Copy of PMB C112 Study Guide (1).pdf

1. Genomics a. Nucleotide sequences are used to pass info across generations b. Microbes evolve all the time - how they evolve i. HGT 1. 3 ways - conjugation, transduction, transformation a. Transformation - bacteria take up DNA...

1. Genomics a. Nucleotide sequences are used to pass info across generations b. Microbes evolve all the time - how they evolve i. HGT 1. 3 ways - conjugation, transduction, transformation a. Transformation - bacteria take up DNA from environment b. Transduction - phage transfers DNA from one cell to another c. Conjugation - 1 bacterium directly transfers DNA to another by means of a pilus 2. HGT has led to presence of genomic islands - regions of the genome that are separate from the rest of the genome due to a different GC count and a specific function like pathogenesis a. Main example is pathogenicity but there are other cases as well ii. Duplication -> divergence in function via mutation c. Importance of genomic content i. We can find potential of gene or protein by sequencing it; sum up sequences to get potential functions of organism 1. But predict via transcriptomics or proteomics 2. Experimentally determine function from there ii. We can also compare organisms and trace back evolutionary changes d. Bacterial genome features i. Generally in Mb range 1. Closed genome - we know protein-coding genes and non protein-coders 2. Open genome - we don’t know all the genes (dark zone) ii. 1 ORF per kb iii. 30-40% of predicted ORF’s are iv. Many genomes have dark regions -not sequenced e. How gene sequence helps i. Comparative genomics - compare organisms -> evolution and relation ii. Functional genomics - understanding potential within genomes iii. Structural genomics - understand 3D components of genome and encoded by genome iv. Assemble sequences to get an idea of what genome is 1. NGS - take small sequences and combine ‘em 2. PacBio - long reads that get connected v. Annotation - describing contents of genome  1. Find ORF by finding ATG (start codon) 2. Then find RBS 3. Determine codon bias of each ORF compared to others in genome a. Some AA’s are coded by more than 1 codon; each organism prefers certain codons over others b. ORF with different codon bias from rest of predicted proteins in genome less likely to produce protein because it’s not preferred vi. BLAST used to compare input sequence to others in database and seeing what happens as a result vii. How genomes enable biological research 1. Reverse genetics - go from gene to function 2. Predictive/comparative genomics - make educated guesses based on other organisms 3. Transcriptomics a. Take RNA sample, erase DNA, erase rRNA and tRNA, do reverse transcription to get opposite strand and then get cDNA -> compare that to database 4. Predict metabolic pathways 2. Measuring growth a. 3 ways to count bacteria i. Direct microscopic counting - get cells per mL ii. Viable colony count - count number of cells capable of growing in a specific medium 1. Only living cells counted 2. Not all organisms can be cultured iii. Turbidity 1. Indirectly measure growth by seeing how turbid culture is - get viable cells per mL 2. But there could be dead cells, and there is no way to distinguish between cells b. DIfferent types of media i. Rich/complex - not chemically defined, and has tons of different complex organic molecules for bacteria to grow on ii. Defined - has specific known ratios of different compounds 1. Minimal - subset where only bare minimum for growth is there iii. What elements are needed by cells - H, O, C, N, P, S, K, Mg, Ca, Fe, traces of Se, Mn, Co, Zn, Cu 1. Marine microbes may require Na, Cl 2. Total cell yield  iv. If bacterial population has enough nutrients, it can undergo exponential growth 1. Double based on generation time v. Key equations for growth 1. N = N0 * 2n; N is final cell number, N0 is initial cell number, n is number of generations 2. g = t/n; g is doubling time, t is elapsed time vi. Batch culture - closed environment where bacterial growth depletes nutrients and alters environment vii. Stationary phase - microbial growth limited because conditions changed 1. Essential nutrient gone or toxic waste product builds up 2. No net change in cell number 3. Energy metabolism/some biosynthetic processes can still continue 4. Stationary phase not a single state at molecular level viii. Lag phase 1. Occurs when a. Bacteria transferred from stationary phase medium to fresh medium b. Cells transformed from rich to minimal medium c. Culture transferred to different temperature/stress conditions 2. What bacteria do in this time a. Sense new environment - adapt to it b. Make new proteins for rapid growth, producing nutrients not in medium, adapting to new condition/stress ix. Heterotrophs vs autotrophs 1. Heterotrophs require organic C source; autotrophs do carbon fixation from CO2 2. Most organisms use NH4+ or NO2-; require nitrogen fixing bacteria to convert N2 to those forms x. Different relationships to oxygen 1. Obligate aerobes - only use oxygen as electron acceptor; nothing else will do 2. Microaerophiles - only live in places of low oxygen concentration 3. Aerotolerant anaerobe - can live with or without oxygen and oxygen doesn’t make a difference to them 4. Facultative anaerobe - can live without oxygen but having oxygen causes reactions to go faster 5. Obligate anaerobe - NEVER NEVER NEVER living with oxygen xi. Why is oxygen shite?  1. Can grab electrons from electron-carrying molecules and become ROS a. Super reactive, super damaging 2. Bacteria have a way to deal with that a. Superoxide dismutase to convert ROS to H2O2; peroxidase to convert that to water and oxygen xii. Temperature - it’s a hot topic 1. Bacteria live in different condition; they adapt to ‘em 2. Psychrophile - optimal in extremely cold temperatures 3. Mesophiles - optimal at some middle range temperature 4. Thermophile - optimal at hot temperatures 5. Hyperthermophile - optimal at extreme heat xiii. How bacteria adapt to heat 1. Problem of proteins denaturing due to heat solved by a. Individual proteins having fewer glycine residues and having salt bridges (much sturdier) b. Chaperone proteins that refold denatured proteins c. Synthesizing solutes that stabilize proteins 2. Problem of membranes being too fluid a. Phospholipids have longer, saturated fatty acid tails i. Linear, pack tightly xiv. How bacteria adapt to cold 1. Problem of proteins not having much thermal action (moving too slowly) a. Make proteins more flexible 2. Problem of lower membrane fluidity a. Short, unsaturated fatty acid tails - don’t pack tightly and keep membrane fluid 3. Problem of ice crystals forming a. Cryoprotectants xv. How bacteria adapt to hypertonic conditions (higher solute concentration outside cell) 1. Synthesize/import compatible molecules to prevent water loss xvi. How bacteria adapt to hypotonic conditions 1. Rigid cell walls that withstand pressure for lysis 2. Mechanosensitive channels activated by high internal pressure; leak solutes out 3. Genetics a. Key definitions  i. Gene - segment of DNA coding for functional product (protein or sometimes RNA) plus regulatory regions surrounding it ii. Open reading frame - stretch of DNA from start to stop codons that encodes a protein iii. Mutation - permanent, heritable change in nucleotide sequence iv. Allele - different variants of same gene v. Genotype - nucleotide sequence of gene in question vi. Phenotype - physical trait that ties back to genotype b. How genetics works i. Find wildtype with normal phenotype ii. Mutagenize organism and look for changed phenotype iii. Sequence genome, trace mutation, and experimentally verify c. How to isolate mutants of interest and identify affected genes i. Selectable mutations - distinct growth advantage in certain conditions (like resistance) ii. Non-selectable mutations - no growth advantage (like becoming auxotrophs, unable to follow some important aspect of life cycle, etc.) d. Selection vs. screen i. Selection - establish conditions where only mutants of interest grow; rest die (like resistance) 1. After mutagenesis, spread 108 survivors on plate and you’ll only get a handful of colonies 2. Super efficient but it isn’t always possible or doable (like if you have something conferring a growth disadvantage) ii. Screen - grow all survivors under permissive conditions 1. Test all survivors for growth in permissive conditions, then test all survivors for growth in nonpermissive conditions (like inability to grow with a specific nutrient or growing at a specific temperature) a. All survivors must first be grown as independent colonies 2. Best when dealing with loss of normal abilities 3. Patching/replica plating - have 1 plate with permissive and 1 plate with nonpermissive condition for screen a. Colonies that grow in permissive but not in nonpermissive conditions are the ones to work with e. Essential genes - genes that are required to be active in all known conditions (like cell wall synthesis, DNA replication, transcription, translation, cell division, chromosome segregation) i. Screen for conditional loss of function mutations  ii. Most common way - screen for temperature sensitive mutation where protein has amino acid change that allows it to work at low (permissive) temperature but not at higher (nonpermissive temperature) 1. Then screen all temperature sensitive mutants for specific desired phenotype at nonpermissive temperature f. Mutation rate - probability that given gene will acquire mutation in a given generation i. Spontaneous mutation - errors in DNA replication ii. Mutagens can increase mutation rate 1. Chemicals a. Like nucleotide base analogs - create mutations when incorporate into DNA during replication i. Similar to nucleotides ii. Can switch between 2 different chemical forms with different base pairing properties iii. Create single single base substitutions during subsequent rounds of replication 2. Radiation a. Bases strongly absorb UV light b. Leads to pyrimidine dimers; blocks replication and triggers RecA-mediated SOS response i. Translesion synthesis is part of this - polymerase can synthesize DNA from pyrimidine dimers 1. But because pyrimidine dimers can’t base pair normally, random bases get added across lesion -> mutation 3. Transposons a. Naturally occurring b. Move from place to place in genome and lead to random mutations c. Transposon has transposable element + inverted repeats d. Transposase recognizes inverted repeats, cut at ends of transposon, and moves it somewhere else e. 1 transposase can’t affect another transposon (1 transposase per transposon) f. Leads to loss of function mutations; not good for essential genes g. Engineered to only hop once i. Transposon removed from transposon, placed in a separate plasmid  ii. Antibiotic resistance gene still in transposon iii. Can’t replicate in host; won’t be passed down iv. Mutated gene easy to identify iii. Microlesions - small changes (base pair substitutions/small indels) iv. Macrolesions - large changes (large indels, duplications, inversions) 4. Genetics 2 a. Genetic exchange - how bacteria acquire new genes from other organisms (and how we introduce genes of choice into other bacteria) b. Natural genetic exchange b/w strains or species - HGT i. Evolutionary force ii. How antibiotic resistance spreads c. What is genetic exchange used for i. Complementation - identify affected genes in mutants ii. Engineer bacteria to have desired properties d. Plasmids get exchanged a lot i. What are plasmids - circular pieces of DNA separate from chromosomes 1. Don’t contain essential genes 2. Less than 1/20 size of chromosome 3. Replicated by normal cell machinery 4. Copy number varies a. Low - 1-5 plasmids b. Medium - 10-50 copies c. High - 100’s 5. Specific host ranges 6. Very common e. Mechanisms of HGT i. Transformation 1. Transfer genetic material from one organism to another - Griffith experiment a. Strep pneumoniae has 2 phenotypes - smooth (pathogenic) or rough (not as effective) i. Inject rough only - no death ii. Inject smooth only - deaths iii. Inject heat killed smooth - no deaths iv. Inject heat killed smooth + rough - deaths because rough strain took up genes for capsule and became virulent 2. DNA (both strands) taken in via DNA-binding protein 3. RecA mediates homologous recombination - donor DNA replaces highly similar recipient strand (integrated into genome)  a. Homologous recombination - physical exchange between two similar DNA molecules (requires 30-1000 nt overlap) i. Won’t work if DNA is entirely foreign 4. Competence - how well bacteria can be transformed a. Can be artificially induced through electroporation or incubating in high calcium media i. Lead to higher cell permeability -> uptake of DNA in circular or linear forms b. This lets non-homologous, replicating plasmids enter cells i. Artificial transformation -> doesn’t require homologous recombination ii. Transduction 1. Generalized transduction - how phages transfer bacterial DNA between cells 2. Phage accidentally packs up part of host DNA 3. Very rare - 10-5 times when transducing particle forms -> injects host bacterial DNA to new recipient a. So select for DNA that you want to get transferred (antibiotic resistance) 4. RecA based homologous recombination can occur if new DNA is homologous enough 5. Not all phages do this because some phages have specific packaging sequence 6. Commonly used to introduce double mutant (combine 2 mutants in chromosome of 1 bacterial strain) a. Have strain with 1 mutation already and infect with phage i. Generally this strain has transposon mutagenesis-based mutation; transposon also carries resistance to antibiotic for selection b. Harvest all phages after cell lysis c. Tiny fraction will have bacterial DNA; even tinier fraction will have mutant gene of interest d. Have this tiny fraction of phages infect strain with other mutation and grow on antibiotic that transposon carries resistance to iii. Conjugation 1. Bacterial sex 2. DNA transfer requiring cell-cell contact via pilus (maintained by F plasmid) - 1 strand goes in and will get replicated a. Donor has F plasmid; recipient doesn’t  3. Requires oriT and tra genes a. Origin of transfer - where mobilization starts b. Tra genes - transfer c. OriV is good for replication in host cell 4. What’s the point of replicating outside chromosome? a. If plasmid can do so - used for complementation, housing transcriptional reporter, expressing fluorescence fusion protein b. If plasmid can’t do so - transposon mutagenesis, homologous recombination to insert all or part of plasmid into host chromosome i. Good for gene knockouts/altering specific host genes) 5. This involves replication of stable plasmid 5. Genetics 3 a. Why would we want to express a particular gene from a plasmid in a bacterial host? i. Move gene into different species/different mutant background ii. Express different allele of gene instead of wildtype allele iii. Complement genomic mutation with WT allele of gene on a plasmid iv. Change regulation of gene (switch it off/on, change expression level) b. Let’s say you have randomly mutagenized organism and identified several mutants of interest with different phenotype - how to identify what mutation is really responsible? i. Complementation - restore WT phenotype by expressing WT allele 1. Transform mutant with plasmid expressing WT allele 2. If WT phenotype returns, you know what mutation(s) are responsible (usually mutation in 1 gene since mutation in multiple genes at the same time becomes unlikely) c. Random mutagenesis i. Used when you don’t know what genes are responsible for phenotype of interest, or if no sequence info of organism known 1. UV, Tn mut, chemicals (Tn mut requires ability to introduce Tn DNA) d. Targeted mutagenesis i. Used when genes of interest are known and we want to directly study them 1. Sequence info, then either do transformation or conjugation ii. How to do targeted gene disruption to knock out a gene (just one of many ways)  1. PCR amplify regions upstream and downstream of ORF of interest and the antibiotic resistance gene 2. Ligate PCR products with plasmid that won’t replicate, allow for homologous recombination between host DNA and this sequence (50-1000 nt chromosome sequence) 3. Transform competent cells, plate on antibiotic to select for anyone that replaced ORF of interest with antibiotic resistance gene a. Only way resistant colonies form is if they replaced their host DNA with this resistance gene 4. This won’t work if you mess with essential genes e. How to target essential genes i. Temp sensitive mutation where you compare activity in low vs. high temperatures ii. Or inducible promoter method 1. Some proteins only need in certain conditions (example - bacteria turn on certain genes when digesting xylose) a. Take one of those genes and put it in front of a random essential gene of interest 2. Now make a depletion strain a. Create plasmid with essential gene driven by inducible promoter b. Transform plasmid into host strain and and keep gene on with appropriate inducer c. Keep plasmid gene on, turn off chromosomal copy through targeted gene disruption d. Now remove inducer to turn plasmid gene off e. See what happens as existing protein gets depleted and see what happens f. Forward vs. reverse genetics i. Forward - make random mutations, screen/select through a lot of mutants to find the one of interest ii. Reverse - target genes of interest and see what happens g. Notes about Tn mut i. Mutagenize species with transposon (because this is random), select mutants that got transposon, select/screen for mutants with desired phenotype 1. Doesn’t guarantee mutation in gene of interest 2. Inverted repeats needed for transposase activity 3. Transposons don’t insert anywhere inverted repeats already are present  4. No homologous recombination involved h. Notes about targeted gene knockout i. Not random technique (phenotype still unknown even if gene is) ii. Recombinant DNA techniques needed to create construct to replace gene of interest iii. Delivery plasmid can’t replicate in host iv. RecA necessary here