MT1 Flashcards
Bacteria size
Very small ( like 1 by 2 microns)
Microbiology definition
The study of organisms too small to be seen clearly with the unaided eye
Bacteria type (pro or eukaryotes?)
Bacteria are prokaryotes whose shapes are determined by a uniquely structured cell wall
Prokaryote
Do not have a membrane-bound nucleus or membrane-bound organelles
Bacterial metabolic functions
Metabolic functions in bacteria occur within different areas inside the same compartment
- the cell membrane contains enzymes for generating ATP from aerobic respiration
- bacteria set up proton gradients for ATP synthesis by translocating H+ out of the cell (very similar to how eukaryotic mitochondria set up proton gradients for ATP synthesis by translocating H+ out of mitochondria
Nucleoid
Means like a nucleus, is specialized for information storage, processing and distribution
- typical bacterial genome is 6 to 10 Mbp
- nucleoid is essentially analogous to eukaryotic nucleus
Bacterial RNA
Bacteria have a single type RNA polymerase in their cytoplasm
Bacterial ribosomes
Bacteria have 70S ribosomes in their cytoplasm
- eukaryotes have slightly larger 80S ribosomes in their cytoplasm
- the 70S ribosome in bacteria are essentially analogous to the 70S ribosomes in mitochondria and chloroplasts
- transcription and translation in bacteria are not always separated in time and in space and can occur in tandem
Prokaryote biomass
The total biomass of prokaryotes vastly exceeds the total biomass of eukaryotes
Gram positive Rogues gallery bacteria
Micrococcus… Micrococcus luteus
Staphylococcus… Staphylococcus epidermidis
Streptococcus… Streptococcus lactis
Endosperm formers… Bacillus cereus
Micrococcus
Gram positive Spherical bacteria Obligate aerobe Common in soils and on human skin Almost always brightly pigmented - ex) Micrococcus luteus = yellow
Staphylococcus
Gram positive
Facultative aerobe
Spherical bacteria that grow in clusters
Simple nutritional requirements (compared to strep)
Streptococcus
Gram positive Spherical bacteria that grow in strips Aerotolerant aerobes Very complex nutritional requirements - streptococcus lactis is used in the production of natural cheeses
Endospore formers
Aerobic endospore forming rods
- Bacillus cereus
Gram negative Rogues gallery bacteria
Pseudomonads… Pseudomonas putida
Enterics… E. Coli
… Enterobacter aerogenes
Pseudomonads
Obligate aerobic rods
- non fermenters utilize oxygen as the terminal electron acceptor
Most are motile via polar flagella
Gram negative
Enterics
Gram negative bacteria
Small, non spore forming rods
Facultative aerobes
Simple nutritional requirements
Usually motile via peritrichous flagella
- ferment glucose under anaerobic conditions
Mixed-acid fermenters and butanediol fermenters
-
Mixed-Acid Fermenter, Enteric bacteria
Mixed acid fermentation of glucose produces large amounts of acid
- Escherichia coli (E. coli) = travelers diarrhea
Butanediol fermenter, enteric bacteria
Butanediol fermentation of glucose produces non-acidic components
- Enterobacter aerogenes
Rogues gallery bacteria found in soil
Pseudomonas putida
- gram negative obligate aerobic rods
Bacillus cereus
- gram positive facultative aerobic endospore forming rods
Rogues gallery bacteria found in food
Streptococcus lactis
- gram positive aerotolerant anaerobic spheres
Rogues gallery bacteria on human flora (=on or in humans)
Skin
- Micrococcus luteus
- gram positive obligate aerobic spheres
- Staphylococcus epidermidis
- gram positive facultative aerobic spheres
Enteric bacteria
- Enterobacter aerogenes
- gram negative facultative aerobic rods
- Escherichia coli (E. Coli)
- gram negative facultative aerobic rods
Distinguishing rogues gallery bacteria in lab (gram positive)
-tests and results
- Gram test
- positive= purple - Shape
- rod or sphere
3a. Spore stain if rod
- pink rods with green spores… BACILLUS CEREUS
3b. Catalase test if sphere
- Catalase positive= fizz, wombat spit
- Catalase negative= does negative
4a. If positive, plate on YGC
- lemon yellow, no zone of clearing= MICROCOCCUS LUTEUS
- white colonies, zones of clearing= STAPHYLOCOCCUS EPIDERMIDIS
4b. If negative= STREPTOCOCCUS LACTIS, but plate on YGC and get small colonies, huge zone of clearing
Distinguishing rogues gallery bacteria in lab (Gram negative)
- tests and results
- Gram Test
- negative= pink - Oxidase test
- oxidase positive= royal Thai blue
- oxidase negative, no color change within 10 seconds
3a. If positive, plate on F Agar
- fluorescence= PSEUDOMONAS PUTIDA
3b. If negative, perform IMVIC Test
- Goes (-,-,+,+)= no red ring, yellow, red over yellow, Malibu blue = ENTEROBACTER AEROGENES
- Goes (+,+,-,-)= red ring, red, no color, red woods green = E. COLI
In the case of a fire…
Turn off all Bunsen burners, edit lab, turn right, use the south stairwell, regroup with the rest of lab outside of the building in the gravel paved bike rack area between Sci lab building and Sci Lec.
In the event of an earthquake…
Turn off all Bunsen burners, get underneath lab bench or doorway, important to exit building as soon as ossicle because a stronger motion may shortly follow, therefore once the motion ceases immediately exit lab, turn right, south stairwell regroup outside in gravel paved area in between Sci Lab and sci lec
Biological spill procedure
- Liquid spill with broken glass
- Liquid spill (ignore glass part, same procedure)
- Tell everyone about both the broken glass and spill
- Block the spill area with a stool or stools
- Lab supervisor will bring clean up material
- Decontaminate spill area with Bacdown
- TA or Dr. Mann will clean up glass
- Cover entire spill with paper towels
- Pour approximately same volume of Bacdown as the spill onto the paper towels
- Soak area for 30 min
- After 30 min, put on gloves and use the things to pick up the paper towels. Place paper towels in autoclave bag
- Use broom and dustpan to sweet residual broken glass
- Place tongs in Rubbermaid autoclave bus pan
- Use additional paper towels to dry up the spill area
Biological spill procedure
- Dry spills
- Tell everyone about spill
- Block spill area with stool or stools
- Lab supervisor will bring clean up materials
- Put in gloves and carefully pick up petri plate lid and bottom, put in autoclave bag
- Cover spill area with paper towels
- Pour Bacdown onto spill area covering it
- Allow to soak for 30 min
- Out on gloves and use tongs to pick up paper towels, lace in autoclave bag. Tongs in Rubbermaid autoclave bus pan
- Use paper towels to dry up the area
Lab Waste Carts
Oscar - used petri plates
Stainless steel bus pan - used glassware
Stainless steel pitches - used sharps (slides, cover slips, microtiter pipettes tips, swabs, toothpicks)
Plastic bus pan - serological pipettes
Streaking procedure
- how many times do you sterilize inoculating loop
4
The Germ Theory
- who proposed it
- studying what when came up with it
Louis Pasteur
Studying Lilie Alcohol Industry, something wrong with beet juice fermentation
- “healthy vats” had lots of alcohol, contained lots of yeast
- “sick vats” lots of gray slime, contained lots of bacteria
- Pasteur proposed the yeast were responsible for the fermentation that produced alcohol and the bacteria in the sick vats were responsible for a different type of fermentation that produced lactic acid
Spontaneous Generation
Thought that microbes arose spontaneously, most scientists didn’t believe this
Pouchet said to have proof, Pasteur proved him wrong
- used 3 different flasks to prove the “vital essence” didn’t cause microbes to grow
Pasteurization
A process developed by Louis Pasteur to kill the bacteria in wine without harming the wine
- slow heatinga t approx. 63 C
- maintained 30 min
Proof that different diseases of wine are caused by bacteria
Germ Theory
- guidelines
- Specific microbes have specific metabolic processes
- Microbes are present in the air
- Specific microbes can cause specific diseases in wine
Robert Koch
Published first proof of Pasteur’s germ theory
- a specific microbe was shown to cause a specific disease
- a microbe from pure culture was shown to cause a specific disease
Invented bacterial pure culture technique
Pasteur and vaccinations
Developed first ever vaccine, to anthrax (chickens)
Developed first ever vaccine for humans, rabies
Koch’s Postulates
Outlines that prove germ theory for human disease
- The microbe must be found in a diseased animal but not in a healthy animal
- The microbe must be isolated and grown in pure culture
- The microbe from this pure culture must produce the same disease when inoculated into healthy animals
- The microbe must be re-isolated from these newly diseased animals
How are bacteria grown in the lab?
In a closed system, a batch culture, no fresh medium is added, therefore nutrients will be depleted and waste products will accumulate
If the growth or an in vitro population is plotted out, there are how many distinct phases characteristic of the growth curve? What are they?
There are four distinct phases
- lag phase= period during which bacteria adapt to their new environment
- logarithmic phase (exponential)= period during which bacteria undergo rapid binary fusion
- stationary phase= period during which death rate equals growth rate
- death phase= period during which death rate exceeds growth rate
Lag phase
Period durning which bacteria undergo rapid binary fusion
No immediate increase in cell mass or numbers
Highly variable time wise
- new cell component and ATP synthesis
- microbes may need to “re-tool” for new medium
Logarithmic phase
Period during which bacteria undergo rapid binary fusion
Maximum growth possible for a particular bacterium under a given set if conditions
- constant rate of growth
- culture is at its most uniform
- best time for biochemical tests (i.e. Identification)
Stationary phase
Period during which death rate equals growth rate
Population growth ceases
- no cell divisions
- nutrient/O2 depletion and accumulation of waste products
Death phase
Period during which death rate exceeds growth rate
Growth curve goes downward
What is the generation time? How is it determined?
It is the doubling time
- the interval between successive binary fusions
- generation times are determined during exponential growth?
What is pure culture
A population of cells arising from a single cell
What is a colony
A macroscopically visible growth or cluster of microbes on a solid medium
- each colony represents a pure culture(or clone)
Colony morphology can sometimes aide in identification of bacteria
- bacteria at the edge of the colony are happy, those in the middle aren’t
What is cryptic growth
The number of cells grown equals the number of cells that die
What are the two types of culture media
Defined(synthetic) media
Undefined (complex or enriched) media
Defined media
This is synthetic media
- all components are known
- they are expensive and a major pain to make
Undefined media
Complex or enriched media - actual chemical composition unknown - contains undefined stuff like: - yeast extract (autolysed yeast) - peptone (protein digest) - blood or serums - beef heart extract most bacteria of medical and industrial interest are quite happy growing in inexpensive, easy to make complex media
What are the pure culture plating techniques
Streak plate isolation
Spread plate isolation
Pour plate isolation
Streak plate isolation
Bacteria are transferred from plate to plate or liquid to liquid using a heat sterilized inoculating loop
- once mastered, streaking is simple and reliable
Spread plate isolation
Bacteria in suspension are pipetted onto a plate and spread uniformly across the plate with a sterile “hockey state” spreader or with a sterile swab
- it is best for moe uniform results when dealing with large numbers of samples
Pour plate isolation
Bacteria in suspension are added to approximately 20 ml of molten agar! which is then outed into a petri plate
- allows detection of both aerobic bacteria (on top of the agar) and microaerophillic bacteria (in the agar)
Psychrophiles
Cold loving bacteria
Obligate Psychrophiles
= has to be in cold
- cannot grow about 20 C
- common in arctic, Antarctic, mountains, and ocean floor
- membranes have lots if unsaturated fatty acids (helps resist freezing)
Facultative Psychrophiles
= prefers to be in cold
- grows best at or below 20 C but can grow at higher
- common in your refrigerator
Mesophiles
- optimum 25-40C
- most bacteria
- most pathogens
Thermophiles
Heat loving bacteria
Facultative Thermophiles
- grow best above 37C but can grow at cooler
- common in compost heaps and “cooler” hot springs
Obligate thermophiles
- cannot grow below 37C
- optimum 50-60C
- common in hot water lines (heating plants, power plants, nuclear reactors), hot springs
- membranes with lots of saturated fatty acids (resists membrane to stick together)
Hyperthermophiles
- optimum near 100C
- archaeobacteria neer deep ocean vents
Obligate aerobes
Requires O2 for growth
Facultative aerobes
Do not require O2 but grow better in its presence (grow better under aerobic conditions)
Microaerophiles
Require small amounts of O2 but are damaged by higher (atmospheric) O2 levels
Aerotolerant Anaerobes
Ignore O2
Facultative anaerobes
Do not require O2 but grow better in its presence
Obligate anaerobes
Do not tolerate the presence of O2
How is bacteria controlled
Sterilization
Disinfection
Sterilization
- the elimination or removal of all living entities
- an absolute term
Disinfect
The elimination or removal of contaminating microbes
- disinfectant = toxic chemicals or agents that eliminate or remove disease-causing microbes from an inanimate object
- antiseptics = usually non toxic chemicals or agents that eliminate or remove disease causing bacteria from the skin or tissue
What are the physical methods of control
Heat, filtration, irradiation , chemicals
Heat as a control for bacteria
Heat denatures microbial proteins
- Direct flame= oldest! most direct form of sterilization
- Dry heat Sterilization= 160-170C for 2-3hours, how labs sterilize glassware
- boiling water = cannot eliminate spores and so cannot sterilize! but conveniently low tech
- autoclave= like a pressure cooker, 250F 15psi 15min
- how most labs sterilize glass, and how clinics
Filtration
Microbes are removed from heat sensitive stuff via microfiltration
- pharmaceuticals, cell culture media, wine, beer (cold filtered)
Types of filters
- membrane filters (made of cellulose acetate, cellulose nitrate, polycarbonate)
- high efficiency particulate air filters (HEPA) (air filters off sterile hoods, hospital operating rooms, industry sterile rooms, and aircraft air conditioning systems
Irradiation
Uv light (200nm)
- induces thymine-thymine dimers in DNA
- used for lab surfaces and small instruments
- uv light in sunlight can disinfect clothes dried outside Ina. Clothesline
- limited use because it doesn’t penetrate glass, cloth, nor paper
Chemical methods of controls
Phenol and phenolics
- disrupt cell membranes and denature proteins
- ex) Lysol, Listernine
Halogens
- oxidize cell components
- ex) bleach iodine, iodophor
Alcohols
- disrupt membranes and allow microbes to be washed away
- ex) ethanol, isopropanol
Soaps and detergents
- disrupt membranes and allow microbes to be washed away
- ex) soaps= ivory , Castile; detergents= dial, Bacdown, Tide
Why shouldn’t you vortex your Pseudomonas tube during Lab 2
Pseudomonas is tile and you’re using its motility to isolate it from all of the other bacteria in the soil that aren’t motile. So keep those un motile bacteria at the bottom
What does the isolation of pseudomonas rely on
It relies on Enrichment to allow that bacterium to come to dominate the sodium Benzoate medium. Not many bacteria besides pseudomonas can grow on sodium Benzoate medium. Lying on motility f pseudomonas in the sodium Benzoate liquid
What does the direct isolation of Bacillus rely on
It relies in the ability for Bacillus spores to resist heat. Since we incubated the plates under aerobic conditions, clostridium spores which germinated could not grow.
Why is the cell wall a critically important structure
It confers shape and protection from osmotic lysis
What is peptidoglycan
Enormous polymer
- two sugar derivatives (NAG and NAM)
- tetra peptide side chains (listed in order)
1. L-Alanine
2. D-Glutamic acid
3. DAP (mesodiaminopimelic acid)
4. D-Alanine
Composed of a backbone of alternating NAM and NAG
- tetra peptide side chains attached to NAM
Transpeptidation results in a large, strong, flexible, network like molecule
- tetrapeptide side chains on separate backbones are linked
Peptidoglycan synthesis
Chains of peptidoglycan subunits are joined by a reaction termed Transpeptidation
- peptidoglycan precursors have pentapeptide side chains
… Peptidoglycan in a cell wall has tetrapeptide Side chains but during “construction” it is pentapeptide
Transpeptidation links
- the sub terminal (next to last) D-Alanine on the pentapeptide side chain of one peptidoglycan chain
… With
- the DAP of the pentapeptide side chain of an adjacent peptidoglycan chain
- removal of the terminal D-Alanines is exergonic and “powers” the Transpeptidation reaction (which occurs outside the cytoplasm)
- Transpeptidation results in a large, strong, flexible, network like molecule
Overall structure of gram positive bacteria
Thick peptidoglycan layers (that’s it, like a water balloon inside a shoebox)
Overall structure of Gram negative bacteria
Outer membrane (inside the cell wall) Thin peptidoglycan layer Periplasm (area between the outer membrane and cell membrane)
Gram positive cell walls
Thick cell wall composed mostly of peptidoglycan
- but may also contain Teichoic acid
- Teichoic acids are only found in gram positive bacteria
Tetrapeptide side chains are linked by peptide interbridges composed of several amino acids
Gram negative cell walls
Thin cell wall composed entirely of peptidoglycan
Tetrapeptide side chains are directly linked
Outer membrane
- it is much less permeable than the cell membrane
- porin proteins join to form transmembrane channels
- porins give gram negative bacteria control over the entry of toxic substances and antibiotics
- it contains Lipopolysaccharides (LPS)
- LPS are found only in the outer membrane of gram negative bacteria
Periplasm
- the area between the outer membrane and cell membrane
- gel like matrix containing hydrolytic enzymes and binding proteins
- initial degradation of nutrients (and antibiotics)
- transport of stuff
Capsule
Diffuse layer outside of the cell wall
- helps resist phagocytosis
- excludes bacteriophage
- protects from desiccation
Usually composed of polysaccharides
- can be homopolymers
Some capsules are composed of polypeptides
- the amino acids can be D stereoisomers
- D stereoisomers are never found in proteins on this planet
Biofilms
Matrix outside of the cell wall
- bio films are populations or ccommunites of microbes attached to a surface and contained within an extra cellular polysaccharide matrix
Matrix formation only occurs when a minimum number (Quorum) of bacteria have colonized a surface
The Cell membrane
Fluid mosaic model similar to eukaryotic cell membrane
- usually lacks sterols
All singing, all dancing structure in bacteria
- site of respiration, photosynthesis, and biosynthesis
- important during cell division
- new cell membrane synthesis substitutes for the spindle apparatus to separate bacterial chromosomes
Internal structures
Cytoplasm
- 4/5 water, 1/5 stuff (enzymes, lipids, proteins, carbs)
Ribosomes
- bacteria have a 70S ribosome
- 30S small subunit (21 proteins + 16S rRNA)
- 50S large subunit (34 proteins + 23S rRNA and 5S rRNA)
Nucleoid
- chromosome
- most bacteria have a single circular, supercooled, double-stranded DNA chromosome in an irregularly shaped area
- Plasmids
- circular,double stranded DNA molecules in the cytoplasm
- can exist and replicate autonomously
- can integrate with the bacterial chromosome
- confer intriguing advantages
- F factors encode for conjugation, a sexual process
- R factors encode for drug resistance
Gas vesicles
- provide buoyancy for (usually) photosynthetic bacteria
Flagella
Thread like locomotion appendage
- positioning is frequently species specific
- monotrichous bacteria (=one hair) have a flagellum at one end of the cell
- amphitrichious bacteria (=both hairs) have a flagellum at both ends of the cell
- lophotrichous bacteria (=tufted hairs) have clustered flagella (usually clustered at one end, like Bunsen and beaker)
- peritrichous bacteria (=surrounding hairs) have numerous flagella covering the cell
Monotrichous bacteria
Have a flagellum at one end of the cell
Amphitrichious bacteria
Have a flagellum at both ends of the cell
Lophotrichous bacteria
Have clustered flagella
Peritirichous bacteria
Have numerous flagella covering the cell
Three components of flagella
Filament
Hook
Basal structure (gram positive lack L and P rings
- L ring (Lipopolysaccharide)
- P ring (peptidoglycan)
- MS ring (membrane and supramembrane)
- C ring (cytoplasm)
- the MS and C rings function together as a reversible turbine rotor
- motor proteins (Mot A and Mot B) form the stator
- L and P rings seem to serve as bearings
Fimbria
Short, hair like appendages
- composed of helically arranged proteins
- aid in attachment to surfaces
(Fimbria and pills are structurally similar, pilli tend to be longer)
(Fimbria are important for pathogenicity factors)
Heterocysts
Heterocysts are “different looking” cells in filamentous Cyanobacteria that are specialized for nitrogen fixation
- nitrogen fixation is the conversion of atmospheric nitrogen (N2) to ammonia (NH3)
Nitrogen fixation involves the oxygen sensitive enzyme nitrogenase
NH3 (Ammonia) is exported to adjacent cells
- Photosynthetic products are imported from adjacent cells
Endospores
Endospores (spores) are resistant, dormant stuctures found in some gram positive bacteria (and only in gram positive bacteria)
- spores are characteristic of Bacillus and clostridium
Spores are highly resistant structures
- resist heat, UV, chemical disinfectants, desiccation
- can last for a very long time (possibly millions of years)
Spores have important medical and industrial implications
- they pose serious contamination and sepsis problems
Bacterial genome gene pools
Core been pool (80-99% of bacterial genes)
- genes that are part of a bacterium’s chromosome
- homogenous G + C content (similar G+C content implies similar DNA and similar taxonomic relatedness)
- encode for essential proteins involved in structure and protein
Flexible gene pool (01-20% of bacterial genes)
- blocks of genes (“genomic islands”) that are bacteriophage-derived, plasmid-derived, or transposon-derived
- 10-100 Kbp clusters of genes with related functions
- pathogenicity islands (PAI)
- very different G+C content from Core Gene Pool
- encode for non essential proteins that may confer the bacterium with specific advantages under different circumstances
Core Gene Pool
Most bacteria have a single circular chromosome
Bacterial chromosome size ranges from ~580Kbp to ~10Kbp
- genome size is species specific
The ae. coli genome is composed of 4640000 base pairs
- 88% of the E. coli genome consists of ~4000 structural genes
- 10% of the genome consists of regulatory sequences
- origin of replication (oriC, 84 min)=where hi directional chromosome replication originates
- terminus of DNA replication (terC, 31 min) = a sequence at which chromosome replication terminates
- promoters (= tell RNA polymerases where to attach to begin transcribing)
- operators (sequences to which depressor molecules bind to prevent RNA polymerases from attaching to a promoter and hence prevent transcription of a gene)
- 01% consists of genes for tRNA and rRNA
- 0.5% consists of non coding repeating sequences
Flexible gene pool
Transposable elements= small pieces of DNA that can move from place to place within the chromosome
- ex) insertion sequences= contain the gene for transposase (the enzyme that inserts transposable elements)
- IS2 (insertion sequence 2)
- ex) transposons= contain the gene for transposase; contain at least one other gene in addition to transposase
- Tn3 (transposon 3)
Plasmids
Are small, circular DNA molecules that can exist independently from the bacterial chromosome whole giving the host bacterium unique functions
- many plasmids can integrate into the chromosome
- integration is the enzyme mediated incorporation of a plasmid into the bacterial chromosome
- excision is the enzyme mediated escape of a plasmid from the bacterial chromosome
- many plasmids can replicate autonomously
- such plasmids have their own origin of replication(ori)
- plasmids can vary in size
- other important plasmids
- resistance factors (R factors) -> code for antibiotic resistance
- virulence plasmids -> code for antibiotic resistance and toxins
Bacteriophage
Biological entities containing either DNA or RNA (never both) that are obligate intercellular parasites of bacteria (=bacteria eater)
- phage are viruses that infect bacteria
- phage are composed of
- genome (=Gene set) -> can be either DNA or RNA
- capsid= protein shell that protects the genome
Replication
- phage attaches to host bacterium and injects its genome
- phage nucleic acid commandeers bacterial transcription, translation and replication machinery
- bacterium is “retooled” to
- transcribe phage nucleic acid into phage mRNA
- translate phage mRNA into phage proteins
- replicate phage nucleic acid
- phage proteins assemble into capsids
- phage nucleic acid is inserted into capsids
- phage lyses host bacterium to release progeny phage to attack other bacteria
Bacterial gene transfer
Transformation
Transduction
Conjugation
Transformation
The uptake of free DNA and the incorporation of this DNA into the genome
- the ability of a bacterium to be transformed is termed competence
Artificial transformation
What’s done in cell and molecular labs
Some Bacteria can be induced to become artificially component and undergo transformation after some specific treatment
- usually involves circular DNA (plasmid DNA)
- the bacterium takes up an extrachromosal genetic element
Natural transformation
Many (possibly most) bacteria become naturally competent and undergo transformation at some point in their cell cycle
- can involve linear DNA (chromosomal segments) or circular DNA (plasmids)
- the bacterium takes up chromosomal segments or plasmids
Why do bacteria bother with transformation?
It may be a large scale DNA repair mechanism
- kind of like how you’d borrow a friends notes if you missed a lecture
It may be a mechanism for exploring the “fitness landscape”
Conjugation
Conjugation is gene transfer and recombination that requires direct cell to cell contact
Conjugation is a primitive but effective sexual process
F-
Bacterium without an F Factor
F+
Bacterium with an extrachromosomal F Factor
Hfr
High frequency recombinant bacterium with an integrated F factor
F’ (F prime)
Bacterium with a formerly integrated F factor that has excised somewhat sloppily and now contains some genes from the bacterial chromosome
What happens when you cross F+ x F-?
F+ x F- = F+ and F+
The F factor has been transferred
What happens when you cross Hfr x F-
Hfr x F- = Hfr and F-
- the entire F factor (which is integrated into the chromosome) is rarely transferred
- large numbers of Donor bacterial genes are transferred (hence the term “high frequency recombinant” (Hfr)
- transfer of bacterial genes is sequential
- transfer of the entire genome would take about 100 min
- to bacteria don’t typically stay in contact for 100 min
- Hfr genes (the integrated F factor) are the last genes to be transferred
- so F- is rarely converted into an Hfr
What do you get when you cross F’x F-?
F’ x F- = F’ and F’
The F factor (F’) has been transferred and some Donor bacterial genes are transferred, too (it’s an F’)
Transduction
It is the transfer of genes from one bacterium to another bacterium via a DNA bacteriophage (and ONLY DNA bacteriophage)
- transduction is a DNA only phenomenon
DNA bacteriophage replication
Phage attaches to bacterium and injects its DNA
Phage DNA commandeers bacterial replication, transcription and translation mechanisms
- bacterium is “retooled” to
- transcribe phage DNA into phage mRNA
- translate phage mRNA into phage proteins
- replicate phage nucleic acid
- phage proteins assemble into capsids
- phage DNA is inserted into capsids
- phage lyses host bacterium to release progeny phage to attack other bacteria
Transduction is basically what?
A packaging mistake
- most phage package DNA based on length, not specific sequences
- bacterial DNA can be packaged into the phage capsids
- so some progeny phage may contain bacterial genes
- when such progeny phage infects another bacterium it will inject bacterial genes
What is the most utilized mode of gene transfer?
Transduction
Who was the first physical to administer tuberculin
Ehrlich
What does chemotherapy mean
We must learn to shoot microbes with magic bullets
What is sleeping sickness caused by
Trypanosomes
What is the significance of Sahachiro Hata?
He knew how to give rabbits Syphilis, and the syphilis spirochete was thought to be related to trypanosomes
- so it seemed logical to test the Atoxyl (previously failed by British scientists and Ehrlich) against Treponema pallidium
- Atoxyl derivative 606 was effective, named Salvarsan
- improved formula 914, neosalvarsan, was used to treat syphilois until penicillin
Antibiotics
Metabolic products of organisms that inhibit other organisms
- antibiotics are naturally produced by many microbes
- inhibit other organisms so there’s less competition for food
Semi synthetic antibiotics
Natural antibiotics that have been chemically modified to make them less susceptible to breakdown by bacteria
Chemo therapeutic agents
(Ehrlich’s definition) organic and inorganic chemicals that are used within the body for eliminating or limiting the growth of microbes
- anti biotic and chemo therapeutic agent are relatively interchangeable, Dr. Mann uses antibiotic
Selective toxicity of antibiotics
Antibiotics just control or eliminate the microbe without haring the patient
Therapeutic dose/effective dose
The level that if maintained over a period of time will result in the control or elimination of a bacterium
Toxic dose/tolerable dose
The level that if maintained over a period of time will silt in damage to the host
Therapeutic index
The ratio of toxic dose to the therapeutic dose
- the larger the number, the less toxic and more effective the agent
Bacteriostatic
Reversible inhibits the growth of a specific bacterium
- rely on the immune system to take up the slack once they’ve forced the bacteria into an “induced” stationary phase
(Literally means to stop)
Bactericidal
Kills specific bacteria
- most bactericidal antibiotics are bacteriostatic at lower concentrations
Minimum inhibitory concentration (MIC)
The lowest concentration of an antibiotic that prevents the growth of a specific bacterium
- MIC is the lowest concentration that is bacteriostatic
Minimum lethal concentration
The lowest concentration of an antibiotic that kills a specific bacterium
-MLC is the lowest concentration that is bactericidal
Narrow spectrum antibiotics
Effective against a limited variety of bacteria
- this can be either a small number of bacteria or a single taxonomic group
Broad spectrum antibiotic
Effective against a wide variety of bacteria
- this is generally a large number of bacteria, frequently including both gram negative and gram positive bacteria
Is the effect, effectiveness and spectrum independent or dependent of an antibiotic’s therapeutic index?
Independent. The higher the therapeutic index the better
Antibiotics that inhibit cell wall synthesis
The integrity of a Bactria depends on their rigid cell walls. Animals do not have cell wall
- Beta-Lactams
- ex) penicillins, cephalosporins
- glycopeptides
- ex) vancomycin
Beta Lactams
Examples= penicillins, cephalosporins
- inhibit Transpeptidation by binding to penicillin binding protons (PBPs) and preventing their Transpeptidase activity
Penicillins
Inhibit cell cell wall synthesis
- Beta Lactams
- bactericidal, narrow spectrum (gram positive cocci)
- streptococci, meningococci, pneumococci
- most (not all) must be given by injection
- excrete via kidneys and remain active in the urine
- suitable for many urinary tract infections
Cephalosporins
Inhibit cell wall synthesis Bacticidal, broad spectrum Beta Lactams - can be given orally or by injection - some are metabolized to less active compounds by the liver but all are excreted via the kidneys
Glycopeptides
Inhibit cell wall synthesis
- ex) vancomycin
- inhibit Transpeptidation by binding to D-Alanine of pentapeptide side chains of peptidoglycan precursors (Gram positive)
- bactericidal. Narrow spectrum (gram positive)
- but EXTREMELY important against Staphyloccal infections (particularly Methicillin-resistant Staphylococcus aureus (MRSA))
- can be administered orally or by injection
- ototoxic (ear damage) and nephrotoxic (kidney damage)
- excreted via kidneys
Antibiotics that inhibit protein synthesis
Bacteria have 70S ribosomes. Animals have 80s ribosomes in their cell cytoplasm (and their 70s ribosomes are sequestered in mito)
- aminoglycosides (ex - streptomycin, neomycin)
- tetracyclines
- macrolides
Aminoglycosides
Inhibits protein synthesis
- ex) streptomycin, neomycin, kanamycin, gentamycin
- binds to the 30s small subunit and inhibit the binding of fMet-tRNA and the formation of an initiation complex
- bactericidal, narrow spectrum (gram negatives)
- must be administered by injection
- ototoxic and nephrotoxic
- excreted via kidneys
Tetracyclines
Inhibit protein synthesis
- ex) tetracycline, chlorotetracycline, oxytetracycline
- bind to the 30s ribosome small subunit and prevent the binding of new tRNAs to the ribosome acceptor site (“A” site)
- bacteriostatic, hora deer spectrum of any antibiotics
- effective against gram positives and gram negatives
- can be administered orally or by injection
- ototoxic, nephrotoxic, and hepatoxic (liver damage)
- tetracyclines interfere with bone development and shouldn’t be given to pregnant women
- excreted via the kidneys
Macrolides
Inhibit protein synthesis
- ex) erythromycin
- binds to the 50s large subunit and blocks transcription as the release of tRNA after peptide broad formation
- bacteriostatic, relatively broad spectrum
- can be administered orally or by injection
- very high therapeutic index, one of the least toxic antibiotics
- concentrated in the liver and excreted in the bile
- powerful for penicillin resistant infections
Antibiotics that inhibit nucleic acid synthesis
Bactria have unique modes of DNA of RNA sue this that involve pathways and or different enzymes
- sulphonamides
- rifamycins
- quinolones
Sulphonamides
Inhibit nucleic acid synthesis (=sulfa drugs)
- ex) sulfanilamide, sulfamethoxazole
- interfere with frolic acid synthesis
- bacteria and Protozoa need to make folic acid so th can synthesize purines and pyrimidines. Animals get folic acid for nucleic acid synthesis from their diet
- bacteriostatic, broad spectrum
- usually administered orally
- metabolized by the liver and excreted via kidneys
- minor problems with allergies (hives and rashes)
- major problems with resistance
Rifamycins
Inhibit nucleic acid synthesis
- ex) rifampin, rifampicin
- binds to RNA polymerase and blocks the synthesis of mRNA
- bactericidal, broad spectrum
- oral administration
- metabolized by the liver and excreted in the bile
- they are red and turn excretions orange
- rifamycins are useful for (and their use in the US is only approved for)
- treating tuberculosis And leprosy
- eliminating meningococci from carriers
Quinolones
Inhibit nucleic acid synthesis
- ex) naldixic acid, ciprofloxacin
- Inhibit the activity of DNA gyrase and prevent DNA supercoiling
- bactericidal, broad spectrum
- can be administered orally or by injection
- excreted via the kidneys
- effective for treating UTIs and travelers diarrhea
