nature primarily in two
oxidation states, ferrous (Fe2+) and ferric
(Fe3+), and bacterial and chemical
transformation of these metals is of
geological and ecological importance.
• Bacterial ferric iron reduction occurs in
anoxic environments and results in the
mobilization of iron from swamps, bogs,
and other iron-rich aquatic habitats.
• Bacterial oxidation of ferrous iron occurs on
a large scale at low pH and is very common
in coal-mining regions, where it results in a
type of pollution called acid mine drainage.
• The principal form of nitrogen on Earth is
nitrogen gas (N2), which can be used as a
nitrogen source only by the nitrogen-fixing
bacteria.
• Ammonia produced by nitrogen fixation or
by ammonification from organic nitrogen
compounds can be assimilated into organic
matter or
• it can be oxidized to nitrate by the nitrifying
bacteria.
• Losses of nitrogen from the biosphere occur
as a result of denitrification, in which nitrate is
converted back to N2.
• Bacteria play major roles in both the
oxidative and reductive sides of the sulfur
cycle.
• Sulfur- and sulfide-oxidizing bacteria
produce sulfate, while sulfate-reducing
bacteria consume sulfate as an electron
acceptor in anaerobic respiration, producing
hydrogen sulfide. Because sulfide is toxic and
also reacts with various metals, sulfate
reduction is an important biogeochemical
process.
• Dimethyl sulfide is the major organic sulfur
compound of ecological significance in nature.
Proteobacteria:
1.- Phototrophes anoxygenic:
a – Purple sulfur: Chromatium, Ectothiorhodospira, Thiocapsa
b – Purple non-sulfur: Rhodospirillum, Rhodomicrobium
2.- Chemolithotrophs:
a - Nitrosifyers & nitrifyers: Nitrosococcus, Nitrobacter
b - Sulfur oxidizers: Thiobacillus, Beggiatoa, Thioploca
c - Iron oxidizers: Leptothrix, Gallionella
d - Hydrogen oxidizers
e - Methane oxidizer
3.- Chemoorganotrophs:
a - Aerobic respirers: Pseudomonads, Acetic A., N-fixers:
Azotobacter, Photobacteria
b - Anaerobic respirers: S - reducers, Desulfovibrio
c - Facultative aerobes: Enteric bacteria, E. coli
d - Fermenters: Zymomonas
e - Pathogens: Neisseria, Campylobacter, Salmonella
Vibrios, Spirilla, Prostecate bacteria, Myxobacteria
Ammonia and nitrite can be used as electron
donors by the nitrifying bacteria. The
ammonia-oxidizing bacteria produce nitrite,
which is then oxidized by the nitrite-oxidizing
bacteria to nitrate. Anoxic NH3 oxidation is
coupled to both N2 and NO3– production in the
anammoxosome.
Thiocapsa roseopersicina
- a sulfide oxidizing,
non-oxygenic phototroph
containing intracellular
sulfur grains and bundled
tubular pigment vesicles
So
Purple bacteria are anoxygenic phototrophs
that grow phototrophically, obtaining carbon
from CO2 + H2S (purple sulfur bacteria) or
organic compounds (purple nonsulfur
bacteria). Purple nonsulfur bacteria are
physiologically diverse and most can grow
as chemoorganotrophs in darkness. The
purple bacteria reside in the alpha, beta, and
gamma subdivisions of the
Proteobacteria.
3 – Cyanobacteria
• Gram-negative bacteria (formerly blue-green ‘algae’)
• Evolutionary origins and paleoecology of:
Oxygenic phototrophy (unique event in evolution)
All chloroplasts in eukaryotes through endosymbiosis
Atmospheric oxygen provided by Cyanobacteria
Most of the global primary production
Stromatolites, organo-sedimentery structures
• Ecological significance today:
Dinitrogen fixation, respond to P-load as ‘algal blooms’
in coastal and interior waters and enrichment of tropical
ocean (Trichodesmium)
Picoplankton contribution to open ocean (Synechococcus,
Prochlorococcus).
Sediment and soil stabilization
Microbial endoliths and bioerosion
Microcystis flos aquae – a bloomForming, gas-vesicle loaded,
Toxic coccoid cyanobacterium
Petalonema alatum – a
Heterocystous, N2-fixing,
Filamentous cyanobacterium
hν
Phycoerythrin
Phycocyanin
Allophycocyanin
Thylakoid membrane
Chlorophyll a
Phycobilisome
Microbial Bioerosion
Microbial bioerosion is carried by phototrophic
cyanobacteria, green and red algae and
organotrophic fungi. They may remove up to 50% of
carbonate along the surfaces of substrates, such as
shells, corals and limestone rocks.
Solentia achromatica
Endolithic cyanobacterium
responsible for destruction
of limestone coasts at the
intertidal zone.
Hyella racemus – a modern
endolithic cyanobacterium
and its
Neoproterozoic counterpart
Eohyella dichotoma
After microbial endoliths
have Successfully
colonized the rock……
Microbial euendoliths are integrated in the
community of prokaryotes and eukaryotes.
Consequently, the combined bioerosion of
microbial endoliths (bio-corrosion) and their
grazers becomes a progressive force that
undercuts limestone coasts, and creates sharp
and bizarre shapes called ‘biokarst’.
Biokarst & bioerosional notch are geologically significant
Modifications of limestones caused by combined biocorrosion
by microbial endoliths and bioabrasion
by heir grazers
detail
Ammonia and nitrite can be used as electron
donors by the nitrifying bacteria. The
ammonia-oxidizing bacteria produce nitrite,
which is then oxidized by the nitrite-oxidizing
bacteria to nitrate. Anoxic NH3 oxidation is
coupled to both N2 and NO3– production in the
anammoxosome.
Iron Bacteria
They are chemolithotrophs able to use ferrous
iron (Fe2+) as sole energy source. Most iron
bacteria grow only at acid pH and are often
associated with acid pollution from mineral and
coal mining. Some phototrophic purple bacteria
can oxidize Fe2+ to Fe3+ anaerobically.
Methanotrophy & Methylotrophy
Methane is oxidized by methanotrophic bacteria.
Methane (CH4 ) is converted to methanol (CH3OH)
by the enzyme methane monooxygenase (MMO).
The electrons needed to drive this first step come
from cytochrome c, and no energy is conserved in
this reaction. A proton motive force is established
from electron flow in the membrane, and this fuels
ATPase. Carbon for biosynthesis comes primarily
from formaldehyde (CH2O), MMO is a membraneassociated enzyme.