Microbiology 303 Exam 3

What is selection? How can you use to find a desired strain?

Media conditions are set up such that only the mutants of interest are able to grow i.e. Adding the antibiotic rifampicin causes only rifampicin resistance bacteria to grow

What is pCRR?

Put the DNA sequence in a special plasmid that has promoter and you can express the gene Will often leave a one extra base—stick end --TOPO isomerase will bind to the end

What happens between Hfr and F- cells?

The Hfr will form the pore between the F- cell --Integrated plasmids can facilitate genome movement --Doesn't usually transfer entire chromosome Hfr—high frequencies of changing phenotypes

How do transposons cause mutations?

--Disrupt genes causes a mutation and could kill host --Have transcription and translation stop sites near ends Selectable markets (drug resistance) --Make them easy to find in the genome --Safe for humans to use

How could to clone a target gene?

1. Create primers that flank the amylase gene 2. Run PCR to amplify the fragment 3. Use TOPO cloning to place the fragment into an expression vector 4. Grow up a large culture and turn on expression

How do genomes change?

1. Errors during DNA replication --Varies per organism 2. Spontaneous chemical changes in bases --E.g. germination of cytosine to produce uracil 3. DNA damage --Agents (mutagens) that damage DNA **All of these can cause mutations, and if the daughter cell inherits that change, it is a mutant **Causes Evolution to occur

With a given change in a genotype, what mutation might have caused the change?

1. Errors during DNA replication --Varies per organism 2. Spontaneous chemical changes in bases --E.g. germination of cytosine to produce uracil 3. DNA damage --Agents (mutagens) that damage DNA Mutagens: --Base analogs: causes faulty base-pairing based upon analog chemical structure --Chemicals reacting with DNA: can chase a single change in base pairs --Radiation: causes pyrimidine dimer formation and cause the DNA chain to break **All of these can cause mutations, and if the daughter cell inherits that change, it is a mutant **Causes Evolution to occur

What are the various techniques in microbial ecology to answer a research question?

1. FISH—fluorescence in situ hybridization 2. Amplicon sequence -PCR amplification of 16S rRNA/sequence 3. Metagenomics—whole sample sequencing 4. Metabolomics --Sequence cDNA libraries of mRNA expression --LC/GC mass spec

How does transformation occur?

1. Free DS DNA in the environment 2. DS DNA is taken up and processed to SS DNA 3. SS DNA is integrated into genome at homologous site, if present Cells that are capable of transformation are called "competent" --Some species are naturally competent and will take DNA up from the environment Artificial competence—in the lab --Inducted transformation: heat shock --Induction transformation: electroshock *Both causes the pores to open and the plasmid to enter

What are the 6 types of ways the cells can repair DNA damage?

1. Photoreactivation 2. Nucleotide excision 3. Base excision 4. Methyl mismatch 5. Recombination 6. Translation bypass synthesis/SOS response

How do you determine the location of a specific gene?

1. Predict ORF with Prodigal 2. Use BLAST to then determine function of genes 3. Look for genes that have similar sequences to known amylases

What are the major products in community physiology? 1. Under aerobic conditions 2. Under anaerobic conditions with no SO4 3. Under anaerobic conditions with SO4

1. Under aerobic conditions · Water and carbon dioxide 2.Under anaerobic conditions with no SO4 · Methane and water 3. Under anaerobic conditions with SO4 · Hydrogen sulfide, water, and carbon dioxide

How is protein expression controlled?

4 major systems --Bacteria-E.Coli --Yeast-Sacchromyces cerevsia --Insect cells --Mammalian cells Control expression often using lac repressor, promoter/operator --Relax the repression or turn on promoter to great a large culture Often have tags for easy purification

What are the parts of the F factor (a conjugable plasmid)?

99.2kb in size --Generally 1-2 copes per cell 1. Tra region encodes transfer function 2. oriT—start site for transfer 3. oriV—for replication 4. Contains transposons and IS (insertion sequences) elements 5. Contains toxin-antitoxin system Transfer takes about 5 minutes

How can CTC and DAPI help with identifying microbes?

CTC—5-cyano-2,3-ditolyl tetrazolium chloride · Oxidized, colorless, soluble · Reduced, fluorescence red, insoluble (precipitates) · Metabolism is reducing environment · **Changes color based upon oxidation state! DAPI stains biofilms and detects DNA 1. DAPI stained biofilm--all blue 2. CTC stained biofilm --All of the cells are activity growing and metabolism 3. DAPI starved for 1 hour biofilm --Cells are all still there 4. CTC stained biofilm for 1 hour --Many of the cells are metabolically inactive Microscopy shows --Most of the things thought were cells were actually cells --Also 1-35% of them were respiring Culture shows much lower numbers 20-100x less --Culture medium is inefficient --Only 10% of microbes are cultures

What is amplicon sequences?

Cans use to get a survey of species 1. Collect all cells 2. Extract DNA 3. Run PCR reaction with 16S rRNA primers 4. Add adapters 5. DNA sequencing --Take these and count **End with a complete survey of the microbes without culturing

What is the difference between a plasmid and chromosome?

Chromosomes: carries essential genes --Copy # varies · Haploid—one copy · Diploid—two copies · Polyploid—many copies --Usually circular --1-12 Mb Plasmids: carries non-essential genes --Copy # varies: 1-100s of copies --Can have different plasmids per cell **Genomes can vary a lot!

What are the parts of a plasmid?

Circular double-stranded DNA --Ori—origin of replication --Amp—antibiotic resistance gene --Restriction enzyme sites Range in size from 2 kb to 1,000 kb Encode accessory functions that are not essential for survival under most conditions

What is the CRISPR mechanism? What is the function in the cell?

Clusters of Regularly Interspersed Short Palindromic Repeats 3 types of systems Cas1—endonuclease activity Cas2—structural role Cas 9—inserts the small short sequences of the virus --Degrades the rest of viral sequence Immune defense against virus for bacteria --Provides future defense Could be used to insert DNA into genome --Need to have part of DNA bind to gRNA (guide RNA) --The genome will get repaired imperfectly & will cause of mutation--the indel · A donor DNA could be inserted instead **Can make mutations in any place of the DNA or insert a donor DNA Much more efficient

How do you predict an ORF function?

Compare predicted amino acid sequence to amino acid sequence of all known and predicted proteins in a database Common database: GenBank --Run by the NCMI, National Center for Biotechnology --Part of the NIH --Contains all publicly available nucleotide sequences Common program to compare sequences: BLAST --Basic Local Alignment Search Tool --Results of a BLAST search · Red bar = your sequence that matches the alignment sequence · Other bars = matches --Proteins have high homology leads to high probability that the protein preforms the same function

How does conjugation occur?

Conjugation—movement of a plasmid (or other DNA) from one live cell to another Many plasmids encode machinery conjugation --Can move DNA between bacteria that are not closely related 1. The organism will the F plasmid will create a pilius 2. Then a pore is created between the cells 3. The F+ cell donor will donate the plasmid to the F- cell 4. Then, both the cells are F+ cells can donate plasmids to other cells

How can you determine the prevalence of horizontal gene transfer just using the DNA sequence of bacteria?

Each microbe has a typical pattern of sequences—a signature --Will have common codons Programs have been written to detect sequences where these signatures suddenly shift --The DNA is from a different organism A recent one, Mean Shift Genomic Island Predicator uses highly accurate predictor

How does the respiratory chain of an iron-oxidizing microbe at the acid mine compare to the respiratory chain of a typical microbe?

Electrons flow in the reserve direction as oxidative phosphorylation to generate NADH, H2O, and ATP --This occurs due to the Cyc2 and Rus complex pumping electrons from the outer membrane into the inner membrane They both use NDH1, B/C1 complex, quinone pool, and ATP synthase.

What are the types of screens?

Enzyme assay --Mutagen: increase in the activity of an enzyme --Plate thousands of colonies --Pick colonies and assay the enzyme --Save the colonies with the mutant and the desired enzyme activity **Very labor intensive Replica plates o Looking for a mutant that can no longer grow on X o Master plate: plate cells onto minimal media + X --Press onto velveteen, which imprint o Test plate: plate cells onto minimal media --Place into velveteen --Incubate --Some of the colonies will not grow onto minimal media · These colonies will be the mutant as they need growth factor X Color indicator plates --Dilute mutagenic colony --Plate onto color indicator plate (i.e. MAC)

What the important uses of genetics and genomics?

GMO crops --Resistance to round-up --Curing of genetic diseases Expression of proteins --Polymerases, research substrate, restriction enzymes --Therapeutics, insulin, human growth hormones --Detergents Sequencing --Disease diagnosis --Pop culture nonsense As tools in research

What are Leptospirillum ferroxidans?

Gram negative Spiral shape Chemolithoautotroph --Oxidized Fe2+ with oxygen --Energy generation Dominant species CO2 fixation by reductive TCA cycle Somewhat similar to Acidithiobacillus ferroxidans --Figured out the mechanisms of the organism via its genome as in module 8

What are the potential benefits and hazards of CRISPR technology?

Great potential to treat genetic diseases --Diabetes, cancer, HIV, Huntingon's disease Great potential for abuse --Any gene can be edited --Off site mutations --Biohackers **Need a public consensus

What is horizonntal gene transfer and the 3 types?

Horizontal Gene Transfer: between individuals 1. Transduction: DNA is moved by virus 2. Transformation: ability to take up free DNA 3. Conjugation: DNA moves between two living cells *Vertical gene transfer: from parent to offspring

Based upon the Luria-Delbruck experiment, when do mutations occur? Due to presence of the antibiotic (induced mutation) or mutations spontaneously arises and antibiotics select for them

If a mutation happens once under selection—induced mutation --Each mutation is independent --Follows a Poisson distribution · C and T : mean = variance **The control followed this pattern If a mutation happens before selection—spontaneous mutation --Each change of variance will not independent (siblings) the results will not follow a Poisson distribution --Depends upon the time of the mutation · C : mean = variance · T : mean variance **The test group followed this pattern The experiment found that the mutations occur spontaneously before the selection occurs

What is whole genome sequecing?

Isolate DNA from environment Break up sequences Assemble chromosomes 1. Collect all cells 2. Extract DNA 3. Shear DNA into uniform small pieces 4. Add adapters 5. DNA sequencing --Assemble genomes Can know who is there and their metabolic capabilities

What is Griffith's classic experiment through rough and smooth cells? What is the relationship between capsule genes and virulence?

Live S cell—mouse dies Heat-kill S cell- mouse lived Live R cells—mouse lived Live R cells + heat-killed S cell—mouse died --Live S cells --Transforming principal that was moving from the heat-killed smooth cells to the rough cells · DNA was being transferred! Have a capsule = virulent Lack capsule = avirulent

What is the microbial role in acid mines. What is the primary microbial groups?

Microbe accelerate iron oxidation rates at low pH five orders of magnitude --Causing the low pH Primary microbial groups are lithotrophs

How does horizontal gene transfer affect the genotype of microbes? How does this affect the bacterial definition of species?

Microbial genomes are dynamic --Core genome—common to all member of species --Pan genome—found in some but not all members of species **Not all species have the same genome!

What is screening? How can you use it to find a desired strain?

Most cells grow, with the desired mutant being detectably different in some fashion Use screens when there is no good selection

What is the primary producer in acid mines?

Mostly chemolithoautotrophs --Energy from chemicals—oxidation of FeS2 and other sulfur compounds --Carbon from CO2 --Nitrogen from nitrogen fixation—mostly nitrogen gas Some growth of chemoheterotrophs off of Dissolved Organic Carbon (DOC) Autotrophic growth --Autotrophs really important since little organic carbon in the environment --Many species --CO2 fixation by Calvin cycle and reverse TCA

How does transduction occur?

Movement of DNA from one cell to another by a virus 1) Virus infects bacterial cell and replicates its genome within, leading to cell lysis 2) Virus takes up own DNA again, with some viruses taking in lysed bacterial DNA segments 3) Bacterial DNA is inserted into new cell by virus and integrates into nucleus **Can occur naturally in a # of species

What kind of mutations do can you screen for?

Nutritional mutants --Selects transposons on rich medium, place on minimal medium and look for absence of growth --Allows you look for the absence of growth Photopigment mutants --Look for colors that are different than wild-type Photosynthetic minus mutants --Grow under photosynthetic conditions and look for deficiencies

How do you find open reading frames?

ORF—open reading frame --Has a start codon, series of codon, and then stop codon --Sequence that should code for a protein --Looking for start codons with a series of amino acids Extra helper rules --Stop codon should appear randomly every 20 amino acids · If there are codons farther than this, it is probably a real protein --Codon preference · Figure if it is codons that the normally usually --Uses preference codons, codes for a real protein kBase—prodigal --A program that incorporates these rules to predict ORF

How doe the produces and consumers differ from the ocean to the forest?

Ocean—phytoplankton bacteria and algae produce biomass --60% is consumed by grazer—protists and invertebrates --40% is consumed by viruses · Recycle elements · Keeps the bacteria and algae under control Forests—plants produce biomass --20% is consumed by grazers --80% is decomposed by fire, fungi, and bacteria

How does oxidation of Fe2S cause the red/orange run off in acid mines?

Oxidation releases sulfuric acid—Microbial action Further chemical oxidation releases Fe (III) --This reacts with water making acid Causes red/orange color --Iron is recycled by bacteria by oxidizing it --The chemical reaction reduces iron and releases sulfuric acid

What is FISH?

Probes can be designed for any sequence --DNA probe is for a region of interest --Has a fluoresce tag on it · Sticks to DNA via hybridization Domain --rRNA has very high sequence conservation · Might be for a whole domain Phylum Species --Use hyper-variable sequences in rRNA 1. Denature DNA 2. Design DNA probe 3. It hybridizes to DNA 4. Visual in fluorescent microscope --Can see what microbes there are and where they are

What is the translation bypass synthesis/SOS response?

Repair: Massive DNA damage; trying to stop death 1. DNA polymerase stops and falls off 2. RecA binds and puts random bases in 3. SSB and PolV (umuCD complex) **Error prone repair

What is the recombination repair?

Repairs: DNA lesions during replication 1. When the replication machinery reaches the DNA lesion, it stops 2. The replication fork is regressed and the damaged area is degraded 3. The DNA undergoes strand exchange and replicant to fill in the missing area 4. The strand resolution occurs and replication can begin again

What is photoreactivation?

Repairs: pyrimidine dimers 1. UV light will hit the DNA and will cause a pyrimidine dimer 2. Photolyase will be bind --Uses light energy to undo damage and repair DNA

What is nucleotide excision repair?

Repairs: replication errors 1. A base will be changed and cause the DNA to be damaged --Causes a bubble in the DNA 2. DNA glycolase will bind to the damaged DNA and removed the wrong base 3. AP endonuclease clips out the region 4. DNA polymerase comes in and repairs the DNA

What is methyl mismatch repair?

Repairs: replication errors 1. MutS recognizes mismatch and binds 2. MutH binds to nearest methylated GATC --IDs parent strand 3. Non-parent strand degraded 4. DNA pol III fills in gap

How do plasmids maintain themselves in a host cell? How do they replicate?

Replication: Can direct their own replication --Uses host machinery to copy --Plasmid-encoded proteins (Rep protein) co-opt the cell's replication proteins --Need ori of replication --Selfish for DNA Segregation: Have to make sure each daughter cell gets a plasmid For-low copy plasmid --Replication is coordinated with chromosome replication For-high copy plasmid --Random partitioning occurs

How is DNA incorporated into the genome?

Self-replication DNA (plasmids) can maintain themselves Linear DNA needs to be integrated into genome by recombination --1. Generalized recombination--high DNA homology --2. Site specific recombination--short seq. of DNA homology **Even linear DNA can be brought in by recombination

What are mobile genetic elements (MGE)?

Sequences of DNA found in all organism that are capable of moving Can increase # in the genome and are another form of selfish DNA Types 1. Insertion sequences—only code for function for moving DNA 2. Transposons—have useful genes for host · i.e. antibiotic resistance

With a given mutation, what repair system will likely fix it?

Single nucleotide exchanges and frame shift mutations occur during replication --They can be repaired by mismatch repair

What is microbial ecology?

The study of how organisms interact with their environment --Microbes influence the ecosystem · Metabolic activity of microbes determines Earth's biochemistry · Microbial activity has macroscopic consequences Concerned with distribution and abundance of organisms in nature --Microbial location and growth is determined by · Metabolic potential · Environment conditions · Other organisms (building communities)

What is metabolomics?

The study of the entire set of small molecules in a biological system --Survey of the metabolites Uses a Mass spec --Capillary electrophoresis --Gas chromatography --Liquid chromatography o Can determine hundreds of compounds in a single run 1. Collect sample 2. Extract metabolites 3. Inject into GC or LC mass spec 4. Identify peaks and make metabolic map

What is toxin-antitoxin system?

Toxin is more stable If plasmid is lost, antitoxin is degraded --Toxin will be then be able to attack its targets · Toxin is now active --Plasmids use to prevent their loss Example targets for toxins --DNA gyrase --mRNA --Translation --Peptidoglycan synthesis

Why is the plasmid pR27 important?

Transposases are outside of IS (insertion sequence) of Tn5 --Once inserted will not move again; only moves once Hyper transposase --High frequency of transpositions OriR6K—is on the transposon --E. Coli specific --Suicide plasmid · Plasmid cannot replicate --Inside transposons and can be used for coloning OriT origin of transfer for conjugation --Allows transfer of tra genes present The mating --Kn selects for Tn --Nal selects against E. Coli --See image for step by steps End result: R. Rubum with the plasmid genes inside the chromosome

What are the microbes physiological role in a community?

Will see a very similar pattern among all communities --each step is done by a different type of bacteria 3 degradation pathways 1. Depolymerize · Polymers --> lipids, fats, sugars, amino acids, and nucleotides 2. Aerobic respires · Convert nucleotides into water and CO2 3. Fermented into acids into · Converted into methane and water OR water, CO2, H2S

How do you assign ORFs to pathways?

kBase incorporates KEGG (Kyotos encyclopedia of genes and genomes) --Has assembled information on biochemical pathways Assign each ORF to EC (enzyme commission) # --EC # gives information of what the enzyme probably does Can ask question of genomes