restoration of normal gene function by adding/deleting bp near original mutation
Revertant Strain
Strain in which wildtype phenotype lost in mutant is restored
Same Site Revertant
2 mutations occur: 1 cancels the other
Second Site Revertant
1. mutation somewhere else in the gene
2. mutation in other gene can restore enzyme function (ex. a produced coenzyme mutates and now fits mutated enzyme)
3. mutation in other gene can result in another metabolic pathway
Jimmy was trying to isolate a histidine auxotroph. He replica plated his culture on a complete medium and on a minimal medium that lacked only the histidine growth factor. How would he identify his auxotroph?
A. it would grow on the complete medium, but not on the minimal
The original DNA coding strand lookls like this: 5'-ATG-3' (codes for f-Met). The mutated DNA looks like this: 5'-AAG-3' (Lys). What kind of mutation is this?
Base Analogs- chemicals that look like the molecules they substitue
Alkylating Agents- cause change to DNA by alkylating it (ex. Ethidium bromide)
What class of mutation is most common?
A. neutral
Radiation as a Mutagen
a. Non-ionizing: UV light is absorbed by nucleotide bases to form pyramidine dimers (2 adjacent pyramidine bases become covalently bonded)
b. Ionizing: short wavelength such as Xray or gamma rays
Biological Mutagens
a. Transposon mutagenesis
b. Mutations that arise from mutation repairs
Transposable Elements
"jumping genes"
1. Transposase- recognizes, cuts, and ligates DNA
2. Short inverted terminal repeats at end of DNA (10-1000 bp long)
2 Types of Transposable Elements
1. Duplicated when inserted at target sequence
2. Not duplicated at target sequence (can be used to cause mutations if you know the target sequence)
Ames Test
Way to test for carcinogens: Use an auxitroph that requires histidine. Do Kirby Baur test using possible carcinogen on disc. If revertants grow, the substance causes mutations.
Biological Mutagens
1. Mechanism to exise single nucleotide may make a mistake
2. Allelic Exchange- based on genetic recombination. Usually a large chain of DNA. Replace HisG w/ tagged DNA (antibiotic). *Don't have to be equal sized pieces of DNA. Since bacteria is haploid, only one copy of gene needs to be replaced.
Bacteriophage Mutations
sometimes DNA can integrate into host DNA.
1. attB sequence in bacteria genome
2. attP sequence in virus genome
By using integrase, lysogenic phages can incorporate an entire plasmid into bacteria genome
In early Earth, reducing compounds came from:
volcanoes, solar radiation, heat
Liquid Soup Theory
Mimicked atmosphere and found that certain biological molecules could be produced (such as CoA)
Thermal Vent Theory
formation of first cell was on a substrate porous enough to collect needed molecules (mimicked semipermeable membrane)
Insertion sequences and transposons both must have ______ for transposition to take place
D. transposase
Where do we find the evidence for early microbial life?
A. stromatolites
Evidence for Microbial Life
1. microfossils- decayed cells filled with calcium carbonate or silica
2. stromatolites- 3.4 billion years old
3. biosignatures- organic molecules found in sedementary rock. could only be formed by microbes
4. Isotope ratios- ratio of certain isotopes is altered by microbes. ex. microbes fix 12CO2 more readily than 13CO2
Early earth was a reducing environment. Chemicals present:
CH4, NH3, H2, CO2
Amphiphilic molecules as first barrier for biological molecules
First have a bilayer vessicle then a protocell
Metabolist Model of Morowitz & Wachterhauser
CO2 based metabolism originated through self sustaining reactions
-catalyzed by metal sulfides
RNA World Theory
RNA is the first molecule to serve as genetic material. Also served as a catalyst (ex. splicing of introns & exons is catalyzed by RNA)
The first cell was likely:
D. all of the above
Cyanobacteria and early earth
Oxygen was not present in the atmosphere until cyanobacteria. Took 1.6 billion years to go from 0% to 20% atmospheric oxygen.
Endosymbiosis
Mitochondria is like coxiella (very small, similar genome).
Chloroplasts likely came from cyanobacteria
Species definition for microbes
share many properties and differ significantly from other groups/strains
1. 16S ribosome sequence must differ by more than 3% from other organisms
2. 70% or greater genomic sequence variability
Strain
a population of microbes descended from a single individual or pure culture
May be 99.9% similar to test strain
Biovars
Morphovars
Serovars
Biovars- strains that are biochemically or physiologically different
Morphovars- Strains that vary in morphology
Serovars- Strains that vary in antigenic properties
Classical Taxonomy
based on colony morphology, cell shape, etc.
Molecular Classification
1. G&C content. Higher concentration=higher melting temperature. Extract DNA and do melting curves.
2. Nucleic acid hybridization: greater percent hybridization = closer related species
3. Nucleic acid sequences of 16S ribosome
Bergey's Manual of Systematic Bacteriology
1920s. There are now 5 volumes.
International Journal of Systematic and Evolutionary Microbiology
Help name novel species
Phylogeny
Clades
Monophyletic Groups
Clades- groups of related organisms
Monophyletic Groups- Group of species that share a common ancestor that is not shared by others
Internal Nodes on a phylogenetic tree represent...
ancestor
The first primitive organism
B. used RNA both as a catalyst and a unit of heredity
Which of the following gas was not present on early earth?
C. oxygen
Beneficial Interactions between humans and microbes
1. Normal flora
2. Food via digestion (vitamin K)
Parasytic Interactions
Harm us by overstimulating immune system or killing our cells
**Microbes trigger chronic disease by triggering immune response
Host factors in infection
1. age
2. stress
3. diet
4. genetics
Nosocomial Infections
associated with hospitals. Many sick people and bacteria with antibiotic resistance
Steps of Harful Interactions w/ Microbes
1. Exposed
2. Adherence
3. Invasion
4. Colonization & growth
5. Result
Exposure
microbe must be able to penetrate barriers (skin, mucous membrane, etc.)
Adherence
Receptor mediated (specific)
ex. N. gonorrhea in urogenital epitherlia has CD66 which binds to opa
-capsule, fimbrae, or pilli may be involved (bind to host cell glycoproteins)
Invasion
penetrate epithelium to initiate pahtogenesis
*if gets in blood or lymph, can get in any other part of body
Proteins for invasion by microbe
ex. Hyaluronidase (breaks down hyaluron in connective tissue)
ex. Colleginase (breaks down colligen)
*allows deeper tissue invasion
S. aureus: function of coagulase and kinase
forms clot around microbe so that antibodies can't detect it
Colonization & Growth
must have appropriate environmental conditions & nutrients
Trace Elements In Human Body
Iron is difficult for microbes to obtain.
Transferrins & lactoferrins keep iron in host body.
Siderophores are used by microbes to take in iron
Bacteriemia vs Septicemia
Bacteremia- presence of transient bacteria in blood
Septicemia- blood poisoning. Presense of pathogens in blood
Virulence Factors
genes involved in pathogenesis, aid in establishment and maintenence of disease
Invasiveness
ability of organism to grow in host tissue in large number that inhibits host function
ex. S. pneumoniae: hemolysis inhibits host lung function
Exotoxins
proteins that cause damage (at site or travel to other parts of body)
AB Toxins (exotoxins)
A=active part (inserted into cell to cause damage)
B=binding part
Cholera exotoxins
AB toxin disrupts electrolyte balance to cause diarrhea and frothing at mouth
-toxin affects adenyl cyclase so that ATP -> cAMP causing Cl- to move to lumen and Na+ movement is blocked. Water moves into intestinal lumen and does not get taken back in.
Diptheria exotoxins
Disrupts protein synthesis by ADP riboslyation of elongation factor 2
-toxin binds to epithelial layer of intestinal tract. Without protein synthesis, cells begin dying.
Virulence refers to:
B. the degree or intensity of pathogenicity
Organisms that are ubiquitously found in nature, but that cause disease only in individuals whose defenses are somehow debilitated are called ____ pathogens
C. opportunistic
Where would you expect to find the lowest level of microbes?
D. the kidney
Which of the following does NOT describe one of Koch's postulates:
E. characterize the gram staining characteristic of the isolated agent
Which of the following is not an adhesion factor for pathogens
A. flagella
Endotoxins
bind to macrophages and B cells
-LPS releases TNF-alpha, interferon, & cytokines
-symptoms: fever, activation of clotting factors, clots in arteries, low blood pressure, shock, death
Bacteria in Blood: endotoxins & antibiotics
If antibiotics kill bacteria in blood, could make things worse. Bacteria can release more endotoxins.
Endotoxins vs exotoxins
Need a lot of endotoxins for disease (micrograms of it) but only need a little bit of exotoxins (picograms)
