Microbial ecology
Microbial ecology: how microorganisms interact with one another and their environment.
Enrichment culture: technique is a means of obtaining microorganisms from natural samples.
Enrichment Culture Technique
Enrichment Culture Technique:
A medium and set of incubation conditions are established (highly selective)
Conditions are selective for desired organism and counter selective for undesired organisms
Inoculum: Sample
Resources and Conditions
The Winogradsky column is a miniature anoxic ecosystem that can be used as a long-term source of bacteria for enrichment culture purposes
Enrichment Culture
Enrichment bias is demonstrated by comparing the results obtained in dilution cultures with classical liquid enrichment.
Some organisms will grow more rapidly and “take over” culture
Isolation in Pure Culture
Once an enrichment culture is established, a pure culture can be obtained using streak plates, agar shakes, or dilution methods.
MPN: Most probable number: method of measuring the numbers of microbes in different media and conditions.
Fluorescent stains
DAPI (4’, 6-diamido2-phenylindole) is a general stain for identifying microorganisms in natural samples.
Some stains can differentiate live versus dead cells- Viability stains.
Fluorescent antibodies that are specific for one or a small group of related cells can be prepared
Fluorescent stains
The green fluorescent protein makes cells autofluorescent and is a means for tracking cells introduced into the environment.
Unlike in pure cultures, morphologically similar cells may actually be quite different genetically in natural samples.
Genetic Stains
A variety of fluorescent-staining methods employ the power of nucleic acid probes and thus are highly specific in their staining properties.
These include phylogenetic staining, chromosome painting, and reverse transcription fluorescent in situ hybridization (FISH).
FISH
The sample DNA is separated into complimentary strands within the DNA double helix structure.
The fluorescently labeled probe of interest is then added to one sample mixture and binds with the sample DNA at the target site.
The probe signal can then be seen through a fluorescent microscope as a colored spot located in the target area/cell/chromosome.
The assay uses fluorescence-labeled peptide nucleic acid (PNA) probes that target the species-specific ribosomal RNA (rRNA) in an organism.
Results are visualized using fluorescence microscopy to see green fluorescing cells.
Other Methods
FISH
Chromosome painting: DNA probe used to ID cells with specific highly conserved genes within groups of bacteria.
ISRT (in situ reverse transcription):
Used to measure gene expression.
Use of a probe that hybridizes with a specific mRNA.
Electrophoresis
PCR
Environmental Genomics
Electrophoresis
Denaturing gradient gel electrophoresis (DGGE) can be used to resolve difference in genes present in the various species inhabiting a natural sample.
Radioisotopes
Isotopes: Different forms of the same element containing the same number of protons and electrons but different numbers of neutrons
Microautoradiography (MAR): Radioisotopes used as measures of microbial activity in a microscopic technique
Isotope fractionation: can reveal the biological origin of various substances
FISH MAR: Technique that allows the metabolic activity to be measured and the organism to be identified
Saturday, March 28, 2009
Chapter 23-Microbial Ecology
Microbial Ecosystems
Chapter 23
Ecological Concepts
Many microbes establish relationships with other organisms (symbioses)
Parasitism
One member is harmed and the other benefits
Mutualism
Both species benefit
Commensalism
One species benefits and the other is neither harmed nor helped
Ecological Concepts
The diversity of microbial species in an ecosystem can be expressed in two ways
Species richness: the total number of different species present
Species abundance: the proportion of each species in an ecosystem
Microbial species richness and abundance is a function of the kinds and amounts of nutrients available in a given habitat
Microbial Ecosystems and Biogeochemical Cycling
Guilds
Metabolically related microbial populations
Sets of guilds form microbial communities that interact with macroorganisms and abiotic factors in the ecosystem
Microbial Ecosystems and Biogeochemical Cycling
Biogeochemistry: the study of biologically mediated chemical transformations
A biogeochemical cycle defines the transformations of a key element that is catalyzed by biological or chemical agents
Typically proceed by oxidation-reduction reactions
Microbes play critical roles in energy transformations and biogeochemical processes that result in the recycling of elements to living systems
Environments and Microenvironments
Difference in the type and quantity of resources and the physiochemical conditions of a habitat define the niche for each microbe
For each organism there exists at least one niche in which that organism is most successful (prime niche)
Microenvironment
The immediate environmental surroundings of a microbial cell or group of cells
Environments and Microenvironments
Physiochemical conditions in a microenvironment are subject to rapid change, both spatially and temporally
Resources in natural environments are highly variable and many microbes in nature face a feast-or-famine existence
Growth rates of microbes in nature are usually well below maximum growth rates defined in the laboratory
Competition and cooperation occur between microbes in natural systems
Biofilms: Microbial Growth on Surfaces
Surfaces are important microbial habitats because
Nutrients adsorb to surfaces
Microbial cells can attach to surfaces
Biofilms: Microbial Growth on Surfaces
Biofilms
Assemblages of bacterial cells adhered to a surface and enclosed in an adhesive matrix excreted by the cells
The matrix is typically a mixture of polysaccharides
Biofilms trap nutrients for microbial growth and help prevent detachment of cells in flowing systems
Biofilms: Microbial Growth on Surfaces
Biofilm formation is initiated by attachment of a cell to a surface followed by expression of biofilm-specific genes
Genes encode proteins that synthesize intercellular signaling molecules and initiate matrix formation
Biofilms: Microbial Growth on Surfaces
Intracellular communication (quorum sensing) is critical in the development and maintenance of a biofilm
The major intracellular signaling molecules are acylated homoserine lactones
Both intra- and interspecies signaling likely occurs in biofilms
Biofilms: Advantages and Control
Bacteria form biofilms for several reasons
Self-defense
Biofilms resist physical forces that sweep away unattached cells, phagocytosis by immune system cells, and penetration of toxins (e.g., antibiotics)
Allows cells to remain in a favorable niche
Allows bacterial cells to live in close association with one another
Biofilms: Advantages and Control
Biofilms are important in human health and commerce
Biofilms have been implicated in several medical and dental conditions
Including periodontal disease, kidney stones, tuberculosis, Legionnaire’s disease, and Staphylococcus infections
In industrial settings, biofilms can slow the flow of liquids through pipelines and can accelerate corrosion of inert surfaces
Few highly effective antibiofilm agents are available
Freshwater Environments
Freshwater environments are highly variable in the resources and conditions available for microbial growth
The balance between photosynthesis and respiration controls the oxygen and carbon cycles
Phytoplankton: oxygenic phototrophs suspended freely in water; include algae and cyanobacteria
Benthic species are attached to the bottom or sides of a lake or stream
Freshwater Environments
The activity of heterotrophic microbes in aquatic systems is highly dependent upon activity of primary producers; oxygenic phototrophs produce organic material and oxygen
Oxygen has limited solubility in water; once consumed in freshwater lakes the deep layers can become anoxic
Oxygen concentrations in aquatic systems is dependent on the amount of organic matter present and the physical mixing of the system
Freshwater Environments
In many temperate lakes the water column becomes stratified during the summer
Freshwater Environments
Rivers
May be well mixed because of rapid water flow
Can still suffer from oxygen deficiencies due to high inputs of
Organic matter from sewage
Agricultural and industrial pollution
Freshwater Environments
Biochemical Oxygen Demand (BOD)
The microbial oxygen-consuming capacity of a body of water
Terrestrial Environments
Soil
The loose outer material of Earth’s surface
Distinct from bedrock
Soil can be divided into two broad groups
Mineral soils
Derived from rock weathering and other inorganic materials
Organic soils
Derived from sedimentation in bogs and marshes
Terrestrial Environments
Soils are composed of
Inorganic mineral matter (~40% of soil volume)
Organic matter (~5%)
Air and water (~50%)
Living organisms
Terrestrial Environments
Most microbial growth takes place on the surfaces of soil particles
Soil aggregates can contain many different microenvironments supporting the growth of several types of microbes
Terrestrial Environments
The availability of water is the most important factor in influencing microbial activity in surface soils
Nutrient availability is the most important factor in subsurface environments
Plants as Microbial Habitats
Rhizosphere
The region immediately outside the root
Zone where microbial activity is usually high
Phyllosphere
The surface of plant leaf
Microbial communities form in both the rhizosphere and phyllosphere of plants
Open Oceans
Compared with most freshwater environ-ments, the open ocean environment is
Saline
Low nutrient; especially with respect to nitrogen, phosphorus, and iron
Cooler
Due to the size of the oceans, the microbial activities taking place in them are major factors in the Earth’s carbon balance
Open Oceans
Nearshore marine waters typically contain higher microbial numbers than the open ocean because of higher nutrient levels
Open Oceans
Most of the primary productivity in the open oceans is due to photosynthesis by prochlorophyte
Prochlorococcus accounts for
> 40% of the biomass of marine phototrophs
~50% of the net primary production
Open Oceans
The planktonic filamentous cyanobacterium Trichodesmium is an abundant phototroph in tropical and subtropical oceans
Small phototrophic eukaryotes, such as Ostreococcus, inhabit coastal and marine waters and are likely important primary producers
Open Oceans
Small planktonic heterotrophic prokaryotes are abundant (105–106 cells/ml) in pelagic marine waters
The most abundant marine heterotroph is Pelagibacter, an oligotroph
Oligotroph: an organism that grows best at very low nutrient concentrations
Open Oceans
Pelagibacter and other marine heterotrophs contain proteorhodopsin, a form of rhodopsin that allows cells to use light energy to drive ATP synthesis
Aerobic anoxygenic phototrophs
Another class of marine microbes that use light energy but do not fix carbon dioxide
Light is used for ATP synthesis via photophosphorylation
Open Oceans
Prokaryote densities in the open ocean decrease with depth
Surface waters contain ~106 cells/ml; below 1,000 m cell numbers drop to 103–105/ml
Bacterial species tend to dominate in surface waters and Archaeal species dominate in deeper waters
Open Oceans
Viruses are the most abundant microorganisms in the oceans (107 virion particles/ml)
Viruses affect prokaryotic populations and are highly diverse
The Deep Sea and Barophilism
> 75% of all ocean water is deep sea, lying primarily between 1000 and 6000 m
Organisms that inhabit the deep sea must deal with
Low temperature
High pressure
Low nutrient levels
Absence of light energy
The Deep Sea and Barophilism
Deep sea microbes are
Psychrophilic (cold-loving) or psychrotolerant
Barophilic (pressure-loving) or barotolerant
Chapter 23
Ecological Concepts
Many microbes establish relationships with other organisms (symbioses)
Parasitism
One member is harmed and the other benefits
Mutualism
Both species benefit
Commensalism
One species benefits and the other is neither harmed nor helped
Ecological Concepts
The diversity of microbial species in an ecosystem can be expressed in two ways
Species richness: the total number of different species present
Species abundance: the proportion of each species in an ecosystem
Microbial species richness and abundance is a function of the kinds and amounts of nutrients available in a given habitat
Microbial Ecosystems and Biogeochemical Cycling
Guilds
Metabolically related microbial populations
Sets of guilds form microbial communities that interact with macroorganisms and abiotic factors in the ecosystem
Microbial Ecosystems and Biogeochemical Cycling
Biogeochemistry: the study of biologically mediated chemical transformations
A biogeochemical cycle defines the transformations of a key element that is catalyzed by biological or chemical agents
Typically proceed by oxidation-reduction reactions
Microbes play critical roles in energy transformations and biogeochemical processes that result in the recycling of elements to living systems
Environments and Microenvironments
Difference in the type and quantity of resources and the physiochemical conditions of a habitat define the niche for each microbe
For each organism there exists at least one niche in which that organism is most successful (prime niche)
Microenvironment
The immediate environmental surroundings of a microbial cell or group of cells
Environments and Microenvironments
Physiochemical conditions in a microenvironment are subject to rapid change, both spatially and temporally
Resources in natural environments are highly variable and many microbes in nature face a feast-or-famine existence
Growth rates of microbes in nature are usually well below maximum growth rates defined in the laboratory
Competition and cooperation occur between microbes in natural systems
Biofilms: Microbial Growth on Surfaces
Surfaces are important microbial habitats because
Nutrients adsorb to surfaces
Microbial cells can attach to surfaces
Biofilms: Microbial Growth on Surfaces
Biofilms
Assemblages of bacterial cells adhered to a surface and enclosed in an adhesive matrix excreted by the cells
The matrix is typically a mixture of polysaccharides
Biofilms trap nutrients for microbial growth and help prevent detachment of cells in flowing systems
Biofilms: Microbial Growth on Surfaces
Biofilm formation is initiated by attachment of a cell to a surface followed by expression of biofilm-specific genes
Genes encode proteins that synthesize intercellular signaling molecules and initiate matrix formation
Biofilms: Microbial Growth on Surfaces
Intracellular communication (quorum sensing) is critical in the development and maintenance of a biofilm
The major intracellular signaling molecules are acylated homoserine lactones
Both intra- and interspecies signaling likely occurs in biofilms
Biofilms: Advantages and Control
Bacteria form biofilms for several reasons
Self-defense
Biofilms resist physical forces that sweep away unattached cells, phagocytosis by immune system cells, and penetration of toxins (e.g., antibiotics)
Allows cells to remain in a favorable niche
Allows bacterial cells to live in close association with one another
Biofilms: Advantages and Control
Biofilms are important in human health and commerce
Biofilms have been implicated in several medical and dental conditions
Including periodontal disease, kidney stones, tuberculosis, Legionnaire’s disease, and Staphylococcus infections
In industrial settings, biofilms can slow the flow of liquids through pipelines and can accelerate corrosion of inert surfaces
Few highly effective antibiofilm agents are available
Freshwater Environments
Freshwater environments are highly variable in the resources and conditions available for microbial growth
The balance between photosynthesis and respiration controls the oxygen and carbon cycles
Phytoplankton: oxygenic phototrophs suspended freely in water; include algae and cyanobacteria
Benthic species are attached to the bottom or sides of a lake or stream
Freshwater Environments
The activity of heterotrophic microbes in aquatic systems is highly dependent upon activity of primary producers; oxygenic phototrophs produce organic material and oxygen
Oxygen has limited solubility in water; once consumed in freshwater lakes the deep layers can become anoxic
Oxygen concentrations in aquatic systems is dependent on the amount of organic matter present and the physical mixing of the system
Freshwater Environments
In many temperate lakes the water column becomes stratified during the summer
Freshwater Environments
Rivers
May be well mixed because of rapid water flow
Can still suffer from oxygen deficiencies due to high inputs of
Organic matter from sewage
Agricultural and industrial pollution
Freshwater Environments
Biochemical Oxygen Demand (BOD)
The microbial oxygen-consuming capacity of a body of water
Terrestrial Environments
Soil
The loose outer material of Earth’s surface
Distinct from bedrock
Soil can be divided into two broad groups
Mineral soils
Derived from rock weathering and other inorganic materials
Organic soils
Derived from sedimentation in bogs and marshes
Terrestrial Environments
Soils are composed of
Inorganic mineral matter (~40% of soil volume)
Organic matter (~5%)
Air and water (~50%)
Living organisms
Terrestrial Environments
Most microbial growth takes place on the surfaces of soil particles
Soil aggregates can contain many different microenvironments supporting the growth of several types of microbes
Terrestrial Environments
The availability of water is the most important factor in influencing microbial activity in surface soils
Nutrient availability is the most important factor in subsurface environments
Plants as Microbial Habitats
Rhizosphere
The region immediately outside the root
Zone where microbial activity is usually high
Phyllosphere
The surface of plant leaf
Microbial communities form in both the rhizosphere and phyllosphere of plants
Open Oceans
Compared with most freshwater environ-ments, the open ocean environment is
Saline
Low nutrient; especially with respect to nitrogen, phosphorus, and iron
Cooler
Due to the size of the oceans, the microbial activities taking place in them are major factors in the Earth’s carbon balance
Open Oceans
Nearshore marine waters typically contain higher microbial numbers than the open ocean because of higher nutrient levels
Open Oceans
Most of the primary productivity in the open oceans is due to photosynthesis by prochlorophyte
Prochlorococcus accounts for
> 40% of the biomass of marine phototrophs
~50% of the net primary production
Open Oceans
The planktonic filamentous cyanobacterium Trichodesmium is an abundant phototroph in tropical and subtropical oceans
Small phototrophic eukaryotes, such as Ostreococcus, inhabit coastal and marine waters and are likely important primary producers
Open Oceans
Small planktonic heterotrophic prokaryotes are abundant (105–106 cells/ml) in pelagic marine waters
The most abundant marine heterotroph is Pelagibacter, an oligotroph
Oligotroph: an organism that grows best at very low nutrient concentrations
Open Oceans
Pelagibacter and other marine heterotrophs contain proteorhodopsin, a form of rhodopsin that allows cells to use light energy to drive ATP synthesis
Aerobic anoxygenic phototrophs
Another class of marine microbes that use light energy but do not fix carbon dioxide
Light is used for ATP synthesis via photophosphorylation
Open Oceans
Prokaryote densities in the open ocean decrease with depth
Surface waters contain ~106 cells/ml; below 1,000 m cell numbers drop to 103–105/ml
Bacterial species tend to dominate in surface waters and Archaeal species dominate in deeper waters
Open Oceans
Viruses are the most abundant microorganisms in the oceans (107 virion particles/ml)
Viruses affect prokaryotic populations and are highly diverse
The Deep Sea and Barophilism
> 75% of all ocean water is deep sea, lying primarily between 1000 and 6000 m
Organisms that inhabit the deep sea must deal with
Low temperature
High pressure
Low nutrient levels
Absence of light energy
The Deep Sea and Barophilism
Deep sea microbes are
Psychrophilic (cold-loving) or psychrotolerant
Barophilic (pressure-loving) or barotolerant
Chapter 20-Microbial Metabolic Diversity
Microbial Diversity
REDOX
Oxidative/reduction reaction
One molecule gives another molecule an e-
Carbon and Energy Sources
Photoautotrophs
Carbon source is carbon dioxide
Energy source is sunlight
Reduction of CO2 to organic compounds
Photoheterotrophs: Use organic carbon as their carbon source
Energy is through a series of oxidation/reductions
Photosynthesis
Photosynthesis: The conversion of light energy to chemical energy.
Photosynthesis involves reactions in which ATP is generated and reactions in which ATP is consumed in the reduction of NADH.
Photoautotroph
Two distinct set of reactions
ATP production and
CO2 reduction to organic compounds
Energy is supplied from ATP
Electrons for the reduction of CO2 come from NADH or NADPH
Produced by electrons originating from various electron donors
Variety of Pigments
Chlorophylls a and b
Bacteriochlorophyll
Carotenoids
Anthocyanins
Phycobilins
Light reaches phototrophic organisms in units of energy called quanta.
Photosynthetic Pigments
In the photosynthetic membrane, chlorophyll or bacteriochlorophyll are associated with proteins to form complexes consisting of 50 to 300 molecules.
Chlorophyll: light sensitive, Mg++ containing porphyrin of photosynthetic organisms that initiates the process of photophosphorylation. Pigment of oxygenic phototrophs.
Bacteriochlorophyll: the chlorophyll pigment of anoxygenic phototrophs.
Photophosphorylation: the production of ATP in photosynthesis
Prokaryotes
No chloroplasts
Pigments are integrated into the internal membrane system
Within invaginations of the cytoplasmic membrane
Or within cytoplasmic membrane itself
Chlorosomes: specialized non-unit membrane enclosed structures.
Chlorophylls
Main pigments in most photoautotrophs
Carotenoids and Phycobilins
Carotenoids:
Most widespread accessory pigments
Hydrophobic pigments embedded in membrane
Photoprotective role: Quench toxic oxygen
Transfers energy to the reaction center which is used to make ATP
Phycobilins
Cyanobacteria and red algae
Accessory Pigments
Light-Dependent Reactions
Pigments absorb light energy, give up electron, which enter electron transfer chains.
Water molecules split, ATP and NADH form, and oxygen is released.
Pigments that give up electrons get replacement electrons.
Photosystem Function: Harvester Pigments
Most pigments in photosystem are harvester pigments
When excited by light energy, these pigments transfer energy to adjacent pigment molecules. Each transfer involves energy loss.
Antenna: chlorophyll molecules harvest light energy and transfer it on to the reaction center of the pigments.
Exciton: mobile forms of energy (photons) that migrate through the antenna pigments to the reaction center
Oxygenic Photosynthesis
In oxygenic photosynthesis, water donates electrons to drive autotrophy (CO2), and oxygen is produced as a by-product.
Two separate light reaction center are involved, photosystems I and II.
Anoxygenic Photosynthesis
Anoxygenic Photosynthesis: Photosynthesis in which O2 is not produced
Photosynthesis begins when exciton energy strikes the bacteriochlorophyll a molecules.
The absorption of energy excites the pigments, converting them to strong electron donors with a low reduction potential.
The energy is then released when electrons are transported through the membrane.
Reverse Electron Flow
Electrons from the quinone pool must be forced backward to reduce NAD to NADH
Light-Independent Reactions
Synthesis part of photosynthesis
Can proceed in the dark
Takes place in the stroma
Calvin-Benson cycle
Autotrophic Fixation: The Calvin Cycle
Most phototrophic and other autotrophic organisms accomplish fixation of CO2 by the Calvin cycle, in which the enzyme ribulose bisphosphate carboxylase (RubisCO) plays a key role.
Calvin-Benson Cycle
Overall reactants
Carbon dioxide
ATP
NADPH oxidized to
Overall products
Glucose
ADP
NADP+
Other Types of Energy Production
Chemolithotrophs oxidize inorganic chemicals as their sole sources of energy and reducing power.
Most chemolithotrophs are also able to grow autotrophically.
Mixotrophic: able to obtain energy from the oxidation of an inorganic compound. They require an organic compound as a carbon source.
Chemolithotrophs: ATP generation is similar to that in chemo-organotrophs, except that the electron donor is inorganic.
Chemolithotrophy-Misc.
Sources of inorganic electron donors: geological, biological or anthropogenic in nature.
The hydrogen bacteria can oxidize H2 compounds
The sulfur bacteria can oxidize reduced sulfur compounds such as H2S and S0
The iron bacteria are chemolithotrophs that use ferrous iron (Fe2+)
Anaerobes reduce CO2 to acetate, usually with H2 as the electron donor
Energy Yields from Oxidation of Inorganic Electron Donors
Sulfur Bacteria
Iron-Oxidizing Bacteria
Electron Flow During Fe2+ Oxidation
Nitrification
NH3 and NO2- are oxidized by nitrifying bacteria during the process of nitrification
Two groups of bacteria work in concert to fully oxidize ammonia to nitrate
Key enzymes are ammonia monooxygenase, hydroxylamine oxidoreductase, and nitrite oxidoreductase
Only small energy yields from this reaction
Growth of nitrifying bacteria is very slow
Oxidation of Ammonia by Ammonia-Oxidizing Bacteria
Oxidation of Nitrite to Nitrate by Nitrifying Bacteria
Anammox
Anammox: anoxic ammonia oxidation
Performed by unusual group of obligate aerobes
Anammoxosome is compartment where anammox reactions occur
Protects cell from reactions occuring during anammox
Hydrazine is an intermediate of anammox
Anammox is very beneficial in the treatment of sewage and wastewater
Anammox
Anaerobic Respiration
The use of an alternate electron acceptor other than O2 for reduction.
Assimilative Metabolism: When an inorganic compound such as NO3, SO4 or CO2 is reduced for biosynthesis
Dissimilative metabolism : The reduced product is excreted into the environment
Fermentation
When inorganic electron acceptors are not present in anoxic environments, carbon is catabolized( broken down) by fermentation.
Fermentations are classified in terms of either the substrate fermented or the fermentation products formed.
Products: Amino acids, organic acids, purines and pyrimidines, alcohols, sugars
Lactic and Mixed-Acid Fermentations
Lactic acid fermentation can occur by homofermentative and heterofermentative pathways
Mixed-Acid Fermentations
Generate acids
Acetic, lactic, and succinic
Sometimes also generate neutral products
E.g., butanediol
Characteristic of enteric bacteria
The Butyric Acid and Butanol/Acetone Fermentation-Clostridia
Entner-Doudoroff Pathway-pathway of sugar carabolism for pseudomonas.
Some Clostridium species ferment amino acids using a complex biochemical pathway known as the Strickland reaction
Propionic Acid fermentation-P. acnes
Non-Substrate-Level Phosphorylation Fermentations
Fermentations of certain compounds do not yield sufficient energy to synthesize ATP
Catabolism of the compound can then be linked to ion pumps that establish a proton or sodium motive force
Nitrification
Nitrate is commonly used as an electron acceptor in anaerobic respiration. Its use requires the enzyme nitrate reductase, which reduces nitrate to nitrite.
Nitrification: the use of ammonia and nitrite as electron donors.
NH4 + NO2 N2 + 2 H20
Ammonia + nitrite Nitrous gas
Denitrification: use of nitrate (NO4) in anaerobic respiration to produce N2
Nitrogen fixation —the reduction of N2 to NH3 (ammonia)
The most widespread inorganic nitrogen compounds in nature are ammonia and nitrate, both formed in the atmosphere.
Methanotrophy, Methylotrophy and Methanogenisis
Methanogenesis is the biological production of CH4 (methane) either from CO2 plus H2 or from methylated compounds
Methanotrophy is the use of CH4 as a carbon and energy source.
Methylotrophs use C1 compounds (or other organic compounds lacking C–C bonds) for energy metabolism and biosynthesis.
Polysaccharides, Organic Acids, Fats
Polysaccharides are abundant in nature and can be broken down, usually by phosphorolysis, into hexose (6) or pentose (5) monomers and used as energy sources
Starch and cellulose are common polysaccharides.
Organic acids are frequently metabolized through the citric acid cycle.
Fats are metabolized via hydrolysis by lipases or phospholipases to free fatty acids.
The fatty acids are oxidized by beta oxidation to acetyl-CoA units, which are subsequently oxidized to CO2 by the citric acid cycle
REDOX
Oxidative/reduction reaction
One molecule gives another molecule an e-
Carbon and Energy Sources
Photoautotrophs
Carbon source is carbon dioxide
Energy source is sunlight
Reduction of CO2 to organic compounds
Photoheterotrophs: Use organic carbon as their carbon source
Energy is through a series of oxidation/reductions
Photosynthesis
Photosynthesis: The conversion of light energy to chemical energy.
Photosynthesis involves reactions in which ATP is generated and reactions in which ATP is consumed in the reduction of NADH.
Photoautotroph
Two distinct set of reactions
ATP production and
CO2 reduction to organic compounds
Energy is supplied from ATP
Electrons for the reduction of CO2 come from NADH or NADPH
Produced by electrons originating from various electron donors
Variety of Pigments
Chlorophylls a and b
Bacteriochlorophyll
Carotenoids
Anthocyanins
Phycobilins
Light reaches phototrophic organisms in units of energy called quanta.
Photosynthetic Pigments
In the photosynthetic membrane, chlorophyll or bacteriochlorophyll are associated with proteins to form complexes consisting of 50 to 300 molecules.
Chlorophyll: light sensitive, Mg++ containing porphyrin of photosynthetic organisms that initiates the process of photophosphorylation. Pigment of oxygenic phototrophs.
Bacteriochlorophyll: the chlorophyll pigment of anoxygenic phototrophs.
Photophosphorylation: the production of ATP in photosynthesis
Prokaryotes
No chloroplasts
Pigments are integrated into the internal membrane system
Within invaginations of the cytoplasmic membrane
Or within cytoplasmic membrane itself
Chlorosomes: specialized non-unit membrane enclosed structures.
Chlorophylls
Main pigments in most photoautotrophs
Carotenoids and Phycobilins
Carotenoids:
Most widespread accessory pigments
Hydrophobic pigments embedded in membrane
Photoprotective role: Quench toxic oxygen
Transfers energy to the reaction center which is used to make ATP
Phycobilins
Cyanobacteria and red algae
Accessory Pigments
Light-Dependent Reactions
Pigments absorb light energy, give up electron, which enter electron transfer chains.
Water molecules split, ATP and NADH form, and oxygen is released.
Pigments that give up electrons get replacement electrons.
Photosystem Function: Harvester Pigments
Most pigments in photosystem are harvester pigments
When excited by light energy, these pigments transfer energy to adjacent pigment molecules. Each transfer involves energy loss.
Antenna: chlorophyll molecules harvest light energy and transfer it on to the reaction center of the pigments.
Exciton: mobile forms of energy (photons) that migrate through the antenna pigments to the reaction center
Oxygenic Photosynthesis
In oxygenic photosynthesis, water donates electrons to drive autotrophy (CO2), and oxygen is produced as a by-product.
Two separate light reaction center are involved, photosystems I and II.
Anoxygenic Photosynthesis
Anoxygenic Photosynthesis: Photosynthesis in which O2 is not produced
Photosynthesis begins when exciton energy strikes the bacteriochlorophyll a molecules.
The absorption of energy excites the pigments, converting them to strong electron donors with a low reduction potential.
The energy is then released when electrons are transported through the membrane.
Reverse Electron Flow
Electrons from the quinone pool must be forced backward to reduce NAD to NADH
Light-Independent Reactions
Synthesis part of photosynthesis
Can proceed in the dark
Takes place in the stroma
Calvin-Benson cycle
Autotrophic Fixation: The Calvin Cycle
Most phototrophic and other autotrophic organisms accomplish fixation of CO2 by the Calvin cycle, in which the enzyme ribulose bisphosphate carboxylase (RubisCO) plays a key role.
Calvin-Benson Cycle
Overall reactants
Carbon dioxide
ATP
NADPH oxidized to
Overall products
Glucose
ADP
NADP+
Other Types of Energy Production
Chemolithotrophs oxidize inorganic chemicals as their sole sources of energy and reducing power.
Most chemolithotrophs are also able to grow autotrophically.
Mixotrophic: able to obtain energy from the oxidation of an inorganic compound. They require an organic compound as a carbon source.
Chemolithotrophs: ATP generation is similar to that in chemo-organotrophs, except that the electron donor is inorganic.
Chemolithotrophy-Misc.
Sources of inorganic electron donors: geological, biological or anthropogenic in nature.
The hydrogen bacteria can oxidize H2 compounds
The sulfur bacteria can oxidize reduced sulfur compounds such as H2S and S0
The iron bacteria are chemolithotrophs that use ferrous iron (Fe2+)
Anaerobes reduce CO2 to acetate, usually with H2 as the electron donor
Energy Yields from Oxidation of Inorganic Electron Donors
Sulfur Bacteria
Iron-Oxidizing Bacteria
Electron Flow During Fe2+ Oxidation
Nitrification
NH3 and NO2- are oxidized by nitrifying bacteria during the process of nitrification
Two groups of bacteria work in concert to fully oxidize ammonia to nitrate
Key enzymes are ammonia monooxygenase, hydroxylamine oxidoreductase, and nitrite oxidoreductase
Only small energy yields from this reaction
Growth of nitrifying bacteria is very slow
Oxidation of Ammonia by Ammonia-Oxidizing Bacteria
Oxidation of Nitrite to Nitrate by Nitrifying Bacteria
Anammox
Anammox: anoxic ammonia oxidation
Performed by unusual group of obligate aerobes
Anammoxosome is compartment where anammox reactions occur
Protects cell from reactions occuring during anammox
Hydrazine is an intermediate of anammox
Anammox is very beneficial in the treatment of sewage and wastewater
Anammox
Anaerobic Respiration
The use of an alternate electron acceptor other than O2 for reduction.
Assimilative Metabolism: When an inorganic compound such as NO3, SO4 or CO2 is reduced for biosynthesis
Dissimilative metabolism : The reduced product is excreted into the environment
Fermentation
When inorganic electron acceptors are not present in anoxic environments, carbon is catabolized( broken down) by fermentation.
Fermentations are classified in terms of either the substrate fermented or the fermentation products formed.
Products: Amino acids, organic acids, purines and pyrimidines, alcohols, sugars
Lactic and Mixed-Acid Fermentations
Lactic acid fermentation can occur by homofermentative and heterofermentative pathways
Mixed-Acid Fermentations
Generate acids
Acetic, lactic, and succinic
Sometimes also generate neutral products
E.g., butanediol
Characteristic of enteric bacteria
The Butyric Acid and Butanol/Acetone Fermentation-Clostridia
Entner-Doudoroff Pathway-pathway of sugar carabolism for pseudomonas.
Some Clostridium species ferment amino acids using a complex biochemical pathway known as the Strickland reaction
Propionic Acid fermentation-P. acnes
Non-Substrate-Level Phosphorylation Fermentations
Fermentations of certain compounds do not yield sufficient energy to synthesize ATP
Catabolism of the compound can then be linked to ion pumps that establish a proton or sodium motive force
Nitrification
Nitrate is commonly used as an electron acceptor in anaerobic respiration. Its use requires the enzyme nitrate reductase, which reduces nitrate to nitrite.
Nitrification: the use of ammonia and nitrite as electron donors.
NH4 + NO2 N2 + 2 H20
Ammonia + nitrite Nitrous gas
Denitrification: use of nitrate (NO4) in anaerobic respiration to produce N2
Nitrogen fixation —the reduction of N2 to NH3 (ammonia)
The most widespread inorganic nitrogen compounds in nature are ammonia and nitrate, both formed in the atmosphere.
Methanotrophy, Methylotrophy and Methanogenisis
Methanogenesis is the biological production of CH4 (methane) either from CO2 plus H2 or from methylated compounds
Methanotrophy is the use of CH4 as a carbon and energy source.
Methylotrophs use C1 compounds (or other organic compounds lacking C–C bonds) for energy metabolism and biosynthesis.
Polysaccharides, Organic Acids, Fats
Polysaccharides are abundant in nature and can be broken down, usually by phosphorolysis, into hexose (6) or pentose (5) monomers and used as energy sources
Starch and cellulose are common polysaccharides.
Organic acids are frequently metabolized through the citric acid cycle.
Fats are metabolized via hydrolysis by lipases or phospholipases to free fatty acids.
The fatty acids are oxidized by beta oxidation to acetyl-CoA units, which are subsequently oxidized to CO2 by the citric acid cycle
Chapter 13 Microbial Genomics
Microbial Genomics
Chapter 13
A Short History of Genomics
Genome
Entire complement of genetic information
Includes genes, regulatory sequences, and noncoding DNA
Genomics
Discipline of mapping, sequencing, analyzing, and comparing genomes
Prokaryotic Genomes: Sizes and ORF Contents
On average a prokaryotic gene is 1,000 bp long
1,000 genes per megabase (Mbp; 1,000,000 bp)
As genome size increases gene content proportionally increases
Prokaryotic Genomes: Sizes and ORF Contents
Prokaryotic genomes range in size from those of large viruses to those of eukaryotic microbes
Unlike prokaryotes, eukaryotic genomes contain a large fraction of non-coding DNA
Prokaryotic Genomes: Sizes and ORF Contents
Smallest cellular genomes to date belong to parasitic or endosymbiotic prokaryotes
Obligate parasites range from 490 kbp (Nanoarchaeum equitans) to 4,400 kbp (Mycobacterium tuberculosis)
Endosymbionts can be even smaller (e.g., 160 bp genome of Carsonella ruddii)
Estimates suggest minimum number of genes for a viable cell is 250–300 genes
Prokaryotic Genomes: Sizes and ORF Contents
Largest prokaryotic genomes comparable to those of some eukaryotes
Sorangium cellulosum (Bacteria)
Largest prokaryotic genome to date at 12.3 Mbp
Largest Archaeal genomes tend to be smaller (~ 5 Mp)
Prokaryotic Genomes: Bioinformatic Analyses
Bioinformatics
Science that applies powerful computational tools to DNA and protein sequences
For the purpose of analyzing, storing, and accessing the sequences for comparative purposes
Prokaryotic Genomes: Bioinformatic Analyses
Complement of genes in a particular organism defines its biology but genomes are also molded by an organism’s lifestyle
Prokaryotic Genomes: Bioinformatic Analyses
Many genes can be identified by sequence similarity to genes found in other organisms (comparative analysis)
Comparative analyses allow for predictions of metabolic pathways and transport systems
Prokaryotic Genomes: Bioinformatic Analyses
Gene Distribution in Prokaryotes
Metabolic genes typically most abundant class
DNA replication and transcription genes make up minor fraction of genome
Nontranslated RNA genes are typically prevalent
I.e., rRNA, tRNA, small regulatory RNAs
Prokaryotic Genomes: Bioinformatic Analyses
Number of genes with role that can be clearly identified in a given genome is 70% or less of total ORFs detected
Hypothetical proteins: uncharacterized ORFs; proteins that likely exist but whose function is presently unknown
Likely encode nonessential genes
In E. coli, many predicted to encode regulatory or redundant proteins
Prokaryotic Genomes: Bioinformatic Analyses
Inaccuracies in some annotations are problematic
As many as 10% of annotated genes are incorrectly annotated
Percentage of an organism’s genes devoted to a specific cell function is to some degree a function of genome size
Prokaryotic Genomes: Bioinformatic Analyses
Gene Distribution in Bacteria and Archaea
Archaea typically devote a higher percentage of their genomes to energy and coenzyme production than do Bacteria
Archaea contain fewer genes for carbohydrate metabolism or cytoplasmic membrane functions than do Bacteria
The Genomes of Eukaryotic Organelles
Mitochondria and chloroplasts contain a small genome
Also contain the necessary machinery for protein synthesis
Including ribosomes, tRNAs, and all other components necessary for translation formation of functional proteins
The Genomes of Eukaryotic Organelles
Known Chloroplast Genomes
Circular DNA molecules
Typically 120–160 kbp
Contain two inverted repeats of 6–76 kbp
Many genes encode proteins for photosynthesis and autotrophy
Introns common; primarily of self-splicing type
The Genomes of Eukaryotic Organelles
Known Mitochondrial Genomes
Diverse structures; some linear
Typically smaller than chloroplast genomes
Primarily encode proteins for oxidative phosphorylation
Use simplified genetic codes rather than “universal” code
Some contain small plasmids
The Genomes of Eukaryotic Organelles
Many genes in the nucleus encode proteins required for organelle function
E.g., translational machinery, energy generation
Eukaryotic Microbial Genomes
The Haploid Yeast Genome
Contains 16 chromosomes, ranging in size from 220 kbp to 2,352 kbp
Entire genome is ~ 13,392 kbp; encodes ~ 6600 ORFs; ~3,500 encode proteins with known function
At least 877 ORFs are essential at least 3,121 are not
Contains a large amount of repetitive DNA
Eukaryotic Microbial Genomes
Smallest eukaryotic cellular genome belongs to Encephalitozoon cuniculi
Intracellular pathogen
Haploid genome contains 11 chromosomes
Genome size 2.9 Mbp; ~ 2,000 genes
Smallest eukaryotic genome belongs to a nucleomorph
Degenerate remains of a eukaryotic endosymbiont
Ranges in size from 0.45 to 0.85 Mbp
Eukaryotic Microbial Genomes
Largest eukaryotic genome belongs to Trichomonas
Parasite
~ 60,000 genes (nearly twice as many as humans)
Microarrays and the Transcriptome
Transcriptome
The entire complement of RNA produced under a given set of conditions
Hybridization techniques can be used in conjunction with genomic sequence data to measure gene expression
Microarrays
Small solid-state supports to which genes or portions of genes are fixed and arrayed spatially in a known pattern
Microarrays and the Transcriptome
DNA segments on arrays are hybridized with mRNA from cells grown under specific conditions and analyzed to determine patterns of gene expression
Arrays are large and dense enough that the transcription pattern of an entire genome can be analyzed
Microarrays and the Transcriptome
What can be learned from microarray experiments?
Global gene expression
Expression of specific groups of genes under different conditions
Expression of genes with unknown function; can yield clues to possible roles
Comparison of gene content in closely related organisms
Identification of specific organisms
Proteomics
Proteomics
Genome-wide study of the structure, function, and regulation of an organism’s proteins
Two-dimensional (2-D) polyacrylamide gel electrophoresis
Technique for the separation, identification, and measurement of all proteins present in a sample
In first (horizontal) dimension, proteins separated by differences in isoelectric points
In second (vertical) dimension, proteins separated by size
Proteomics
Proteins with > 50% sequence identity typically have similar functions
Proteins with > 70% sequence identity almost certainly have similar functions
Protein domains
Distinct structural modules within proteins
Have characteristic functions that can reveal much about a protein’s role, even in the absence of complete sequence homology
Nucleic Acid and Amino Acid Sequence Similarities
Metabolomics
Metabolome
The complete set of metabolic intermediates and other small molecules produced in an organism
Mass spectrometry is one of the primary techniques for monitoring metabolites
Gene Families, Duplications, and Deletions
Homologous: related in sequence to an extent that implies common genetic ancestry
Gene families: groups of gene homologs
Paralogs: genes within an organism whose similarity to one or more genes in the same organism is the result of gene duplication
Orthologs: genes found in one organism that are similar to those in another organism but differ because of speciation
Gene Families, Duplications, and Deletions
Gene duplications thought to be mechanism for evolution of most new genes
Deletions can eliminate gene no longer needed
Gene analysis in the three domains of life suggests that many genes present in all organisms have common evolutionary roots
Mobile DNA: Transposons and Insertion Sequences
Horizontal Gene Transfer
The transfer of genetic information between organisms, as opposed to vertical inheritance from parental organism(s)
May be extensive in nature
May cross phylogenetic domain boundaries
Mobile DNA: Transposons and Insertion Sequences
Detecting Horizontal Gene Flow
Presence of genes typically found only in distantly related species
Presence of a DNA with GC content or codon bias that differs significantly from remainder of genome
Mobile DNA: Transposons and Insertion Sequences
Horizontally transferred genes typically encode non-core metabolic functions
Horizontal Gene Transfer and Genome Stability
Transposons may transfer DNA between different organisms
Transposons may also mediate large-scale chromosomal changes within a single organism
Presence of multiple insertion sequences (IS)
Recombination among identical IS can result in chromosomal rearrangements
E.g., deletions, inversions, or translocations
Horizontal Gene Transfer and Genome Stability
Integrons
Genetic elements that collect and express genes carried on mobile segments of DNA (cassettes)
Of those known, most carry genes for antibiotic resistance
Evolution of Virulence: Pathogenicity Islands
Chromosomal Islands
Region of bacterial chromosome of foreign origin that contains clustered genes for some extra property such as virulence or symbiosis
Pathogenicity islands: chromosomal islands containing genes for virulence
Evolution of Virulence: Pathogenicity Islands
Chromosomal islands believed to have a “foreign” origin based on several observations
Extra regions often flanked by inverted repeats
Base composition and codon usage in chromosomal islands often differ from rest of genome
Often found in some strains of a species but not others
Evolution of Virulence: Pathogenicity Islands
Chromosomal islands contribute specialized functions not essential to growth
Virulence
Biodegradation of recalcitrant compounds
E.g., hydrocarbons and herbicides
Symbiosis
Evolution of Virulence: Pathogenicity Islands
The “pan”/ “core” concept: bacterial species consist of two components
Core genome: shared by all strains of the species
Pan genome: includes all the optional extras present in some but not all strains of the species
Detecting Uncultured Microorganisms
Metagenome
The total gene content of the organisms present in an environment
Several environments have been surveyed by large-scale metagenome projects
E.g., acid mine run-off waters,deep sea sediments, fertile soils
Viral Genomes in Nature
Viruses are more prevalent than bacteria in the environment
Most are bacteriophages and have populations that turn over rapidly
Most of the genetic diversity on Earth thought to reside in viruses
Most virus genes are uncharacterized and show little or no sequence similarity to known genes
Chapter 13
A Short History of Genomics
Genome
Entire complement of genetic information
Includes genes, regulatory sequences, and noncoding DNA
Genomics
Discipline of mapping, sequencing, analyzing, and comparing genomes
Prokaryotic Genomes: Sizes and ORF Contents
On average a prokaryotic gene is 1,000 bp long
1,000 genes per megabase (Mbp; 1,000,000 bp)
As genome size increases gene content proportionally increases
Prokaryotic Genomes: Sizes and ORF Contents
Prokaryotic genomes range in size from those of large viruses to those of eukaryotic microbes
Unlike prokaryotes, eukaryotic genomes contain a large fraction of non-coding DNA
Prokaryotic Genomes: Sizes and ORF Contents
Smallest cellular genomes to date belong to parasitic or endosymbiotic prokaryotes
Obligate parasites range from 490 kbp (Nanoarchaeum equitans) to 4,400 kbp (Mycobacterium tuberculosis)
Endosymbionts can be even smaller (e.g., 160 bp genome of Carsonella ruddii)
Estimates suggest minimum number of genes for a viable cell is 250–300 genes
Prokaryotic Genomes: Sizes and ORF Contents
Largest prokaryotic genomes comparable to those of some eukaryotes
Sorangium cellulosum (Bacteria)
Largest prokaryotic genome to date at 12.3 Mbp
Largest Archaeal genomes tend to be smaller (~ 5 Mp)
Prokaryotic Genomes: Bioinformatic Analyses
Bioinformatics
Science that applies powerful computational tools to DNA and protein sequences
For the purpose of analyzing, storing, and accessing the sequences for comparative purposes
Prokaryotic Genomes: Bioinformatic Analyses
Complement of genes in a particular organism defines its biology but genomes are also molded by an organism’s lifestyle
Prokaryotic Genomes: Bioinformatic Analyses
Many genes can be identified by sequence similarity to genes found in other organisms (comparative analysis)
Comparative analyses allow for predictions of metabolic pathways and transport systems
Prokaryotic Genomes: Bioinformatic Analyses
Gene Distribution in Prokaryotes
Metabolic genes typically most abundant class
DNA replication and transcription genes make up minor fraction of genome
Nontranslated RNA genes are typically prevalent
I.e., rRNA, tRNA, small regulatory RNAs
Prokaryotic Genomes: Bioinformatic Analyses
Number of genes with role that can be clearly identified in a given genome is 70% or less of total ORFs detected
Hypothetical proteins: uncharacterized ORFs; proteins that likely exist but whose function is presently unknown
Likely encode nonessential genes
In E. coli, many predicted to encode regulatory or redundant proteins
Prokaryotic Genomes: Bioinformatic Analyses
Inaccuracies in some annotations are problematic
As many as 10% of annotated genes are incorrectly annotated
Percentage of an organism’s genes devoted to a specific cell function is to some degree a function of genome size
Prokaryotic Genomes: Bioinformatic Analyses
Gene Distribution in Bacteria and Archaea
Archaea typically devote a higher percentage of their genomes to energy and coenzyme production than do Bacteria
Archaea contain fewer genes for carbohydrate metabolism or cytoplasmic membrane functions than do Bacteria
The Genomes of Eukaryotic Organelles
Mitochondria and chloroplasts contain a small genome
Also contain the necessary machinery for protein synthesis
Including ribosomes, tRNAs, and all other components necessary for translation formation of functional proteins
The Genomes of Eukaryotic Organelles
Known Chloroplast Genomes
Circular DNA molecules
Typically 120–160 kbp
Contain two inverted repeats of 6–76 kbp
Many genes encode proteins for photosynthesis and autotrophy
Introns common; primarily of self-splicing type
The Genomes of Eukaryotic Organelles
Known Mitochondrial Genomes
Diverse structures; some linear
Typically smaller than chloroplast genomes
Primarily encode proteins for oxidative phosphorylation
Use simplified genetic codes rather than “universal” code
Some contain small plasmids
The Genomes of Eukaryotic Organelles
Many genes in the nucleus encode proteins required for organelle function
E.g., translational machinery, energy generation
Eukaryotic Microbial Genomes
The Haploid Yeast Genome
Contains 16 chromosomes, ranging in size from 220 kbp to 2,352 kbp
Entire genome is ~ 13,392 kbp; encodes ~ 6600 ORFs; ~3,500 encode proteins with known function
At least 877 ORFs are essential at least 3,121 are not
Contains a large amount of repetitive DNA
Eukaryotic Microbial Genomes
Smallest eukaryotic cellular genome belongs to Encephalitozoon cuniculi
Intracellular pathogen
Haploid genome contains 11 chromosomes
Genome size 2.9 Mbp; ~ 2,000 genes
Smallest eukaryotic genome belongs to a nucleomorph
Degenerate remains of a eukaryotic endosymbiont
Ranges in size from 0.45 to 0.85 Mbp
Eukaryotic Microbial Genomes
Largest eukaryotic genome belongs to Trichomonas
Parasite
~ 60,000 genes (nearly twice as many as humans)
Microarrays and the Transcriptome
Transcriptome
The entire complement of RNA produced under a given set of conditions
Hybridization techniques can be used in conjunction with genomic sequence data to measure gene expression
Microarrays
Small solid-state supports to which genes or portions of genes are fixed and arrayed spatially in a known pattern
Microarrays and the Transcriptome
DNA segments on arrays are hybridized with mRNA from cells grown under specific conditions and analyzed to determine patterns of gene expression
Arrays are large and dense enough that the transcription pattern of an entire genome can be analyzed
Microarrays and the Transcriptome
What can be learned from microarray experiments?
Global gene expression
Expression of specific groups of genes under different conditions
Expression of genes with unknown function; can yield clues to possible roles
Comparison of gene content in closely related organisms
Identification of specific organisms
Proteomics
Proteomics
Genome-wide study of the structure, function, and regulation of an organism’s proteins
Two-dimensional (2-D) polyacrylamide gel electrophoresis
Technique for the separation, identification, and measurement of all proteins present in a sample
In first (horizontal) dimension, proteins separated by differences in isoelectric points
In second (vertical) dimension, proteins separated by size
Proteomics
Proteins with > 50% sequence identity typically have similar functions
Proteins with > 70% sequence identity almost certainly have similar functions
Protein domains
Distinct structural modules within proteins
Have characteristic functions that can reveal much about a protein’s role, even in the absence of complete sequence homology
Nucleic Acid and Amino Acid Sequence Similarities
Metabolomics
Metabolome
The complete set of metabolic intermediates and other small molecules produced in an organism
Mass spectrometry is one of the primary techniques for monitoring metabolites
Gene Families, Duplications, and Deletions
Homologous: related in sequence to an extent that implies common genetic ancestry
Gene families: groups of gene homologs
Paralogs: genes within an organism whose similarity to one or more genes in the same organism is the result of gene duplication
Orthologs: genes found in one organism that are similar to those in another organism but differ because of speciation
Gene Families, Duplications, and Deletions
Gene duplications thought to be mechanism for evolution of most new genes
Deletions can eliminate gene no longer needed
Gene analysis in the three domains of life suggests that many genes present in all organisms have common evolutionary roots
Mobile DNA: Transposons and Insertion Sequences
Horizontal Gene Transfer
The transfer of genetic information between organisms, as opposed to vertical inheritance from parental organism(s)
May be extensive in nature
May cross phylogenetic domain boundaries
Mobile DNA: Transposons and Insertion Sequences
Detecting Horizontal Gene Flow
Presence of genes typically found only in distantly related species
Presence of a DNA with GC content or codon bias that differs significantly from remainder of genome
Mobile DNA: Transposons and Insertion Sequences
Horizontally transferred genes typically encode non-core metabolic functions
Horizontal Gene Transfer and Genome Stability
Transposons may transfer DNA between different organisms
Transposons may also mediate large-scale chromosomal changes within a single organism
Presence of multiple insertion sequences (IS)
Recombination among identical IS can result in chromosomal rearrangements
E.g., deletions, inversions, or translocations
Horizontal Gene Transfer and Genome Stability
Integrons
Genetic elements that collect and express genes carried on mobile segments of DNA (cassettes)
Of those known, most carry genes for antibiotic resistance
Evolution of Virulence: Pathogenicity Islands
Chromosomal Islands
Region of bacterial chromosome of foreign origin that contains clustered genes for some extra property such as virulence or symbiosis
Pathogenicity islands: chromosomal islands containing genes for virulence
Evolution of Virulence: Pathogenicity Islands
Chromosomal islands believed to have a “foreign” origin based on several observations
Extra regions often flanked by inverted repeats
Base composition and codon usage in chromosomal islands often differ from rest of genome
Often found in some strains of a species but not others
Evolution of Virulence: Pathogenicity Islands
Chromosomal islands contribute specialized functions not essential to growth
Virulence
Biodegradation of recalcitrant compounds
E.g., hydrocarbons and herbicides
Symbiosis
Evolution of Virulence: Pathogenicity Islands
The “pan”/ “core” concept: bacterial species consist of two components
Core genome: shared by all strains of the species
Pan genome: includes all the optional extras present in some but not all strains of the species
Detecting Uncultured Microorganisms
Metagenome
The total gene content of the organisms present in an environment
Several environments have been surveyed by large-scale metagenome projects
E.g., acid mine run-off waters,deep sea sediments, fertile soils
Viral Genomes in Nature
Viruses are more prevalent than bacteria in the environment
Most are bacteriophages and have populations that turn over rapidly
Most of the genetic diversity on Earth thought to reside in viruses
Most virus genes are uncharacterized and show little or no sequence similarity to known genes
Wednesday, March 25, 2009
Lab exam 2 review with answers for Tues. 3/31
What are organisms grown on? agar
What is nutrient agar? Beef extract and peptone
What are the purposes of growth on agar? Colony morphology, amount, isolation in mixed culture
What is agar? Sea weed-kelp derivative
What is the best temperature for culture growth of most microbes? 37oC
What is sterilization of prepared agar? 121oC for 15 min
What are the two methods of agar inoculation? Loop dilution and streak method
Describe the two methods. Pour plate vs. streak plate
The streak method depends on inoculation into how many areas of the agar? 4 What is accomplished by using the streak method? To gradually dilute colonies What should be the outcome? Pure culture
What two methods of determination of bacterial populations were performed in class? Standard Plate Count/Serial dilutions and Turbidity
What are major differences between the two methods? Use of agar; use of spectrophotometer
How is the outcome of the standard count different from the turbidity method? Colony number versus turbidity reading
How does the spectrophotometer work? Light shown through specimen recorded by photodetector
What is the difference between turbidity ( absorbance) and percent transmission? Turbidity: light absorbed %P: light transmitted
As the percent transmission increases, what is observed about the organisms in the broth? Fewer numbers
Answer the same question for absorbance. More organisms
What is the optimum growth temperature for most bacteria? 37oC
What is room temperature? 25oC
At what temperatures, would you expect to have growth and in what amount? 5C: +-; 23C: +; 37C: ++; 42C: +\-
What happens when the concentration of solutes in the growth media exceeds that in the organism? Plasmolysis (hypertonic)
What if the solute is lower? hypotonic
What special medium was used for oxygen testing? Thioglycollate. What unique substance did it contain? Oxidation-reduction agent.
What is the thermal death point? Temp that kills in 10 min. What is thermal death time? Time to kill at particular temp.
What is the purpose of U.V light? sterilization How is the damage produced? Thymine dimers Can the damage be repaired? Yes What is the procedure for using U.V. light? In the petri dish, the U.V. light affects what? Colonies on plate
What is the difference between disinfectant and antiseptic? Inanimate Objects vs. skin Antiseptics lower concentration of bugs
Be able to recognize aerobic, anaerobic, facultative anaerobic and microaerophilic.
The pour-plate disk method produces results called the colony forming units/ml? How is the result determined? Colony count-number of colonies on plate times dilution factor.
What is the susceptibility testing method used in lab called? Disk diffusion-Kirby Bauer.
What is the medium used to perform antimicrobial susceptibility? Mueller Hinton
What values are used to determine the susceptibility result? Mm size of zone of inhibition How is it obtained? Measure zone of inhibition
Know how to perform a serial dilution. Know Absorbance and Percent Transmission. Know Thermal Death Point and Death Time. Know Growth in types of Oxygen environments and termperatures.
Know Osmolality Experiment: Hypertonic, hypotonic and plasmolysis
Know pour plate, streak plate and broth loop dilution tube plate
Know how to make a dilution
What is nutrient agar? Beef extract and peptone
What are the purposes of growth on agar? Colony morphology, amount, isolation in mixed culture
What is agar? Sea weed-kelp derivative
What is the best temperature for culture growth of most microbes? 37oC
What is sterilization of prepared agar? 121oC for 15 min
What are the two methods of agar inoculation? Loop dilution and streak method
Describe the two methods. Pour plate vs. streak plate
The streak method depends on inoculation into how many areas of the agar? 4 What is accomplished by using the streak method? To gradually dilute colonies What should be the outcome? Pure culture
What two methods of determination of bacterial populations were performed in class? Standard Plate Count/Serial dilutions and Turbidity
What are major differences between the two methods? Use of agar; use of spectrophotometer
How is the outcome of the standard count different from the turbidity method? Colony number versus turbidity reading
How does the spectrophotometer work? Light shown through specimen recorded by photodetector
What is the difference between turbidity ( absorbance) and percent transmission? Turbidity: light absorbed %P: light transmitted
As the percent transmission increases, what is observed about the organisms in the broth? Fewer numbers
Answer the same question for absorbance. More organisms
What is the optimum growth temperature for most bacteria? 37oC
What is room temperature? 25oC
At what temperatures, would you expect to have growth and in what amount? 5C: +-; 23C: +; 37C: ++; 42C: +\-
What happens when the concentration of solutes in the growth media exceeds that in the organism? Plasmolysis (hypertonic)
What if the solute is lower? hypotonic
What special medium was used for oxygen testing? Thioglycollate. What unique substance did it contain? Oxidation-reduction agent.
What is the thermal death point? Temp that kills in 10 min. What is thermal death time? Time to kill at particular temp.
What is the purpose of U.V light? sterilization How is the damage produced? Thymine dimers Can the damage be repaired? Yes What is the procedure for using U.V. light? In the petri dish, the U.V. light affects what? Colonies on plate
What is the difference between disinfectant and antiseptic? Inanimate Objects vs. skin Antiseptics lower concentration of bugs
Be able to recognize aerobic, anaerobic, facultative anaerobic and microaerophilic.
The pour-plate disk method produces results called the colony forming units/ml? How is the result determined? Colony count-number of colonies on plate times dilution factor.
What is the susceptibility testing method used in lab called? Disk diffusion-Kirby Bauer.
What is the medium used to perform antimicrobial susceptibility? Mueller Hinton
What values are used to determine the susceptibility result? Mm size of zone of inhibition How is it obtained? Measure zone of inhibition
Know how to perform a serial dilution. Know Absorbance and Percent Transmission. Know Thermal Death Point and Death Time. Know Growth in types of Oxygen environments and termperatures.
Know Osmolality Experiment: Hypertonic, hypotonic and plasmolysis
Know pour plate, streak plate and broth loop dilution tube plate
Know how to make a dilution
Tuesday, March 24, 2009
Chapter 18-Eukaryotes
Eukaryotic Microbes
The eukaryotic microbes: algae, fungi, slime moulds and protozoa.
Eukaryotic microorganisms differ from Bacteria and Archaea.
These differences include:
cell size
internal structure
genetic arrangement
evolutionary history.
Eukaryotic Organelles
The endoplasmic reticulum
The golgi apparatus
Lysosomes
The peroxisome
3 energy organelles
Mitochondria
Respiration and oxidative phosphorylation are localized in mitochondria.
Organelle is surrounded by two membranes, inner and outer.
Outer membrane important in permeability
Cristae form from the invagination of inner membrane.
ATP synthesis occurs in the cristae
Hydrogenosome
Present in some anaerobic eukaryotic organisms that lack mitochondria
The organelle lack cristae and citric acid cycle enzymes
Metabolism of organisms is fermentative
Acetate excreted from organelle into cytoplasm.
Chloroplast
The chloroplast is the site of photosynthetic energy production and CO2 fixation in eukaryotic phototrophs (algae).
Like mitochondria, chloroplasts have a permeable outermost membrane, a much less permeable inner membrane, and an intermembrane space.
Chloroplast
The inner membrane surrounds the lumen of the chloroplast, but it is not folded into cristae like the inner membrane of the mitochondrion.
Instead, chlorophyll and all other components needed for photosynthesis are located in a series of flattened membrane discs called thylakoids.
Endosymbiotic Theory
Mitochondria, chloroplasts and hydrogenosomes originated from the stable incorporation of chemoorganotrophic and phototrophic symbionts from bacteria.
Aerobic bacterium established residency within the cytoplasm of a primitive eukaryote
Mitochondria vs. Chloroplast
Both contain DNA
Eukaryotic nucleus contains bacterially derived genes
Both contain their own ribosomes
Some antibiotics inhibit mitochondria and chloroplast activity
rRNA sequencing has shown that both originated from bacteria
Eukaryotic Organelles
Golgi Apparatus
involved in protein modification and secretion
Endoplasmic Reticulum
Smooth; synthesis of lipids and some carbohydrate metabolism
Rough: producer of glycoproteins and produces new membrane material. Some protein production and post-translations modification.
Lysosome
Peroxisomes
Small vesicles – similar to lysosomes
Arise by dividing of preexisting peroxisomes
Produce hydrogen peroxide (H2O2) as a byproduct
Oxidize a wide variety of chemicals
Detoxify harmful chemicals such as alcohol
Motility Organelles
In addition, proteinaceous tubes called microfilaments and microtubules are present, forming the cell's cytoskeleton.
Flagella and cilia are organelles of motility that have extensive microtubular structure.
Microfilaments
Thinnest elements
Composed of actin
Take part in movement, formation, and maintenance of cell shape
Microtubules
Largest elements
Composed of tubulin
Arise from microtubule organizing centers (MTOCs)
Involved in shape, motility,
cell division
Eukaryotic Replication DNA Protein Primer
The ends of linear genetic elements present a problem to the replication machinery that circular genetic elements do not.
Some prokaryotic plasmids and viral linear elements solve this problem by using a protein primer .
DNA replicates 5’-3’ (where OH resides)
Protein primer attaches to 5’ end
Replication occurs
Eukaryotic Chromosome
The DNA molecule of a typical chromosome contains:
a linear array of genes (encoding proteins and RNAs)
much noncoding DNA.
Included in the noncoding DNA are long stretches that make up the centromere and long stretches at the ends of the chromosome, the telomeres
Telomeres are crucial to the life of the cell. They keep the ends of the various chromosomes in the cell from accidentally becoming attached to each other.
Telomerases
Telomerase recognizes the tips of chromosomes also know as telomeres. The DNA sequences of telomeres consist of numerous repeats of a 6 to 8 base long sequence, [TTGGGG].
Telomerase consists of a protein and short RNA molecule that is complementary to the TTGGGG repeat in the telomere. This complementary RNA sequence base pairs with the telomere allowing Telomerase to add additional complementary bases to the 3' terminus of the telomere.
When telomeric length shortens to a critical point the cell dies.
Mitosis vs. Meiosis
Eukaryotic microorganisms can mate and exchange DNA during sexual reproduction.
Mitosis ensures appropriate segregation of the chromosomes during asexual cell division.
Haploid cells formed by meiosis can fuse to form a diploid zygote.(sexual reproduction)
RNA Processing
RNA processing: the processing of eukaryotic pre-mRNAs, is unique and involves three distinct steps:
Capping: addition of methylated guanine nucleotide. Facilitates translation
Splicing: removal of introns by spliceosome
Tailing: adding of poly A tail. Role unclear
Ribozyme
Self-splicing introns: work like enzymes
Excise themselves from RNA while joining exons together.
Catalyze reaction only once unlike protein enzymes
Protozoa
Protozoa are unicellular organisms that lack cell walls and obtain nutrients by ingesting other microbes (phagocytosis), or macromolecules in solution (pinocytosis)
They lack pigments and may be motile.
There are four groups, distinguished by their type of motility and their life cycles.
Mastigophora (flagellates) are motile through the use of flagella,
Sarcodina (amoebas) are motile with amoeboid movement,
Ciliophora use cilia for movement
Sporozoa are non-motile. Each group contains representatives that cause important human diseases. hapter
Sarcodines
The sarcodines include Amoeba—which are naked in the vegetative phase—and foraminifera—amoebae that secrete a shell during vegetative growth.
Flagellates
Flagellates are all motile by the activity of flagella.
Ciliates
Ciliates are protozoa that, in some stage of their life cycle, possess cilia, structures that function in motility.
Sporozoa
Sporozoa are a large group of obligately parasitic protozoa. These parasites can cause severe diseases, such as malaria
Slime moulds
Slime molds are non phototrophic eukaryotic microorganisms that live on decaying plant matter by phagocytizing microorganisms present on the surfaces.
There are two groups of slime molds:
cellular slime molds such as Dictyostelium that undergo a life cycle in which the cells exist independently as single amoebalike cells
acellular slime molds where the vegetative forms are naked masses of protoplasm called plasmodia
Slime Molds
Acellular slime molds are masses of motile protoplasm.
Cellular Slime Moulds
Cellular slime molds are masses of individual cells that aggregate to form fruiting bodies that release spores
Fungi
Fungi are chemoorganotrophs, lack chlorophyll, and have simple nutritional requirements as compared to bacteria.
Fungi can be differentiated from prokaryotes because they are much larger, contain a nucleus, vacuoles, and mitochondria. Fungi have a cell wall and are non-motile. Fungi produce spores.
The three important groups of fungi are:
Yeast
moulds
mushrooms
Yeasts
Yeasts are unicellular fungi usually occurring as spheres, ovals or cylinders. They favor environments rich in sugars, such as plant surfaces. They are the causative agents of a number of important diseases. Asexual division in yeasts involves budding.
Algae
Algae are phototrophic Eukarya that contain chlorophyll and carotenoid pigments within a chloroplast. The chloroplast itself has its roots in the Bacteria.
The eukaryotic microbes: algae, fungi, slime moulds and protozoa.
Eukaryotic microorganisms differ from Bacteria and Archaea.
These differences include:
cell size
internal structure
genetic arrangement
evolutionary history.
Eukaryotic Organelles
The endoplasmic reticulum
The golgi apparatus
Lysosomes
The peroxisome
3 energy organelles
Mitochondria
Respiration and oxidative phosphorylation are localized in mitochondria.
Organelle is surrounded by two membranes, inner and outer.
Outer membrane important in permeability
Cristae form from the invagination of inner membrane.
ATP synthesis occurs in the cristae
Hydrogenosome
Present in some anaerobic eukaryotic organisms that lack mitochondria
The organelle lack cristae and citric acid cycle enzymes
Metabolism of organisms is fermentative
Acetate excreted from organelle into cytoplasm.
Chloroplast
The chloroplast is the site of photosynthetic energy production and CO2 fixation in eukaryotic phototrophs (algae).
Like mitochondria, chloroplasts have a permeable outermost membrane, a much less permeable inner membrane, and an intermembrane space.
Chloroplast
The inner membrane surrounds the lumen of the chloroplast, but it is not folded into cristae like the inner membrane of the mitochondrion.
Instead, chlorophyll and all other components needed for photosynthesis are located in a series of flattened membrane discs called thylakoids.
Endosymbiotic Theory
Mitochondria, chloroplasts and hydrogenosomes originated from the stable incorporation of chemoorganotrophic and phototrophic symbionts from bacteria.
Aerobic bacterium established residency within the cytoplasm of a primitive eukaryote
Mitochondria vs. Chloroplast
Both contain DNA
Eukaryotic nucleus contains bacterially derived genes
Both contain their own ribosomes
Some antibiotics inhibit mitochondria and chloroplast activity
rRNA sequencing has shown that both originated from bacteria
Eukaryotic Organelles
Golgi Apparatus
involved in protein modification and secretion
Endoplasmic Reticulum
Smooth; synthesis of lipids and some carbohydrate metabolism
Rough: producer of glycoproteins and produces new membrane material. Some protein production and post-translations modification.
Lysosome
Peroxisomes
Small vesicles – similar to lysosomes
Arise by dividing of preexisting peroxisomes
Produce hydrogen peroxide (H2O2) as a byproduct
Oxidize a wide variety of chemicals
Detoxify harmful chemicals such as alcohol
Motility Organelles
In addition, proteinaceous tubes called microfilaments and microtubules are present, forming the cell's cytoskeleton.
Flagella and cilia are organelles of motility that have extensive microtubular structure.
Microfilaments
Thinnest elements
Composed of actin
Take part in movement, formation, and maintenance of cell shape
Microtubules
Largest elements
Composed of tubulin
Arise from microtubule organizing centers (MTOCs)
Involved in shape, motility,
cell division
Eukaryotic Replication DNA Protein Primer
The ends of linear genetic elements present a problem to the replication machinery that circular genetic elements do not.
Some prokaryotic plasmids and viral linear elements solve this problem by using a protein primer .
DNA replicates 5’-3’ (where OH resides)
Protein primer attaches to 5’ end
Replication occurs
Eukaryotic Chromosome
The DNA molecule of a typical chromosome contains:
a linear array of genes (encoding proteins and RNAs)
much noncoding DNA.
Included in the noncoding DNA are long stretches that make up the centromere and long stretches at the ends of the chromosome, the telomeres
Telomeres are crucial to the life of the cell. They keep the ends of the various chromosomes in the cell from accidentally becoming attached to each other.
Telomerases
Telomerase recognizes the tips of chromosomes also know as telomeres. The DNA sequences of telomeres consist of numerous repeats of a 6 to 8 base long sequence, [TTGGGG].
Telomerase consists of a protein and short RNA molecule that is complementary to the TTGGGG repeat in the telomere. This complementary RNA sequence base pairs with the telomere allowing Telomerase to add additional complementary bases to the 3' terminus of the telomere.
When telomeric length shortens to a critical point the cell dies.
Mitosis vs. Meiosis
Eukaryotic microorganisms can mate and exchange DNA during sexual reproduction.
Mitosis ensures appropriate segregation of the chromosomes during asexual cell division.
Haploid cells formed by meiosis can fuse to form a diploid zygote.(sexual reproduction)
RNA Processing
RNA processing: the processing of eukaryotic pre-mRNAs, is unique and involves three distinct steps:
Capping: addition of methylated guanine nucleotide. Facilitates translation
Splicing: removal of introns by spliceosome
Tailing: adding of poly A tail. Role unclear
Ribozyme
Self-splicing introns: work like enzymes
Excise themselves from RNA while joining exons together.
Catalyze reaction only once unlike protein enzymes
Protozoa
Protozoa are unicellular organisms that lack cell walls and obtain nutrients by ingesting other microbes (phagocytosis), or macromolecules in solution (pinocytosis)
They lack pigments and may be motile.
There are four groups, distinguished by their type of motility and their life cycles.
Mastigophora (flagellates) are motile through the use of flagella,
Sarcodina (amoebas) are motile with amoeboid movement,
Ciliophora use cilia for movement
Sporozoa are non-motile. Each group contains representatives that cause important human diseases. hapter
Sarcodines
The sarcodines include Amoeba—which are naked in the vegetative phase—and foraminifera—amoebae that secrete a shell during vegetative growth.
Flagellates
Flagellates are all motile by the activity of flagella.
Ciliates
Ciliates are protozoa that, in some stage of their life cycle, possess cilia, structures that function in motility.
Sporozoa
Sporozoa are a large group of obligately parasitic protozoa. These parasites can cause severe diseases, such as malaria
Slime moulds
Slime molds are non phototrophic eukaryotic microorganisms that live on decaying plant matter by phagocytizing microorganisms present on the surfaces.
There are two groups of slime molds:
cellular slime molds such as Dictyostelium that undergo a life cycle in which the cells exist independently as single amoebalike cells
acellular slime molds where the vegetative forms are naked masses of protoplasm called plasmodia
Slime Molds
Acellular slime molds are masses of motile protoplasm.
Cellular Slime Moulds
Cellular slime molds are masses of individual cells that aggregate to form fruiting bodies that release spores
Fungi
Fungi are chemoorganotrophs, lack chlorophyll, and have simple nutritional requirements as compared to bacteria.
Fungi can be differentiated from prokaryotes because they are much larger, contain a nucleus, vacuoles, and mitochondria. Fungi have a cell wall and are non-motile. Fungi produce spores.
The three important groups of fungi are:
Yeast
moulds
mushrooms
Yeasts
Yeasts are unicellular fungi usually occurring as spheres, ovals or cylinders. They favor environments rich in sugars, such as plant surfaces. They are the causative agents of a number of important diseases. Asexual division in yeasts involves budding.
Algae
Algae are phototrophic Eukarya that contain chlorophyll and carotenoid pigments within a chloroplast. The chloroplast itself has its roots in the Bacteria.
Saturday, March 21, 2009
Chapters 15 and 17
Proteobacteria
Largest group: The Proteobacteria
The Proteobacteria consist of five clusters (a,b,c,d,e) containing several genera. Greek letters: alpha, beta, gamma, delta, or epsilon
You do not have to know individual species
Proteobacter
Purple phototrophic bacteria
Nitrifying Bacteria
Sulfur and Iron-oxidizing
Hydrogen-Oxidizing
Methanotrophs and methylotrophs
Pseudomonas
Nitrogen-fixing
Phylum 1
Purple bacteria
Nitrifying (soil and water)
Sulfur and Iron-oxidizing
Hydrogen-Oxidizing Bacteria
Methanotrophs and methylotrophs
Pseudomonads
Nitrogen-fixing
Neisseria et al
Enterics
Vibrios and photobacterium
Rickettsias
Spirilla
Sheathed Proteobacteria
Budding and Prosthecate/Stalked Bacteria
Gliding Myxobacteria
Sulfate and sulfur- reducing Proteobacteria
Phyla 2 and 3
Non-sporulating, Gram-positive bacteria;Lactic Acid Bacteria and relatives
Staphylococcus;Micrococcus
Lactic Acid Bacteria; Lactobacillus
Streptococcus and other coccus
Listeria
Endospore-Forming, Gram-Positive Bacteria: Bacillus, Clostridium and relatives
Cell-Wall Less, Gram-Positive Bacteria: the Mycoplasma
Gram-Positive Bacteria: Corynebacteriua and Proprionic Acid Bacteria (Propionibacterium)
Mycobacterium
Streptomyces and other Actinomycetes
Human Inhabitants
Enteric rods: E. coli, Salmonella and Shigella, Proteus, Enterobacter, Klebsiella and Serratia
Vibrios
Rickettsias
Campylobacter and Helicobacter
Staphylococcus and Micrococcus
Lactobacillus
Streptococcus
Listeria
Bacillus
Clostridium
Mycoplasma
Corynebacteria
Mycobacteria
Actinomycetes
Chlamydia
Bacteriodes
Non-fermentative gram-negative rods
Taxonomy
Phenotypic
Gram stain
Morphology
Metabolism
Biochemical reactions
Genotypic
16rRNA:genes for 16S rRNA and related molecules are amplified, treated with one or more restriction enzymes, separated by electrophoresis and then probed with complementary rRNA.
G:C Ratios
Multilocus Sequence Typing
DNA:DNA Hybridization
Types of Metabolism
Phototrophic: obtains energy from light
Chemolithotrophic: oxidize inorganic compounds for energy
Chemoorganotrophic: obtains energy from the oxidation of organic compounds.
Autotrophic: use CO2 as nutrient source
Methanotrophs: use methane for energy
Nitrogen-fixing: reduction of nitrogen gas to ammonia
Homofermentative: fermentation of glucose or other sugar to lactic acid
Heterofermentative: fermentation of glucose or another sugar to a mix of reduced products.
Facultative: grows in either the presence or absence of an environmental factor
Oxygen and Temperature Response
Oxygen Response
Aerobic
Anaerobic
Facultative Anaerobic
Temperature Response
Mesophile
Thermophile
Role in Ecosystem
Decomposition: the breakdown of organisms, and the release of nutrients back into the environment.
Nitrogen Cycling: Plants rely on nitrogen from the soil and cannot acquire it from the gaseous nitrogen in the atmosphere.
Nitrogen Fixation: These bacteria convert gaseous nitrogen into nitrates or nitrites as part of their metabolism. The resulting products are released into the environment and used by plants. Some plants, house the bacteria in their own tissues.
Denitrifying bacteria turn nitrates into nitrogen gas or nitrous oxide.
Methane decomposed to ammonia
Utilization of sulfur and Iron as electron donors. Changes in soil or water content.
Cell Division in Bacteria
Binary Fission: The cell grows to twice its size, Duplicates its DNA and other cellular constituents, and lays down a cross-wall called a septum that separates the cells.
Polar Growth: the cell grows from one end and the two cells that result from cell division are similar in size.
Simple Budding:,the cell wall grows from one end of the bacterium, producing a smaller cell that separates off and. grows.
Buds-Hyphae-swarmer: The cell buds at the end of extensions called hyphae. As the bud increases in size, it forms a flagellum. A septum forms between the bud and the hypha,and the cells separate.
The daughter cell, called a swarmer, matures, lengthens, and eventually loses its flagellum,forms a hypha, and begins the cell division process again.
Stalk: The cell creates two distinct cells during division. One has a Stalk from which the cell grows. The cell divides by unequal binary fission,producing a second, slightly smaller swarmer cell with a flagellum. The swarmer cell swims off, and when it comes to rest, it lose its flagellum and forms a stalk,again.
Phyla of Bacteria
Purple Phototrophic Bacteria
Purple Phototrophic Bacteria
Anoxygenic photosynthesis. They contain pigments - purple, red and brown.
They are divided into two groups: use of hydrogen sulfide, H2S, as an electron donor for carbon dioxide reduction. (Redox Reactions)
Purple sulfur bacteria: normally respire anaerobically in aquatic environments.
Purple Non-sulfur bacteria: Most species are aerobic and utilize a variety of carbon sources.
Sulfate and Sulfur Reducing Proteobacteria
About 90 species of bacteria from over 20 genera are known which are anaerobic obligate sulphur or sulphate reducers .
Most species live in aquatic environments.
Nitrifying Bacteria
Most species are obligatory chemolithotrophs.
These bacteria can be divided into two groups, those that:
oxidize ammonia to nitrite
oxidize the nitrite to nitrate.
Nitrifying bacteria are widespread in soil and aquatic environments where they are an important part of the nitrogen cycle.
Sulfur and Iron Oxidizing Bacteria
Beggiatoa is a filamentous gliding bacterium which oxidizes sulfur commonly found in sulfur springs, sewage works and hydrothermal vents, and other environmental areas.
Thioploca, Thiotrix and Leucathrix are also filamentous sulphur-oxidizing bacteria that aggregate within a star or rosette with their filaments in a central core. Primarily found in marine habitats where they can form thick mats.
Hydrogen-Oxidizing Bacteria-A wide variety of bacteria can grow with:
H2 as the sole electron donor and O2 as the electron acceptor
Methanotrophs
These are aerobic methane oxidizing bacteria (methane to methanol). An important aspect of the carbon cycle. CH4-CH3OH
Some Methanotrophs are also Methylotrophs meaning that they are limited to using single carbon sources, thus they cannot utilize even simple sugars to grow.
Pseudomonas and the Pseudomonads
Chemoorganotrophic
Aerobic rods
Nitrogen-fixing
Phylogenetically closely related.
Metabolize glucose via the Entner- doudoroff pathway
Opportunists
Acetic Acid Bacteria
they partially oxidize various organic compounds, particularly ethanol, into acetic acid. Used commercially to make vinegar.
Free-living Aerobic Nitrogen Fixers
These are an ecologically important group of bacteria that live in the soil or in water.
Use gaseous nitrogen (N2) from the atmosphere and combine it with carbon and hydrogen, to make organic molecules.
Nearly all the organic molecules in the world are derived from bacterially fixed nitrogen.
Other Gram Negative Bacteria
This is a group of related bacteria that don't fit into any other group.
They contain both free-living species and species found living inside animals.
Among this latter division is the genus Neisseria including the species Neisseria gonorrhoeae which is responsible for the human sexually transmitted disease gonorrhoea.
Other species of Neisseria plus species of Kingella, Moraxella and Acinetobacter may be pathogenic (disease-causing) at times.
Enteric Bacteria
A large group of facultative aerobic rods. Human faeces generally comprise 30% (dry weight) of dead bacteria
Enteric bacteria are separated by the type of fermentation products produced by anaerobic fermentation of glucose.
Some cause gastroenteritis; others are opportunists
Bioluminescent and Related Bacteria
Gram-negative, facultatively aerobic curved rods with a fermentative metabolism. Both aquatic.
Two species of Vibrio cause disease in humans. V. cholorae causes cholera. V. parahemolyticus, causes gastroenteritis due to ingestion of contaminated seafood.
Photobacterium emit light mediated by the enzyme luciferase, an oxidation reaction. Bioluminescent bacteria are mostly associated with fish.
Rickettsia
Rickettsias are obligate intracellular parasites of eucaryotic cells. They have leaky membranes and are unable to obtain nutrients in an extracellular habitat.
Rickettsias occur in nature in the gut of arthropods (ticks, fleas, lice, etc.). They are transmitted to vertebrates by an arthropod bite and produce typhus fever, Rocky Mountain Spotted Fever, Q fever and canine ehrlichiosis.
Spirilla
Spirilla are chemoorganotrophic prokaryotes widespread in the environment.
Campylobacter and Helicobacter which are pathogenic to humans cause acute enteritis, chronic gastritis and peptic ulcers.
Spirilla
Bdellovibrio are predators on other bacteria. They attack by dissolving a hole in their prey's cell wall feeding on the cytoplasm.
Ancylobacter, a ring-shaped bacterium and Magnetospirillum magnetobacterium, a curved rod shaped bacterium, contain 5 to 40 magnetic particles magnetosomes and they allow the organism to align itself in relationship to magnetic fields.
Sheathed Bacteria
Sheathed filamentous bacteria in which individual cells form chains within an outer layer called the sheath. When nutrients are low, the individual cells develop a flagella tuft and are called swarmers
Gliding Myxobacteria
The fruiting myxobacteria are gliding bacteria that aggregate to form complex masses of cells called fruiting bodies.
Myxobacteria are chemoorganotrophic soil bacteria that live by consuming dead organic matter or other bacterial cells.
Gram-positive Bacteria
Gram-positive Bacteria are a large phylogenetic group that contains rods and cocci, sporulating and nonsporulating species.
The lactic acid bacteria are used in dairy production and are human commensals
Endospore Producers
Production of endospores is a hallmark of the key genera Bacillus and Clostridium.
These bacteria are major agents for the degradation of organic matter in soil, and a few species are pathogenic.(C. tetani and botulinum)
Mycoplasma
Lack cell walls and contain a very small genome.
Several are pathogenic for humans, other animals, and plants.
Walking pneumonia and neonatal infections
Small Gram-Positive Rods
Nonsporulating gram positive bacteria; lactic acid bacterial et al, coryneform and propionic Acid Bacteria
Swiss Cheese
The propionic acid bacteria were first discovered in Swiss cheese, where their fermentative production of CO2 results in the characteristic holes. shows the enzymatic reactions leading from glucose to propionic acid.
Actinobacteria: Mycobacterium
The genus Mycobacterium consists of rod-shaped organisms that are acid-fast.
The surface of the cell has unique lipids called mycolic acids, found only in the genus Mycobacterium
Main Pathogen: M. tuberculosis
Actinomycetes: Streptomyces
The streptomycetes are a large group of filamentous, gram-positive bacteria that form spores at the end of aerial filaments.
Other species: Nocardia causes brain infections and pneumoniae.
Cyanobacteria
Cyanobacteria comprise a large and mixed group of phototrophic Bacteria.
Cyanobacteria are oxygenic phototrophs.
Oxygen in Earth's atmosphere is thought to have originated from cyanobacterial photosynthesis
Chlamydia
Obligate intracellular parasites
The life cycle contains two cell types:
The elementary body: non-multiplying,transmit infection
The reticulate body: noninfectious replication form
Sexually-Transmitted Disease
Trachoma
Planctomyces
The Planctomyces group contains stalked, budding bacteria.
The Verrucomicrobia are distinguished by their multiple prosthecate cells
Verrocomicrobia Phylum 7
Verrocomicrobia
Bacteroides: Phylum 8
The genus Bacteroides contains obligate anaerobic species.
Bacteroides are normally found in the intestinal tract of humans and animals, and can cause infection.
They are the largest group of bacterial in the human colon.
Phylum 9 and 10
Cytophaga
Green Sulfur Bacteria
Spirochete-Phylum 11
Tightly coiled, motile, helical prokaryotes that contain both free-living and pathogenic species.
Treponema pallidum: syphillis
Borrelia: Lyme Disease
Stalked Bacteria
The new cell wall that separates the cytoplasm into two sections starts from a single point. This is called 'Polar Growth'.
A simple hyphae forms a bud at the distant end of the cell. This bud grows to be a new daughter cell. The daughter cell may develop a flagellum and swims away.
Deinococcus: Phylum 12
Deinococcus radiodurans is the most radiation resistant of all known organisms.
Green non-sulfur bacteria: Phylum 13
Chloroflexus
Branching Hyperthermophilic Bacteria: Phylum 14-16
Grow at temperatures > 80C
Netrospira: Phylum 17
Chemolithotrophs to Chemoorganotrophs
Mesophiles to Thermophiles
Characteristics of Different Organisms
Nitrogen oxidizing
Iron oxidizing
Use of metals for electron acceptors
Budding Bacteria
Budding and prosthecate bacteria are appendaged cells that form stalks or prosthecae used for attachment or nutrient absorption and are primarily aquatic.
Archaea
Archaea were once known as archaebacteria and live in extreme environments.
The Archaea can be divided into four groups: the methanogens, the halophiles, the hyperthermophiles and the genus Thermoplasma
Types of Archaea
Extremely Halophilic
Methane-Producing
Thermophilic/Acidophilic
Hyperthermophilic
Cold Dwelling
Hyperthermophiles from volcanic habitats
Hyperthermophiles for submarine volcanic habitats
Environments
Soil
Mineral deposits
Aquatic areas
Volcanic areas
Animals
Archaeal Cell Walls
Archaeal cell walls do not contain muramic acid and D-amino acids, the building blocks of peptidoglycan.
Particular species may contain pseudopeptidoglycan, polysaccharide, glycoprotein, or protein in their cell walls.
Archaeal Membranes
The Archaeal membranes differ from Bacterial membranes in that they contain ether-linked lipids bonded to glycerol.
Glycerol diethers and diglycerol tetraethers are the major types of lipids present in the cell membrane.
The Archaea also contain large amounts of non-polar lipids.
Metabolism in Archae
Chemoorganotrophic: use organic compounds as energy sources for growth.
Chemolithotrophy: anaerobic growth with H2 being a common electron donor and Sulfur, Nitrite, Iron or Oxygen as electron acceptor.
Autotrophy: Use of CO2 for energy source;widespread in the Archaea
Methanogens
Methanogens are obligate anaerobes
Anaerobic environments include marine and fresh-water sediments, bogs and deep soils, intestinal tracts of animals and humans, and sewage treatment facilities
Methanogens
Methanogens have a type of metabolism that can use H2 as an energy source and CO2 as a carbon source for growth.
In the process of making cell material from H2 and CO2, the methanogens produce methane (CH4) in a unique energy-generating process.
The end product (methane gas) accumulates in their environment.
Halophilic Archaea
Extremely halophilic Archaea require large amounts of NaCl for growth.
These organisms accumulate high levels of KCl in their cytoplasm as a compatible solute.
These salts affect cell wall stability and enzyme activity. The light-mediated proton pump bacteriorhodopsin helps extreme halophiles make ATP
Use of Bacteriorhodopsin
Certain species of Halobacterium can synthesize ATP using light energy.
The process uses a membrane protein called bacteriorhodopsin.
The absorption of light by retinal associated with this protein is used to pump protons across the cell membrane.
The resulting proton motive force can drive ATP synthesis via a membrane-bound ATPase.
Hyperthermophiles
“Hyperthermophiles" require temperatures of 80 degrees to 150 degrees for growth.
Most of these Archaea require elemental sulfur for growth. Some are anaerobes that use sulfur as an electron acceptor for respiration in place of oxygen.
Sulfur-oxidizers grow at low pH (less than pH 2), partly because they acidify their own environment by oxidizing SO (sulfur) to SO4 (sulfuric acid).
Temperature Limits
Although hyperthermophiles live at very high temperatures, in some cases above the boiling point of water, there are temperature limits beyond which no living organism can survive. This limit is likely 140ºC to 150°C
Molecular chaperones
Assist in the folding process.
Fold newly synthesized proteins
Refolding of partially denatured proteins.
Chaperones =heat shock proteins
Refold before proteases destroy them
Reverse DNA Gyrase.
All hyperthermophiles produce a DNA topoisomerase called reverse DNA gyrase.
Reverse gyrase introduces positive supercoils into DNA (in contrast to the negative supercoils introduced by DNA gyrase, found in all nonhyperthermophilic prokaryotes).
Supercoils
The structure of supercoils. (a) Positive supercoils - the front segment of a DNA molecule cross over the back segment from left to right. (b) Negative supercoils. (c) The positive supercoil in bacteria during DNA replication.
DNA-Binding Proteins
Proteins may also function to maintain double stranded DNA.
Histones: wind and compact DNA into nucleosome-like structure.
Early Life Forms
Hyperthermophilic Archaea and Bacteria are likely the closest living relatives to early life forms that remain today.
Hydrogen catabolism may have been the first energy-yielding metabolism of cells.
Pyrodictium and Pyrolobus
Pyrodictium and Pyrolobus are examples of prokaryotes whose growth temperature optimum lies above 100ºC. The optimum for Pyrodictium is 105ºC and for Pyrolobus is 106ºC.
Cells of Pyrodictium are irregularly disc-shaped and grow in culture in a mycelium-like layer attached to crystals of elemental sulfur.
Nanoarchaeum
Nanoarchaeum is a small, parasitic, early-branching member of the Archaea. Its genome is the smallest of all known organisms.
Nanoarchaeum lacks genes for all but core molecular processes and thus depends on its host, Ignicoccus, for most of its cellular needs.
Largest group: The Proteobacteria
The Proteobacteria consist of five clusters (a,b,c,d,e) containing several genera. Greek letters: alpha, beta, gamma, delta, or epsilon
You do not have to know individual species
Proteobacter
Purple phototrophic bacteria
Nitrifying Bacteria
Sulfur and Iron-oxidizing
Hydrogen-Oxidizing
Methanotrophs and methylotrophs
Pseudomonas
Nitrogen-fixing
Phylum 1
Purple bacteria
Nitrifying (soil and water)
Sulfur and Iron-oxidizing
Hydrogen-Oxidizing Bacteria
Methanotrophs and methylotrophs
Pseudomonads
Nitrogen-fixing
Neisseria et al
Enterics
Vibrios and photobacterium
Rickettsias
Spirilla
Sheathed Proteobacteria
Budding and Prosthecate/Stalked Bacteria
Gliding Myxobacteria
Sulfate and sulfur- reducing Proteobacteria
Phyla 2 and 3
Non-sporulating, Gram-positive bacteria;Lactic Acid Bacteria and relatives
Staphylococcus;Micrococcus
Lactic Acid Bacteria; Lactobacillus
Streptococcus and other coccus
Listeria
Endospore-Forming, Gram-Positive Bacteria: Bacillus, Clostridium and relatives
Cell-Wall Less, Gram-Positive Bacteria: the Mycoplasma
Gram-Positive Bacteria: Corynebacteriua and Proprionic Acid Bacteria (Propionibacterium)
Mycobacterium
Streptomyces and other Actinomycetes
Human Inhabitants
Enteric rods: E. coli, Salmonella and Shigella, Proteus, Enterobacter, Klebsiella and Serratia
Vibrios
Rickettsias
Campylobacter and Helicobacter
Staphylococcus and Micrococcus
Lactobacillus
Streptococcus
Listeria
Bacillus
Clostridium
Mycoplasma
Corynebacteria
Mycobacteria
Actinomycetes
Chlamydia
Bacteriodes
Non-fermentative gram-negative rods
Taxonomy
Phenotypic
Gram stain
Morphology
Metabolism
Biochemical reactions
Genotypic
16rRNA:genes for 16S rRNA and related molecules are amplified, treated with one or more restriction enzymes, separated by electrophoresis and then probed with complementary rRNA.
G:C Ratios
Multilocus Sequence Typing
DNA:DNA Hybridization
Types of Metabolism
Phototrophic: obtains energy from light
Chemolithotrophic: oxidize inorganic compounds for energy
Chemoorganotrophic: obtains energy from the oxidation of organic compounds.
Autotrophic: use CO2 as nutrient source
Methanotrophs: use methane for energy
Nitrogen-fixing: reduction of nitrogen gas to ammonia
Homofermentative: fermentation of glucose or other sugar to lactic acid
Heterofermentative: fermentation of glucose or another sugar to a mix of reduced products.
Facultative: grows in either the presence or absence of an environmental factor
Oxygen and Temperature Response
Oxygen Response
Aerobic
Anaerobic
Facultative Anaerobic
Temperature Response
Mesophile
Thermophile
Role in Ecosystem
Decomposition: the breakdown of organisms, and the release of nutrients back into the environment.
Nitrogen Cycling: Plants rely on nitrogen from the soil and cannot acquire it from the gaseous nitrogen in the atmosphere.
Nitrogen Fixation: These bacteria convert gaseous nitrogen into nitrates or nitrites as part of their metabolism. The resulting products are released into the environment and used by plants. Some plants, house the bacteria in their own tissues.
Denitrifying bacteria turn nitrates into nitrogen gas or nitrous oxide.
Methane decomposed to ammonia
Utilization of sulfur and Iron as electron donors. Changes in soil or water content.
Cell Division in Bacteria
Binary Fission: The cell grows to twice its size, Duplicates its DNA and other cellular constituents, and lays down a cross-wall called a septum that separates the cells.
Polar Growth: the cell grows from one end and the two cells that result from cell division are similar in size.
Simple Budding:,the cell wall grows from one end of the bacterium, producing a smaller cell that separates off and. grows.
Buds-Hyphae-swarmer: The cell buds at the end of extensions called hyphae. As the bud increases in size, it forms a flagellum. A septum forms between the bud and the hypha,and the cells separate.
The daughter cell, called a swarmer, matures, lengthens, and eventually loses its flagellum,forms a hypha, and begins the cell division process again.
Stalk: The cell creates two distinct cells during division. One has a Stalk from which the cell grows. The cell divides by unequal binary fission,producing a second, slightly smaller swarmer cell with a flagellum. The swarmer cell swims off, and when it comes to rest, it lose its flagellum and forms a stalk,again.
Phyla of Bacteria
Purple Phototrophic Bacteria
Purple Phototrophic Bacteria
Anoxygenic photosynthesis. They contain pigments - purple, red and brown.
They are divided into two groups: use of hydrogen sulfide, H2S, as an electron donor for carbon dioxide reduction. (Redox Reactions)
Purple sulfur bacteria: normally respire anaerobically in aquatic environments.
Purple Non-sulfur bacteria: Most species are aerobic and utilize a variety of carbon sources.
Sulfate and Sulfur Reducing Proteobacteria
About 90 species of bacteria from over 20 genera are known which are anaerobic obligate sulphur or sulphate reducers .
Most species live in aquatic environments.
Nitrifying Bacteria
Most species are obligatory chemolithotrophs.
These bacteria can be divided into two groups, those that:
oxidize ammonia to nitrite
oxidize the nitrite to nitrate.
Nitrifying bacteria are widespread in soil and aquatic environments where they are an important part of the nitrogen cycle.
Sulfur and Iron Oxidizing Bacteria
Beggiatoa is a filamentous gliding bacterium which oxidizes sulfur commonly found in sulfur springs, sewage works and hydrothermal vents, and other environmental areas.
Thioploca, Thiotrix and Leucathrix are also filamentous sulphur-oxidizing bacteria that aggregate within a star or rosette with their filaments in a central core. Primarily found in marine habitats where they can form thick mats.
Hydrogen-Oxidizing Bacteria-A wide variety of bacteria can grow with:
H2 as the sole electron donor and O2 as the electron acceptor
Methanotrophs
These are aerobic methane oxidizing bacteria (methane to methanol). An important aspect of the carbon cycle. CH4-CH3OH
Some Methanotrophs are also Methylotrophs meaning that they are limited to using single carbon sources, thus they cannot utilize even simple sugars to grow.
Pseudomonas and the Pseudomonads
Chemoorganotrophic
Aerobic rods
Nitrogen-fixing
Phylogenetically closely related.
Metabolize glucose via the Entner- doudoroff pathway
Opportunists
Acetic Acid Bacteria
they partially oxidize various organic compounds, particularly ethanol, into acetic acid. Used commercially to make vinegar.
Free-living Aerobic Nitrogen Fixers
These are an ecologically important group of bacteria that live in the soil or in water.
Use gaseous nitrogen (N2) from the atmosphere and combine it with carbon and hydrogen, to make organic molecules.
Nearly all the organic molecules in the world are derived from bacterially fixed nitrogen.
Other Gram Negative Bacteria
This is a group of related bacteria that don't fit into any other group.
They contain both free-living species and species found living inside animals.
Among this latter division is the genus Neisseria including the species Neisseria gonorrhoeae which is responsible for the human sexually transmitted disease gonorrhoea.
Other species of Neisseria plus species of Kingella, Moraxella and Acinetobacter may be pathogenic (disease-causing) at times.
Enteric Bacteria
A large group of facultative aerobic rods. Human faeces generally comprise 30% (dry weight) of dead bacteria
Enteric bacteria are separated by the type of fermentation products produced by anaerobic fermentation of glucose.
Some cause gastroenteritis; others are opportunists
Bioluminescent and Related Bacteria
Gram-negative, facultatively aerobic curved rods with a fermentative metabolism. Both aquatic.
Two species of Vibrio cause disease in humans. V. cholorae causes cholera. V. parahemolyticus, causes gastroenteritis due to ingestion of contaminated seafood.
Photobacterium emit light mediated by the enzyme luciferase, an oxidation reaction. Bioluminescent bacteria are mostly associated with fish.
Rickettsia
Rickettsias are obligate intracellular parasites of eucaryotic cells. They have leaky membranes and are unable to obtain nutrients in an extracellular habitat.
Rickettsias occur in nature in the gut of arthropods (ticks, fleas, lice, etc.). They are transmitted to vertebrates by an arthropod bite and produce typhus fever, Rocky Mountain Spotted Fever, Q fever and canine ehrlichiosis.
Spirilla
Spirilla are chemoorganotrophic prokaryotes widespread in the environment.
Campylobacter and Helicobacter which are pathogenic to humans cause acute enteritis, chronic gastritis and peptic ulcers.
Spirilla
Bdellovibrio are predators on other bacteria. They attack by dissolving a hole in their prey's cell wall feeding on the cytoplasm.
Ancylobacter, a ring-shaped bacterium and Magnetospirillum magnetobacterium, a curved rod shaped bacterium, contain 5 to 40 magnetic particles magnetosomes and they allow the organism to align itself in relationship to magnetic fields.
Sheathed Bacteria
Sheathed filamentous bacteria in which individual cells form chains within an outer layer called the sheath. When nutrients are low, the individual cells develop a flagella tuft and are called swarmers
Gliding Myxobacteria
The fruiting myxobacteria are gliding bacteria that aggregate to form complex masses of cells called fruiting bodies.
Myxobacteria are chemoorganotrophic soil bacteria that live by consuming dead organic matter or other bacterial cells.
Gram-positive Bacteria
Gram-positive Bacteria are a large phylogenetic group that contains rods and cocci, sporulating and nonsporulating species.
The lactic acid bacteria are used in dairy production and are human commensals
Endospore Producers
Production of endospores is a hallmark of the key genera Bacillus and Clostridium.
These bacteria are major agents for the degradation of organic matter in soil, and a few species are pathogenic.(C. tetani and botulinum)
Mycoplasma
Lack cell walls and contain a very small genome.
Several are pathogenic for humans, other animals, and plants.
Walking pneumonia and neonatal infections
Small Gram-Positive Rods
Nonsporulating gram positive bacteria; lactic acid bacterial et al, coryneform and propionic Acid Bacteria
Swiss Cheese
The propionic acid bacteria were first discovered in Swiss cheese, where their fermentative production of CO2 results in the characteristic holes. shows the enzymatic reactions leading from glucose to propionic acid.
Actinobacteria: Mycobacterium
The genus Mycobacterium consists of rod-shaped organisms that are acid-fast.
The surface of the cell has unique lipids called mycolic acids, found only in the genus Mycobacterium
Main Pathogen: M. tuberculosis
Actinomycetes: Streptomyces
The streptomycetes are a large group of filamentous, gram-positive bacteria that form spores at the end of aerial filaments.
Other species: Nocardia causes brain infections and pneumoniae.
Cyanobacteria
Cyanobacteria comprise a large and mixed group of phototrophic Bacteria.
Cyanobacteria are oxygenic phototrophs.
Oxygen in Earth's atmosphere is thought to have originated from cyanobacterial photosynthesis
Chlamydia
Obligate intracellular parasites
The life cycle contains two cell types:
The elementary body: non-multiplying,transmit infection
The reticulate body: noninfectious replication form
Sexually-Transmitted Disease
Trachoma
Planctomyces
The Planctomyces group contains stalked, budding bacteria.
The Verrucomicrobia are distinguished by their multiple prosthecate cells
Verrocomicrobia Phylum 7
Verrocomicrobia
Bacteroides: Phylum 8
The genus Bacteroides contains obligate anaerobic species.
Bacteroides are normally found in the intestinal tract of humans and animals, and can cause infection.
They are the largest group of bacterial in the human colon.
Phylum 9 and 10
Cytophaga
Green Sulfur Bacteria
Spirochete-Phylum 11
Tightly coiled, motile, helical prokaryotes that contain both free-living and pathogenic species.
Treponema pallidum: syphillis
Borrelia: Lyme Disease
Stalked Bacteria
The new cell wall that separates the cytoplasm into two sections starts from a single point. This is called 'Polar Growth'.
A simple hyphae forms a bud at the distant end of the cell. This bud grows to be a new daughter cell. The daughter cell may develop a flagellum and swims away.
Deinococcus: Phylum 12
Deinococcus radiodurans is the most radiation resistant of all known organisms.
Green non-sulfur bacteria: Phylum 13
Chloroflexus
Branching Hyperthermophilic Bacteria: Phylum 14-16
Grow at temperatures > 80C
Netrospira: Phylum 17
Chemolithotrophs to Chemoorganotrophs
Mesophiles to Thermophiles
Characteristics of Different Organisms
Nitrogen oxidizing
Iron oxidizing
Use of metals for electron acceptors
Budding Bacteria
Budding and prosthecate bacteria are appendaged cells that form stalks or prosthecae used for attachment or nutrient absorption and are primarily aquatic.
Archaea
Archaea were once known as archaebacteria and live in extreme environments.
The Archaea can be divided into four groups: the methanogens, the halophiles, the hyperthermophiles and the genus Thermoplasma
Types of Archaea
Extremely Halophilic
Methane-Producing
Thermophilic/Acidophilic
Hyperthermophilic
Cold Dwelling
Hyperthermophiles from volcanic habitats
Hyperthermophiles for submarine volcanic habitats
Environments
Soil
Mineral deposits
Aquatic areas
Volcanic areas
Animals
Archaeal Cell Walls
Archaeal cell walls do not contain muramic acid and D-amino acids, the building blocks of peptidoglycan.
Particular species may contain pseudopeptidoglycan, polysaccharide, glycoprotein, or protein in their cell walls.
Archaeal Membranes
The Archaeal membranes differ from Bacterial membranes in that they contain ether-linked lipids bonded to glycerol.
Glycerol diethers and diglycerol tetraethers are the major types of lipids present in the cell membrane.
The Archaea also contain large amounts of non-polar lipids.
Metabolism in Archae
Chemoorganotrophic: use organic compounds as energy sources for growth.
Chemolithotrophy: anaerobic growth with H2 being a common electron donor and Sulfur, Nitrite, Iron or Oxygen as electron acceptor.
Autotrophy: Use of CO2 for energy source;widespread in the Archaea
Methanogens
Methanogens are obligate anaerobes
Anaerobic environments include marine and fresh-water sediments, bogs and deep soils, intestinal tracts of animals and humans, and sewage treatment facilities
Methanogens
Methanogens have a type of metabolism that can use H2 as an energy source and CO2 as a carbon source for growth.
In the process of making cell material from H2 and CO2, the methanogens produce methane (CH4) in a unique energy-generating process.
The end product (methane gas) accumulates in their environment.
Halophilic Archaea
Extremely halophilic Archaea require large amounts of NaCl for growth.
These organisms accumulate high levels of KCl in their cytoplasm as a compatible solute.
These salts affect cell wall stability and enzyme activity. The light-mediated proton pump bacteriorhodopsin helps extreme halophiles make ATP
Use of Bacteriorhodopsin
Certain species of Halobacterium can synthesize ATP using light energy.
The process uses a membrane protein called bacteriorhodopsin.
The absorption of light by retinal associated with this protein is used to pump protons across the cell membrane.
The resulting proton motive force can drive ATP synthesis via a membrane-bound ATPase.
Hyperthermophiles
“Hyperthermophiles" require temperatures of 80 degrees to 150 degrees for growth.
Most of these Archaea require elemental sulfur for growth. Some are anaerobes that use sulfur as an electron acceptor for respiration in place of oxygen.
Sulfur-oxidizers grow at low pH (less than pH 2), partly because they acidify their own environment by oxidizing SO (sulfur) to SO4 (sulfuric acid).
Temperature Limits
Although hyperthermophiles live at very high temperatures, in some cases above the boiling point of water, there are temperature limits beyond which no living organism can survive. This limit is likely 140ºC to 150°C
Molecular chaperones
Assist in the folding process.
Fold newly synthesized proteins
Refolding of partially denatured proteins.
Chaperones =heat shock proteins
Refold before proteases destroy them
Reverse DNA Gyrase.
All hyperthermophiles produce a DNA topoisomerase called reverse DNA gyrase.
Reverse gyrase introduces positive supercoils into DNA (in contrast to the negative supercoils introduced by DNA gyrase, found in all nonhyperthermophilic prokaryotes).
Supercoils
The structure of supercoils. (a) Positive supercoils - the front segment of a DNA molecule cross over the back segment from left to right. (b) Negative supercoils. (c) The positive supercoil in bacteria during DNA replication.
DNA-Binding Proteins
Proteins may also function to maintain double stranded DNA.
Histones: wind and compact DNA into nucleosome-like structure.
Early Life Forms
Hyperthermophilic Archaea and Bacteria are likely the closest living relatives to early life forms that remain today.
Hydrogen catabolism may have been the first energy-yielding metabolism of cells.
Pyrodictium and Pyrolobus
Pyrodictium and Pyrolobus are examples of prokaryotes whose growth temperature optimum lies above 100ºC. The optimum for Pyrodictium is 105ºC and for Pyrolobus is 106ºC.
Cells of Pyrodictium are irregularly disc-shaped and grow in culture in a mycelium-like layer attached to crystals of elemental sulfur.
Nanoarchaeum
Nanoarchaeum is a small, parasitic, early-branching member of the Archaea. Its genome is the smallest of all known organisms.
Nanoarchaeum lacks genes for all but core molecular processes and thus depends on its host, Ignicoccus, for most of its cellular needs.
Sunday, March 1, 2009
ANNOUNCEMENT
Remember, participation is important in class. That means arriving on time, paying attention, and attending both lab and lecture. If you are not doing these things, you will be counted off points at the end of the term. Dr. K
Study Aid for Exam 2
General Properties of Viruses
Virus Characteristics
A virion is the extracellular form of a virus and contains either an RNA or a DNA genome.
Only nucleic acid and protein are present, with the nucleic acid on the inside.
The virus genome is introduced into a new host cell by infection.
The virus redirects the host metabolism to support virus replication.
The whole unit, genome and capsid, is called the nucleocapsid.
Classification of Viruses
By Genome: Either RNA or DNA
By Number of strands: Either single strand or double strand for both DNA and RNA
Retroviruses contain single stranded RNA which is changed to double stranded DNA by the enzyme reverse transcriptase
Capsid and Membrane
Capsids consist of capsomeres
Some Viruses have membranes composed of lipid bilayer from host and protein from virus
Membrane makes initial contact with host cell receptor.
Plaques are clear zones that develop on lawns of host cells. Each plaque results from infection by a single virus particle.
Viral Replication
The virus life cycle can be divided into five stages:
attachment (adsorption)
penetration (injection)
protein and nucleic acid synthesis
assembly and packaging
virion release.
Attachment
Bacteriophage
Virus that attacks bacterial cell
Structure
Head
Collar
Tail
Tail pins and fibers
Replication
For example, a virus that has a single-stranded RNA genome with the same orientation as its mRNA is said to be a positive-strand RNA virus.
A virus whose single-stranded RNA genome is complementary to its mRNA is said to be a negative-strand RNA virus.
Viral Genome Replication
Double stranded DNA: transcription of - strand mRNA to + to DNA
Positive single stranded mRNA: transcription of + mRNA
Minus single stranded mRNA: transcription of - stranded mRNA to +
Retrovirus: single stranded + mRNA that is transcribed to DNA by reverse transcriptase
Virulent Bacteriophages
A virion of attaches to a host cell and the DNA penetrates the cytoplasm.
The viral genes redirect the host machinery to the reproduce viral nucleic acid and protein. Virus then assembles and releases new virions by lysing the cell
Temperate Bacteriophage
Host cells can harbor viral genomes without harm if the expression of the viral genes can be controlled. This is the situation found in lysogens and the virus is called a provirus or prophage.
If control is lost, the virus enters the lytic pathway, produces new virions, and lyses the host cell.
Retroviruses
Retroviruses are RNA viruses that replicate through a DNA intermediate. The retrovirus HIV causes AIDS.
The retrovirus contains an enzyme, reverse transcriptase, that copies the information from its RNA genome into DNA, a process called reverse transcription
Retrovirus The DNA becomes integrated into the host chromosome in the same way as it does in a temperate virus.
The retrovirus DNA can be transcribed to yield mRNA (and new genomic RNA), or it may remain in a latent state.
Viroids and Prions
Viroids are small, circular, single-stranded RNA molecules that do not encode proteins and are completely dependent on host-encoded enzymes.
Prions consist of protein but have no nucleic acid.
Prions and viroids are the smallest known pathogens.
Influenza
The flu genomes is highly variable, especially with respect to the envelope components, hemagglutinin (HA) and neuraminadase (NA)
The changing antigens guarantee that there will be susceptible people-Antigenic shift and drift
Antigenic Shift: When two genetically different strains infect the same cell and the RNA in reassorted.
Antigenic Drift: alteration of hemaglutinin and neuraminidase
Chapter 10
Mutation
Mutation is a heritable change in DNA sequence that can lead to a change in phenotype (outer characteristics)
By definition, a mutant differs from its parental strain in genotype, the nucleotide sequence of the genome.
Types of Mutation
A point mutation, occur due to base-pair substitutions leading to a phenotypic change
Types of mutations:
Missense: Changes in amino acid sequence causing inactive polypeptide
Nonsense: Formation of stop codon
Silent: Polypeptide not affected
Wild Type: Non-mutated genome
Insertion/Deletion: Shifts order of codons
Mutagens
Radiation
Ionizing radiation – breaks in chromosomes
Nonionizing radiation – induces thymine dimers
Chemical Mutagens
SOS Repair
A complex cellular mechanism called the SOS regulatory system is activated as a result of some types of DNA damage
Initiates a number of DNA repair processes, both error-prone and high-fidelity
Genetic Recombination
Homologous recombination: when closely related DNA sequences from two distinct genetic elements are combined in a single element.
Recombination that occur in prokaryotes involve DNA transfer during the processes of transformation, transduction, and conjugation
Transformation: Process by which free DNA is incorporated into a recipient cell and brings about genetic change.
Competence: the ability to receive DNA and become transformed. A state in which cells are able to take up free DNA released by other bacteria
Transfection: when bacteria can be transformed with DNA extracted from a bacterial virus rather than from another bacterium.
Transduction
Transduction involves the transfer of host genes from one bacterium to another by bacterial viruses.
Generalized transduction: defective virus particles incorporate fragments of the cell's chromosomal DNA randomly. The efficiency is low.
Specialized Transduction: when DNA becomes integrated into the host DNA at a specific site.
Conjugation
Involves cell to cell contact. A donor and a recipient cell.
Rolling cell replication
F plasmid: unintegrated fertility plasmid
Hfr plasmid: A chromosome-integrated F plasmid.
Transposition
Segments of DNA that move from one location to another in the same or different molecule
Microbial Growth Control
Sterilization
Sterilization: killing of all organisms, including viruses. Heat is the most widely used method of sterilization.
The temperature must eliminate the most heat-resistant organisms, usually bacterial endospores
Pasteurization: does not sterilize but reduces microbial load, inhibiting the growth of spoilage microorganisms.
Autoclave
An autoclave permits application of steam heat under pressure at temperatures above the boiling point of water, killing endospores
Temperature is 121oC using steam under pressure
Antimicrobials
These agents are termed bacteriocidal, fungicidal, and viricidal agents, killing bacteria, fungi, and viruses, respectively.
Cidal: kills organism but cell lysis does not occur
Static: Injures organism. Frequent inhibitors of protein synthesis.
Lytic: induces killing by cell lysis
Testing Methods
A broth dilution assay: a standard concentration of the organism tested against different concentrations of the antimicrobial in broth. Antimicrobial activity is measured by determining the smallest amount of agent needed to inhibit the growth of the test organism
Disk-Diffusion assay: a standardized concentration of the organism is spread over Mueller-Hinton medium; paper disks containing concentrations of various antimicrobials are added to the surface of the medium. After 24 hr of incubation, the zone of inhibition around each disk is measured and represents whether the organism is susceptible or resistant.
Chemical Agents for External Use
Sterilants, sterilizers or sporicides: kill all forms of microbes. Compounds used to decontaminate nonliving material.
Cold sterilization: process that uses chemicals for sterilization
Disinfection: the elimination of organisms from inanimate objects or surfaces. May not kill endospores
External Agents
Sanitizers: reduce but may not eliminate, microbial numbers.
Antiseptics and germicides: chemical agents that kill or inhibit growth or organisms. Non-toxic to living tissues.
Agent Classification
Classification:
Structure,
mechanism of action
Spectrum of antimicrobial activity.
Two Categories
Synthetic agents and antibiotics
Antimicrobial Action
Selective toxicity: Method of action of antimicrobial agent
Inhibition of Cell Wall Synthesis
Inhibition of Protein Synthesis
Disruption of Cytoplasmic Membranes
Inhibition of Metabolic Pathways
Inhibition of Nucleic Acid Synthesis
Naturally Occurring Antimicrobials
Produced by wide range of fungi and bacteria
Inhibits or kills other microorganisms
Semisynthetic: structurally modified by pharmaceutical company
Broad Spectrum: Effective against both Gram-positive and Gram-negative organisms
Narrow Spectrum: Effective against only certain groups of organisms.
Inhibition of Protein Synthesis
Prokaryotic ribosomes are 70S (30S+50S)
Eukaryotic ribosomes are 80S (40S+60S)
Effect organism at level of translation
Initiation
Elongation
Inhibition of Cell Wall Synthesis
Beta-lactam agents: all contain beta lactam ring structure. Penicillins and Cephalosporins
Interferes with the peptides that link carbohydrates of peptidoglycan
Cell bursts from osmotic pressure
Microbial Resistance
Preexisting Resistance - resistant before exposure to antibiotics
Intrinsic resistance - born bad
Genetic mutation of chromosome
Transfer of genetic material: transformation, transduction, conjugation
Resistance Mechanisms
Reduced cell permeability
Efflux of agent from organism
Inactivation of agent
Alteration of target
Development of resistant biochemical pathway
Industrial Microbiology
Industrial Microbiology
Industrial microbiology uses microorganisms, typically grown on a large scale, to produce commercial products.
Biocatalysis: The actual reactions carried out by microorganisms in industrial microbiology
Microbial Biotechnology: Methods for gene manipulation used to yield new microbial products
Properties of Organism
Capable of large scale inexpensive liquid culture
Should produce spores or other reproductive cell for inoculation into large fermentors.
Must grow rapidly
Produce the desired product in short time period
Should not be pathogenic
Must be amenable to genetic manipulation
Metabolites
Primary metabolites are produced during active cell growth (exponential phase)
Secondary metabolites are produced near the onset of stationary phase
Secondary Metabolites
Growth and reproduction not essential
Dependent on growth conditions
Produced as a group of closely related compounds
Can get overproduction of secondary metabolites
Characteristics of Large-Scale Fermentations
Fermentation: any large-scale microbial process whether or not it is biochemically a fermentation.
Industrial fermentors can be divided into two major classes:
anaerobic processes
aerobic processes (majority of processes)
Industrial Microbiology
Grow organisms
Make products from gene-directed metabolites.
Antimicrobials
Must be produced in large scale industrial fermentors.
Must be purified
Need high yielding strains
Involves mutagenesis of the initial culture
Plating of mutant types and testing of mutants for antibiotic production
If penicillin fermentation is carried out without addition of side-chains, natural penicillins are produced.
A side chain can be added to the broth toproduce desired penicillin: biosynthetic penicillin.
All of these antibiotics are typical secondary metabolites.
Microbial biotransformation employs microorganisms to biocatalyze a specific step or steps in a strictly chemical synthesis.
Brewing and Distilling
Alcoholic beverages are produced by yeast and sugar
Vinegar Production
The active ingredient in vinegar is acetic acid, which is produced by acetic acid bacteria oxidizing an alcohol-containing fruit juice.
Adequate aeration is the most important consideration in vinegar process.
Genetic Engineering and Biotechnology
Biotechnology is the use of living organisms to carry out chemical processes for commercial use.
Genetic engineering is based on molecular cloning, in which a double-stranded DNA fragment from any source is recombined with a vector and introduced into a suitable host.
Cloning vectors include plasmids and bacteriophages.
Definitions
Cloning Vector - Element into which genes can be recombined and replicated
Genetic Map -The arrangement of genes on a chromosome
Genotype -The precise genetic makeup of an organism; both chromosome and plasmids
Molecular Cloning -Isolation and incorporation of a fragment of DNA into a vector where it can be replicated
Shuttle Vector -A cloning vector that can stably replicate in two different organisms.
Expression Vector - a cloning vector that contains the necessary regulatory sequences to allow transcription and translation of cloned genes to express the protein coded by the gene.
Integrating Vector -a cloning vector that becomes integrated into a host chromosome
Nucleic Acid Probe -a strand of nucleic acid that can be labeled and used to hybridize to a complementary DNA molecule
Reporter Gene -A gene incorporated into a vector because the product it encodes is easy to detect
Transfection –The introduction of DNA into mammalian cells.
Reverse Translation -using a codon table and amino acid sequence of a protein to obtain a possible sequence of the mRNA or gene that encoded the protein.
Reverse Transcriptase - enzyme which copies DNA from an RNA template
Repressor Protein -A regulatory protein that binds to specific sites on DNA and blocks transcription
Recombination -The process by which parts or all of the DNA from two separate sources are exchanged or brought together into a single DNA molecule
Wild Type -The bacterial strain isolated from nature
Molecular Cloning
Cloning: purpose is to isolate multiple copies of specific genes in pure form.
Steps in Cloning:
Isolation and fragmentation of the source DNA
Joining the DNA fragments to a cloning vector with DNA ligase
Introduction and maintenance of the clone DNA in a host organism using a vector like a plasmid
Site directed mutagenesis: insertion of synthetic DNA at precisely determined sites in genes in vitro.
Uses of cloned genes
To produce a protein product, like human growth hormone.
To prepare many copies of the gene itself
To determine the gene's nucleotide sequence
An ideal host:
Grows rapidly in an inexpensive culture medium.
Host=nonpathogenic
Capable of taking up DNA
Genetically stable in culture
Has appropriate enzymes to allow replication of the vector.
Prokaryotic Vectors
Plasmid
Bacteriophage
Can be placed in host by transformation
transduction or conjugation
The cloned gene can be harvested or the protein product of the gene can be made and collected.
Transfection of Eukaryotic Cells
Phagocytosis –precipitating DNA for take-up by cell
Electroporation –exposing host cells to pulsed electrical fields in the presence of cloned DNA. The electris treatment opens small pores in host membrane.
Particle Gun –Fires nucleic acid-coated particles into target cells.
Microinjection-Injection of DNA into host using micropipets.
Finding the Right Gene Clone
Special procedures are needed to detect
the foreign gene in the cloning host.
If the gene is expressed, the presence of the gene is detected using hybridization (reaction with specific radiolabeled nucleic acid strand complementary to cloned gene)
Hybridization=nucleic acid probe.
The protein produced by cloned gene can be identified using a tagged antibody
Grow cloned cells in culture. To find colonies/cells with the cloned DNA or protein, use nucleic acid hybridization (DNA) or antibody assay (protein)
Hybridization depends on base-pairing between the gene and a complementary sequence.
The probe or antibody will bond specifically to complementary gene or protein.
Specialized Vectors
Shuttle vector:
Allows cloned DNA to be moved between unrelated organisms (eukaryotic and prokaryotic)
Is a cloning vector that can stably replicate in two different organisms.
Expression vectors:
Vector that can be used to clone the desired gene but can also contain the necessary regulatory sequences to manipulate the expression of the gene
Translation of Cloned Gene
Expression vectors must ensure mRNA translation
Bacterial ribosome binding site; must be engineered into eukaryotic vector
The correct codons must be present either naturally, or by synthetic DNA or site-directed mutagenesis
Eukaryotic Vectors
Eukaryotic Vectors should have:
Origins of Replication-specific sequence that is recognized by specific initiation proteins, including protein that opens up helix
Selectable Marker-factor that allows identification of mutated cells (i.e. antibiotic resistance)
Multiple cloning sites- a short segment of DNA containing many different restriction enzyme cut sites.
Virus vectors or yeast artificial chromosome
Integrating Vectors-designed to maintain clones in very low but stable copy numbers
Reporter genes
Are incorporated into vectors because they encode proteins that are readily detected. Can be used to signal the presence or absence of a particular genetic element or its location. Can also be fused to other genes or to the promoter of other genes so that expression can be studied.
Reverse Translation
Using the amino acid sequence of a protein to synthesize an oligonucleotide probe that encodes it.
The codons that represent the amino acid of the protein are obtained.
The nucleotides that represent the codons are selected for use in designing the probe.
Protein Folding
Cloned gene made to contain a fusion protein in the vector.
Fusion protein can be engineered to contain bacterial signal peptide that enables transport of the clone across the cytoplasmic membrane
Cloned protein is released from the fusion protein by special enzymes
Microbial Evolution and Synthesis
Exam Questions
Study the Working Glossary on page 300
Early Earth
Early Earth was anoxic and much hotter than the present Earth
UV energy was dominant
The first biochemical compounds were made by abiotic syntheses that set the stage for the origin of life.
The first life forms may have been self-replicating RNAs (RNA life).
Primitive Life: Energy and Carbon Metabolism
Primitive metabolism was anaerobic and likely chemolithotrophic (oxidation of inorganic compounds), exploiting the abundant sources of sulfides present
Carbon metabolism may have included autotrophy. (Use of CO2 for metabolite)
Mitochondria and chloroplasts, the principal energy-producing organelles of eukaryotes, arose from the symbioticassociation of prokaryotes of the domain Bacteria within eukaryotic cells, a processcalled endosymbiosis
Mitochondria arose from Proteobacteria
Phylogeny
The phylogeny of microorganisms is their evolutionary relationships.
Comparisons of sequences of ribosomal RNA and differences in nucleotide or amino acid sequence of similar macromolecules are a function of their evolutionary distance.
Ribosomal Database
SSU (small subunit) RNA: the sequencing of the 16S(prokaryote) or 18S(eukaryote) ribosomal unit.
Phylogenetic trees based on ribosomal RNA have now been prepared for all the major prokaryotic and eukaryotic groups.
A huge database of rRNA sequences exists. The Ribosomal Database Project (RDP) contains a large collection of such sequences, now numbering over 100,000.
RNA Sequencing
Ribosomal RNA sequencing involves the amplification of the gene encoding 16S ribosomal RNA, sequencing it, and analyzing the sequence in reference to other bacterial 16S sequences.
16 S subunit is part of bacterial 30 S and 50 S subunits
The evolutionary distance is calculated as the percentage of nonidentical sequences between the RNAs of any two organisms
Domains
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
The third, Eukarya, evolved into the modern eukaryotic cell
Characteristics of the Domains of Life
The domains of living organisms were originally defined by ribosomal RNA sequencing, but 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.
Taxonomy
Bacterial taxonomy places emphasis on analyses of phenotypic properties of the organism.
Taxonomy: The science of classification.
Consists of two major subdisciplines, identification and nomenclature
Phylogeny: evolution of organisms.
Groups of genera (singular: genus) are collected into families, families into orders, orders into classes, classes into phyla (singular: phylum), and so on up to the highest-level taxon, the domain.
Speciation
The model for speciation is based solely on the assumption of vertical (mother to daughter) gene flow.
However, bacterial speciation is also affected to some degree by lateral (horizontal) gene transfer.
Lateral flow is the transfer of genes between species by conjugation, transduction, and transformation
Nomenclature and Bergey's Manual
Following the binomial system of nomenclature, prokaryotes are given genus and species name.
Virus Characteristics
A virion is the extracellular form of a virus and contains either an RNA or a DNA genome.
Only nucleic acid and protein are present, with the nucleic acid on the inside.
The virus genome is introduced into a new host cell by infection.
The virus redirects the host metabolism to support virus replication.
The whole unit, genome and capsid, is called the nucleocapsid.
Classification of Viruses
By Genome: Either RNA or DNA
By Number of strands: Either single strand or double strand for both DNA and RNA
Retroviruses contain single stranded RNA which is changed to double stranded DNA by the enzyme reverse transcriptase
Capsid and Membrane
Capsids consist of capsomeres
Some Viruses have membranes composed of lipid bilayer from host and protein from virus
Membrane makes initial contact with host cell receptor.
Plaques are clear zones that develop on lawns of host cells. Each plaque results from infection by a single virus particle.
Viral Replication
The virus life cycle can be divided into five stages:
attachment (adsorption)
penetration (injection)
protein and nucleic acid synthesis
assembly and packaging
virion release.
Attachment
Bacteriophage
Virus that attacks bacterial cell
Structure
Head
Collar
Tail
Tail pins and fibers
Replication
For example, a virus that has a single-stranded RNA genome with the same orientation as its mRNA is said to be a positive-strand RNA virus.
A virus whose single-stranded RNA genome is complementary to its mRNA is said to be a negative-strand RNA virus.
Viral Genome Replication
Double stranded DNA: transcription of - strand mRNA to + to DNA
Positive single stranded mRNA: transcription of + mRNA
Minus single stranded mRNA: transcription of - stranded mRNA to +
Retrovirus: single stranded + mRNA that is transcribed to DNA by reverse transcriptase
Virulent Bacteriophages
A virion of attaches to a host cell and the DNA penetrates the cytoplasm.
The viral genes redirect the host machinery to the reproduce viral nucleic acid and protein. Virus then assembles and releases new virions by lysing the cell
Temperate Bacteriophage
Host cells can harbor viral genomes without harm if the expression of the viral genes can be controlled. This is the situation found in lysogens and the virus is called a provirus or prophage.
If control is lost, the virus enters the lytic pathway, produces new virions, and lyses the host cell.
Retroviruses
Retroviruses are RNA viruses that replicate through a DNA intermediate. The retrovirus HIV causes AIDS.
The retrovirus contains an enzyme, reverse transcriptase, that copies the information from its RNA genome into DNA, a process called reverse transcription
Retrovirus The DNA becomes integrated into the host chromosome in the same way as it does in a temperate virus.
The retrovirus DNA can be transcribed to yield mRNA (and new genomic RNA), or it may remain in a latent state.
Viroids and Prions
Viroids are small, circular, single-stranded RNA molecules that do not encode proteins and are completely dependent on host-encoded enzymes.
Prions consist of protein but have no nucleic acid.
Prions and viroids are the smallest known pathogens.
Influenza
The flu genomes is highly variable, especially with respect to the envelope components, hemagglutinin (HA) and neuraminadase (NA)
The changing antigens guarantee that there will be susceptible people-Antigenic shift and drift
Antigenic Shift: When two genetically different strains infect the same cell and the RNA in reassorted.
Antigenic Drift: alteration of hemaglutinin and neuraminidase
Chapter 10
Mutation
Mutation is a heritable change in DNA sequence that can lead to a change in phenotype (outer characteristics)
By definition, a mutant differs from its parental strain in genotype, the nucleotide sequence of the genome.
Types of Mutation
A point mutation, occur due to base-pair substitutions leading to a phenotypic change
Types of mutations:
Missense: Changes in amino acid sequence causing inactive polypeptide
Nonsense: Formation of stop codon
Silent: Polypeptide not affected
Wild Type: Non-mutated genome
Insertion/Deletion: Shifts order of codons
Mutagens
Radiation
Ionizing radiation – breaks in chromosomes
Nonionizing radiation – induces thymine dimers
Chemical Mutagens
SOS Repair
A complex cellular mechanism called the SOS regulatory system is activated as a result of some types of DNA damage
Initiates a number of DNA repair processes, both error-prone and high-fidelity
Genetic Recombination
Homologous recombination: when closely related DNA sequences from two distinct genetic elements are combined in a single element.
Recombination that occur in prokaryotes involve DNA transfer during the processes of transformation, transduction, and conjugation
Transformation: Process by which free DNA is incorporated into a recipient cell and brings about genetic change.
Competence: the ability to receive DNA and become transformed. A state in which cells are able to take up free DNA released by other bacteria
Transfection: when bacteria can be transformed with DNA extracted from a bacterial virus rather than from another bacterium.
Transduction
Transduction involves the transfer of host genes from one bacterium to another by bacterial viruses.
Generalized transduction: defective virus particles incorporate fragments of the cell's chromosomal DNA randomly. The efficiency is low.
Specialized Transduction: when DNA becomes integrated into the host DNA at a specific site.
Conjugation
Involves cell to cell contact. A donor and a recipient cell.
Rolling cell replication
F plasmid: unintegrated fertility plasmid
Hfr plasmid: A chromosome-integrated F plasmid.
Transposition
Segments of DNA that move from one location to another in the same or different molecule
Microbial Growth Control
Sterilization
Sterilization: killing of all organisms, including viruses. Heat is the most widely used method of sterilization.
The temperature must eliminate the most heat-resistant organisms, usually bacterial endospores
Pasteurization: does not sterilize but reduces microbial load, inhibiting the growth of spoilage microorganisms.
Autoclave
An autoclave permits application of steam heat under pressure at temperatures above the boiling point of water, killing endospores
Temperature is 121oC using steam under pressure
Antimicrobials
These agents are termed bacteriocidal, fungicidal, and viricidal agents, killing bacteria, fungi, and viruses, respectively.
Cidal: kills organism but cell lysis does not occur
Static: Injures organism. Frequent inhibitors of protein synthesis.
Lytic: induces killing by cell lysis
Testing Methods
A broth dilution assay: a standard concentration of the organism tested against different concentrations of the antimicrobial in broth. Antimicrobial activity is measured by determining the smallest amount of agent needed to inhibit the growth of the test organism
Disk-Diffusion assay: a standardized concentration of the organism is spread over Mueller-Hinton medium; paper disks containing concentrations of various antimicrobials are added to the surface of the medium. After 24 hr of incubation, the zone of inhibition around each disk is measured and represents whether the organism is susceptible or resistant.
Chemical Agents for External Use
Sterilants, sterilizers or sporicides: kill all forms of microbes. Compounds used to decontaminate nonliving material.
Cold sterilization: process that uses chemicals for sterilization
Disinfection: the elimination of organisms from inanimate objects or surfaces. May not kill endospores
External Agents
Sanitizers: reduce but may not eliminate, microbial numbers.
Antiseptics and germicides: chemical agents that kill or inhibit growth or organisms. Non-toxic to living tissues.
Agent Classification
Classification:
Structure,
mechanism of action
Spectrum of antimicrobial activity.
Two Categories
Synthetic agents and antibiotics
Antimicrobial Action
Selective toxicity: Method of action of antimicrobial agent
Inhibition of Cell Wall Synthesis
Inhibition of Protein Synthesis
Disruption of Cytoplasmic Membranes
Inhibition of Metabolic Pathways
Inhibition of Nucleic Acid Synthesis
Naturally Occurring Antimicrobials
Produced by wide range of fungi and bacteria
Inhibits or kills other microorganisms
Semisynthetic: structurally modified by pharmaceutical company
Broad Spectrum: Effective against both Gram-positive and Gram-negative organisms
Narrow Spectrum: Effective against only certain groups of organisms.
Inhibition of Protein Synthesis
Prokaryotic ribosomes are 70S (30S+50S)
Eukaryotic ribosomes are 80S (40S+60S)
Effect organism at level of translation
Initiation
Elongation
Inhibition of Cell Wall Synthesis
Beta-lactam agents: all contain beta lactam ring structure. Penicillins and Cephalosporins
Interferes with the peptides that link carbohydrates of peptidoglycan
Cell bursts from osmotic pressure
Microbial Resistance
Preexisting Resistance - resistant before exposure to antibiotics
Intrinsic resistance - born bad
Genetic mutation of chromosome
Transfer of genetic material: transformation, transduction, conjugation
Resistance Mechanisms
Reduced cell permeability
Efflux of agent from organism
Inactivation of agent
Alteration of target
Development of resistant biochemical pathway
Industrial Microbiology
Industrial Microbiology
Industrial microbiology uses microorganisms, typically grown on a large scale, to produce commercial products.
Biocatalysis: The actual reactions carried out by microorganisms in industrial microbiology
Microbial Biotechnology: Methods for gene manipulation used to yield new microbial products
Properties of Organism
Capable of large scale inexpensive liquid culture
Should produce spores or other reproductive cell for inoculation into large fermentors.
Must grow rapidly
Produce the desired product in short time period
Should not be pathogenic
Must be amenable to genetic manipulation
Metabolites
Primary metabolites are produced during active cell growth (exponential phase)
Secondary metabolites are produced near the onset of stationary phase
Secondary Metabolites
Growth and reproduction not essential
Dependent on growth conditions
Produced as a group of closely related compounds
Can get overproduction of secondary metabolites
Characteristics of Large-Scale Fermentations
Fermentation: any large-scale microbial process whether or not it is biochemically a fermentation.
Industrial fermentors can be divided into two major classes:
anaerobic processes
aerobic processes (majority of processes)
Industrial Microbiology
Grow organisms
Make products from gene-directed metabolites.
Antimicrobials
Must be produced in large scale industrial fermentors.
Must be purified
Need high yielding strains
Involves mutagenesis of the initial culture
Plating of mutant types and testing of mutants for antibiotic production
If penicillin fermentation is carried out without addition of side-chains, natural penicillins are produced.
A side chain can be added to the broth toproduce desired penicillin: biosynthetic penicillin.
All of these antibiotics are typical secondary metabolites.
Microbial biotransformation employs microorganisms to biocatalyze a specific step or steps in a strictly chemical synthesis.
Brewing and Distilling
Alcoholic beverages are produced by yeast and sugar
Vinegar Production
The active ingredient in vinegar is acetic acid, which is produced by acetic acid bacteria oxidizing an alcohol-containing fruit juice.
Adequate aeration is the most important consideration in vinegar process.
Genetic Engineering and Biotechnology
Biotechnology is the use of living organisms to carry out chemical processes for commercial use.
Genetic engineering is based on molecular cloning, in which a double-stranded DNA fragment from any source is recombined with a vector and introduced into a suitable host.
Cloning vectors include plasmids and bacteriophages.
Definitions
Cloning Vector - Element into which genes can be recombined and replicated
Genetic Map -The arrangement of genes on a chromosome
Genotype -The precise genetic makeup of an organism; both chromosome and plasmids
Molecular Cloning -Isolation and incorporation of a fragment of DNA into a vector where it can be replicated
Shuttle Vector -A cloning vector that can stably replicate in two different organisms.
Expression Vector - a cloning vector that contains the necessary regulatory sequences to allow transcription and translation of cloned genes to express the protein coded by the gene.
Integrating Vector -a cloning vector that becomes integrated into a host chromosome
Nucleic Acid Probe -a strand of nucleic acid that can be labeled and used to hybridize to a complementary DNA molecule
Reporter Gene -A gene incorporated into a vector because the product it encodes is easy to detect
Transfection –The introduction of DNA into mammalian cells.
Reverse Translation -using a codon table and amino acid sequence of a protein to obtain a possible sequence of the mRNA or gene that encoded the protein.
Reverse Transcriptase - enzyme which copies DNA from an RNA template
Repressor Protein -A regulatory protein that binds to specific sites on DNA and blocks transcription
Recombination -The process by which parts or all of the DNA from two separate sources are exchanged or brought together into a single DNA molecule
Wild Type -The bacterial strain isolated from nature
Molecular Cloning
Cloning: purpose is to isolate multiple copies of specific genes in pure form.
Steps in Cloning:
Isolation and fragmentation of the source DNA
Joining the DNA fragments to a cloning vector with DNA ligase
Introduction and maintenance of the clone DNA in a host organism using a vector like a plasmid
Site directed mutagenesis: insertion of synthetic DNA at precisely determined sites in genes in vitro.
Uses of cloned genes
To produce a protein product, like human growth hormone.
To prepare many copies of the gene itself
To determine the gene's nucleotide sequence
An ideal host:
Grows rapidly in an inexpensive culture medium.
Host=nonpathogenic
Capable of taking up DNA
Genetically stable in culture
Has appropriate enzymes to allow replication of the vector.
Prokaryotic Vectors
Plasmid
Bacteriophage
Can be placed in host by transformation
transduction or conjugation
The cloned gene can be harvested or the protein product of the gene can be made and collected.
Transfection of Eukaryotic Cells
Phagocytosis –precipitating DNA for take-up by cell
Electroporation –exposing host cells to pulsed electrical fields in the presence of cloned DNA. The electris treatment opens small pores in host membrane.
Particle Gun –Fires nucleic acid-coated particles into target cells.
Microinjection-Injection of DNA into host using micropipets.
Finding the Right Gene Clone
Special procedures are needed to detect
the foreign gene in the cloning host.
If the gene is expressed, the presence of the gene is detected using hybridization (reaction with specific radiolabeled nucleic acid strand complementary to cloned gene)
Hybridization=nucleic acid probe.
The protein produced by cloned gene can be identified using a tagged antibody
Grow cloned cells in culture. To find colonies/cells with the cloned DNA or protein, use nucleic acid hybridization (DNA) or antibody assay (protein)
Hybridization depends on base-pairing between the gene and a complementary sequence.
The probe or antibody will bond specifically to complementary gene or protein.
Specialized Vectors
Shuttle vector:
Allows cloned DNA to be moved between unrelated organisms (eukaryotic and prokaryotic)
Is a cloning vector that can stably replicate in two different organisms.
Expression vectors:
Vector that can be used to clone the desired gene but can also contain the necessary regulatory sequences to manipulate the expression of the gene
Translation of Cloned Gene
Expression vectors must ensure mRNA translation
Bacterial ribosome binding site; must be engineered into eukaryotic vector
The correct codons must be present either naturally, or by synthetic DNA or site-directed mutagenesis
Eukaryotic Vectors
Eukaryotic Vectors should have:
Origins of Replication-specific sequence that is recognized by specific initiation proteins, including protein that opens up helix
Selectable Marker-factor that allows identification of mutated cells (i.e. antibiotic resistance)
Multiple cloning sites- a short segment of DNA containing many different restriction enzyme cut sites.
Virus vectors or yeast artificial chromosome
Integrating Vectors-designed to maintain clones in very low but stable copy numbers
Reporter genes
Are incorporated into vectors because they encode proteins that are readily detected. Can be used to signal the presence or absence of a particular genetic element or its location. Can also be fused to other genes or to the promoter of other genes so that expression can be studied.
Reverse Translation
Using the amino acid sequence of a protein to synthesize an oligonucleotide probe that encodes it.
The codons that represent the amino acid of the protein are obtained.
The nucleotides that represent the codons are selected for use in designing the probe.
Protein Folding
Cloned gene made to contain a fusion protein in the vector.
Fusion protein can be engineered to contain bacterial signal peptide that enables transport of the clone across the cytoplasmic membrane
Cloned protein is released from the fusion protein by special enzymes
Microbial Evolution and Synthesis
Exam Questions
Study the Working Glossary on page 300
Early Earth
Early Earth was anoxic and much hotter than the present Earth
UV energy was dominant
The first biochemical compounds were made by abiotic syntheses that set the stage for the origin of life.
The first life forms may have been self-replicating RNAs (RNA life).
Primitive Life: Energy and Carbon Metabolism
Primitive metabolism was anaerobic and likely chemolithotrophic (oxidation of inorganic compounds), exploiting the abundant sources of sulfides present
Carbon metabolism may have included autotrophy. (Use of CO2 for metabolite)
Mitochondria and chloroplasts, the principal energy-producing organelles of eukaryotes, arose from the symbioticassociation of prokaryotes of the domain Bacteria within eukaryotic cells, a processcalled endosymbiosis
Mitochondria arose from Proteobacteria
Phylogeny
The phylogeny of microorganisms is their evolutionary relationships.
Comparisons of sequences of ribosomal RNA and differences in nucleotide or amino acid sequence of similar macromolecules are a function of their evolutionary distance.
Ribosomal Database
SSU (small subunit) RNA: the sequencing of the 16S(prokaryote) or 18S(eukaryote) ribosomal unit.
Phylogenetic trees based on ribosomal RNA have now been prepared for all the major prokaryotic and eukaryotic groups.
A huge database of rRNA sequences exists. The Ribosomal Database Project (RDP) contains a large collection of such sequences, now numbering over 100,000.
RNA Sequencing
Ribosomal RNA sequencing involves the amplification of the gene encoding 16S ribosomal RNA, sequencing it, and analyzing the sequence in reference to other bacterial 16S sequences.
16 S subunit is part of bacterial 30 S and 50 S subunits
The evolutionary distance is calculated as the percentage of nonidentical sequences between the RNAs of any two organisms
Domains
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
The third, Eukarya, evolved into the modern eukaryotic cell
Characteristics of the Domains of Life
The domains of living organisms were originally defined by ribosomal RNA sequencing, but 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.
Taxonomy
Bacterial taxonomy places emphasis on analyses of phenotypic properties of the organism.
Taxonomy: The science of classification.
Consists of two major subdisciplines, identification and nomenclature
Phylogeny: evolution of organisms.
Groups of genera (singular: genus) are collected into families, families into orders, orders into classes, classes into phyla (singular: phylum), and so on up to the highest-level taxon, the domain.
Speciation
The model for speciation is based solely on the assumption of vertical (mother to daughter) gene flow.
However, bacterial speciation is also affected to some degree by lateral (horizontal) gene transfer.
Lateral flow is the transfer of genes between species by conjugation, transduction, and transformation
Nomenclature and Bergey's Manual
Following the binomial system of nomenclature, prokaryotes are given genus and species name.
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