Popular in Microbiology 106
Popular in Eberly College of Science
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Date Created: 12/28/14
122814 1113 PM 121960 You and microbiology 10x more bacterial genes than yours consist of 25 lbs of bacteria bacteria talk to one another to become better pathogens and to colonize surfaces microbes too small to be seen interact with humans with food polypetides 0 catalysts Domains Bacteria Archaea Eukarya Split by comparing DNA Viruses Aren39t alive don39t replicate outside host cell Little to no biochemical activity outside host cell Inert and nonreactive outside of host cell 122814 1113 PM Chapter 6 24 26 29 3 methods for finding density 1 average the cells multiply by 10 get number per milliliter multiply by 1000 or 1000000 depending on dilution rate make table and count 2 at a time amount of time per generation multiply by 2 question is how many doublings do we have how long does it take to double the pop 30 minutes to double start with 10 5 cells find how much time it takes until you find the final number 51 questions 1 or 2 from each homework section 7 questions from each chapter 3 questions that he made up lots of true false study red and blue Not much specific chapter 6 cultivating microorganisms 122814 1113 PM Nutritional requirements of microorganisms Large amounts of carbon nitrogen phosphorus sulfur and oxygen Various micronutrients o Siderophores are required to sequester iron Acquisition of nutrients o Autotrophs assimilate carbon from inorganic sources 0 Heterotrops assimilate carbon in preexisting organic form 0 Aquistion of nitrogen o Needed for amino acids and nucleic acids 0 Ammonia is common form part of glutamate Factors affecting microbial growth Effects of nutritional factors on microbial growth 0 Prototrophs can synthesize all needed macromolecular precursors from a single carbon source and inorganic molecules 0 Auxotrophs cannot synthesize all needed precursors from a single carbon source Trp mutant of E Coli cannot produce tryptophan Nutrient concentration 0 One key nutrient available in lowest amount with dictate how much growth can occur over time it39s the limiting factor Effects of oxygen on microbial growth 0 Aerobes grow in presence of oxygen Obligate aerobes require oxygen 0 Anaerobic growth occurs without oxygen Obligate anaerobes cannot grow when oxygen is present Facultative anaerobes can use oxygen but can also grow in the absence of oxygen e Coli Effects of pH on growth 0 Acidophies pH lt55 o Neutrophis pH 5585 o Alkalophiles pH gt85 Water availability 0 Most bacteria requite ax gt9 no bacterial growth in honey Growing microorganisms in the laboratory 0 Media for microbial growth 0 All cells in colony from one cell 0 1 million to see a colony o colony morphology related to extracellular matrix 0 media can be complex unknown composition or defined seective crystal violet and bile salts inhibit gram positives 0 allows isolation of microbes with specific properites differentia dye nuetural red shows lactose fermators 0 allows certain microbes to be recognized based on visual reactions in the medium unculturable bacteria Metagenomics 0 DNA is isolated form environmental sample and sequenced 0 Genetic comtent can be examined and compared to those from different area even if they cant be grown 0 Info must be comfiemd in cultured organisms Measuring microbial population growths Direct counts 0 Special slide with etched gride Hemocytometer or Petroff Hausser counting chamber Cant differentiate living vs dead cells Viable living cell counting serial dilutions and CFUS o Coony forming unites CFUs per mL of intial culture is calculated by multiplying number of colonies by inverse of dilution factor 0 Vortex each tube 0 Filter apparatus can concentrate cells Turbidity o Spectrophotometer sends light through culture gives rough measure of cell denisity Microbial growth curve 0 Lag phase microbes are gearing up for steady growth 0 Log microbes are replicating at a constant and steady exponential rate 0 Stationary replication has halted due to lack or nutrients and excessive wastes or rate of replication is now equal to death rate 0 Death phase nutrients are depleted waste levels high cells dying at a steady exponential rate Highly overrated more cells produced then die o Generation time td time to double the population in exponen alphase 0 Maximum specific growth rate u measure of how quickly cells grow uIn 2td Depends on strain temperature and medium 0 Doubling time td n2u Continuous culture 0 Chemostat flows in fresh medium and takes out old meadium and microbes to keep culture in continuous operation 0 Rate at which fresh medium pumped in dictates rate at which cells divide Eliminating microbes and preventing growth NylonTeflon filters with por size of 2 4 um can keep out most eukaralya and bacterial cells Temperature heat 0 Usually 121 C for 15 minutes Temperature lower heat 0 Pasteurization low temp heating to reduce microbe numbers D value 0 Decimal reduction time time required to kill 90 of the target organism under specific conditions 7 DNA replication and gene expression 122814 1113 PM Role of DNA Griffith experiment 0 19205 o S and R strains of S pneumonia something could transform nonpathogenic R stain into pathogenic S strain when mixed together Avery Macleod and McCarthy 0 19405 o DNA RNA or protein is transformation only DNA could transform R cells into S cells HershyChase 1952 o Labeled radioactive DNA and protein only labled Phage DNA went into bacterial cells 0 Structure of DNA o Franklin Watson and Crick early 1950s o Bacteria single circular chromosome o Archaea singular circular chromosome packaged around histone proteins o Eukarya multiple linear chromosomes packaged around histone proteins o Histones Help compact DNA DNA replication Origins of replication o Bacteria dnaA binds to oriC DnaB recutis DNaC dnaG primaselay down initial RNA primers for DNA polymerase to work 0 Eukaryal replication Multiple origins of replication on each chromosome Very similar to bacteria different proteins Intiaition and elongation 2 DNA pol adds nucleotides 2 Continuous leading strand and lagging strang formed Termination 0 Reach ter sites telomerase is recruited Transcription Initiation and Elongation o Bacteria Sigma factors bound to RNA pol core enzyme direct the combinded holoenzyme to promotor Once RNA pol is situatioed sigma factor dissociates Translation Stop codons o UAA UAG UGA Chapter 8 Viral replication Strategies 122814 1113 PM Recognition of host cells Each virus has range of cells it can infect 0 Range dictated by interaction between viral attachment proteins and host cells receptor proteins 0 Host cell receptors are often molecules critical for cellular function and cannot be easily eliminated CD4 immune cell interactions HIV virus Viral entry and uncoating Enveloped viruses influenza 0 Fusion of receptor and attachment proteins Envelope of attachment fusion peptide 0 Endocytosis o Acidification of endosome exposes viral membrane fusion pep de o Membrane fusion release of viral nucleocapsides into cytoplasm 0 HIV Binding to CD4 alters conformation of gp120 permitting binding to co receptor Additional conformational changes expose fusion peptide facilitates membrane fusion Bind to fusion peptide in gp41 becomes expsed and interacts with host cell membrane 2 Absence of fuzeon gp41 proteins fold brining viral envelope and plasma membrane together 2 Fuzeon gp41 is blocked and fusion inhibited Non enveoped viruses 0 Reovirus Viral binding to report on cell surface triggers receptor mediated endocytosis Bacteriophages o Fail fibers facilitate attachment 0 Virus injects its nucleic acids into host cells 0 Viral genes enter cytoplasm leaving viral capsid on cell surface Plant viruses tomato yellow leaf o Require damaged induced by insects Viral uncoating 0 Different methods 0 Genome must be inserted into the host cell Viral replication Classificatations depend on genome type 0 3 DNA virus groups 0 4 RNA virus groups typical eukaryal DNA viruses 0 entry uncoating DNA enters nucleous replication of viral DNA transcription of Viral DNA translation of viral mRNA viral proteins enter nucleous assembly exit cells atypical eukaryal DNA 0 they bring their own RNA polymerase so they do not have to enter nucleous positive sense RNA viruses o bring their own RNA dependent RNA polymerase so they don39t have to enter nucleus o replicate genome on own retroviruses HIV o brings reverse transcriptase an RNA dependent DNA polymerace 0 entry reverse transcription intergration of viral DNA transcription genome replication translation of viral proteins assembly exit bacteriophages 0 insert genome o lytic DNA replication capsid synthesis and assembly cell lyce and release phages o Lysogenic DNA integrates in cellular genome prophate DNA replicates with cellular DNA 0 Lysogenic or lytic depend on lambda phage Viral assembly and egress Assembly o Many viral components are self assembling make fast and cheap with energy Egress exit o Enveloped virsuses bud off taking some plasma membrane with them 0 Naked virus Iyse cell o Pant move from cell to cell through connections Antiviral drugs AZT for HIV o Look like nucleosides for viral polymerases but prevent from lengthening Tamiflu o Prevents activity of enzyme that helps new virus detach from infected cell Chapter 9 Bacterial genetic analysis 122814 1113 PM Genetic potential of microbes evolution biotechnology Bacteria as subjects of genetic study One chromosome easy detection of mutations Nutritional mutants auxotrophs used 0 Allowed study of one gene based on inability to utilize or produce a particular nutrient o Reversion was a problem Spontaneous mutation that corrects a metabolic abnormaility back to wild type from To avoid problem double and triple auxotroph mutant strains were used 2 Decreases possibility of a spontaneous reversion masking desired results 2 Also delete whole gene rather than use point mutations Organization of bacterial genomes 0 Single chromosome and plasmid Termed the replicon o Plasmids cant use identical copy number control mechanisms in the same cell plasmid incompatibility One cell is not replicated Mutations mutants and strains Mutants and mutations o Microbial geneticists work to compare wild type strains and mutant strains of bacteria 0 Genotype of an organism decribes its collection of alleles of a given set of genes General designation rules 2 Gene is given three etter abbreviation in italics followed by capital letter to separate genes in same pathway acY and acZ 2 Protein given same three etter designation but first letter capitalized and no italics LacZ 0 Changes in genes are often visible by changes in phenotype or growth patterns 0 Detection of mutants Phenotypic selection 2 Use of growth medium that will inhibit microbes lacking the desired genes Phenotypic screening 2 Duplicate plates are created first under full nutritional support second lacks a nutrient 2 Colony grows on fully supported by not second mutation has occurred 2 Duplicate plates are created by replica plating Imprint colonies from master plate onto velvet Transfer colonies to replica plates Incubate and compare growth Can be created by patching o Transferring colonies to a gridded plate 0 Usually more accurate then velvet plate o Pick colonies from aster with tooth pick put picked colony on gridded plate and compare Evolution in test tube 0 Mutation is not bad or take long Lenskis work showed evolution of E Coli in 75 days 2 1mL of cells into 99 mL fresh medium everyday 2 increased in fitness compared to other e coli 2 developed ability to utilize citrate after 31000 generates in medium wid type cannot use citrate 0 mutations and selction can mutations occur spontaneously 2 Esther Lederberg 1950s used replica plating to show spontaneous mutation without select Cells never exposed to strep developed resistance mutations can arise in absence of selective agen 392 Luria and Delbruk 1943 Nobel prize Showed variable resistance to phage infection arises in bacteria without selective pressure Different colony numbers show mutations were random Restriction enzymes vectors and cloning Restriction enzymes Res 0 Allow cutting of specific DNA pieces required to moving or copying DNA Recognizes restriction site and cuts it 0 Similar ends of cut DNA paired together 0 Paired ends can be tied together by DNA ligase Cloning vectors 0 RES allow researchers to stich together fragments of useful DNA into recombinant molecules 0 Recombinant molecules can be used to clone or make copies of bacterial gene of interest 0 Vectors are used to insert recombinant DNA molecules into recipient host bacterial cell 0 Plasmid cloning vectors Discovered by Cohen at Stanford 2 Cut fragments from 2 plasmids carrying antibiotic resistance genes with same RE ligase together 2 Insert recombinant plasmid into bacteria strain exhibited traits from both plasmids X ga system visual bluewhite colony growth 2 Bue acZ functional glacatodimase 2 White nonfunctional o Shutte vector plasmids have multiple types of origins Expands range of host cell types the plasmids can be inserted too 0 Phage vectors mix viral DNA with fragment of interest 0 Cosmids are phage genomes that omit nearly all of the phage DNA leaving more room for the fragment Only critical cos packaging sites are left mult cloning site and antibiotic selection marker Recombination and DNA transfer Homologous recombination o 2 idential fragment of DNA line up and exchange pieces 0 enymes RecBCD and RecA mediate 0 single crossover 2 circular moecues integration 0 double crossover 2 circular moecues DNA swap non homoogous recombination 0 DNA with little to no similarity Prevalent in viruses and transposable elements 0 Use of recombination in bacterial genetics Knockout mutations to study alterations in phenotypes 2 Incorporates plasmid DNA into functional gene disrupting it 2 Suicide plasmids that cant replicate in host cell Transformation uptake of naked DNA 0 Intro of extracellular DNA directly into organism 0 Doesn39t need cell to cell contact Electrical pulse can be used to create temporary holes in plasma membrane electroporation DNA binds to receptor on cell one strand pulled through DNA translocase DNA integrated into chromosome Conjugation DNA transfer via direct contact 0 F plasmid carries gene to form sex pilus between cells 0 Can turn f cell into f cell capable of conjugating with another f cell 0 Nick singe strand break in DNA 0 Hfr DNA transfer F plasmic can integrate into host chromosome 2 Pass genetic info like Hfr high frequency of recombination 2 F plasmid cells host cell DNA to incorporation site across sex pilus over time can map location of genes 0 F Plasmids Can integrate into host chromosome El Generation of F prime plasmids Incorporated F plasmid excises itself Excision is inaccurate some host cell DNA is excised as well When f plasmid conjugate it sends host cell DNA to the recipient o Triparental conjugation Insertes can be too large to be used in plasmid El Using recombinant plasmid lacking required tra gene and helper plasmids with gene conjugation can still occure Move room in recombinant plasmid for desired DNA fragment 0 Transposition movement of DNA via mobile genetic elements Can move within and between genomes Mechanisms El Requires the transposase and resolvase genes Transposons contain other genes in addition to those needed for transposition Repicative copies element and moves copy to another location Non repicative cuts and pastes the element into new location 0 Transduction Mechanisms El Sometimes during phage replication a virus will accidently package a fragment of host cell DNA Odd virus then delievrs that fragment instead of DNA when infecting next cells Usually unable to replicate Homologous recombination must still occur Co transduction frequency was used to map bacterial genomes Can be used to modify bacteria for good purposes Common vaginal tract micrboes to expres and secrete chemokine with anti HIV activity Only performed in vitro Chapter 10 Microbial Genomics 122814 1113 PM Genome sequencing DNA sequencing o Genomics spurred by development of recombinant DNA protocols DNA sequencing methods 0 Sanger or dideoxy sequencing Cloning of gene DNA synthesis electrophoresis Places dideoxynucleotides into DNA synthesis mixture process terminated since doesn39t have free hydroxyl group needed for DNA synthesis Gel electrophoresis can separate fragements and detect labeled nucleotide on end providing sequence 700 10000 basepairs cheaply in hours longer sequences obtained by primer walking using repeated rounds of sequencing with primers complementary to the end of last segment sequenced 0 Next generation sequencing pyrosequencing Detects addition of nucleotide to end of synthesized strand of DNA by production of light faster and easier then Sanger 4 dNTPs added at once one makes light to know sequence then wash and start again 0 454 sequencing highthroughput pyrosequecning method 2 DNA doesn39t have to be cloned 2 Many reactions carried out at once 2 million rns 2 4 dNPs are added one at a time as continuous flow 1 makes light to know sequence and then start again 0 whole genome shotgun sequencing attemps to sequence entire genome in one setup DNA fragments sheared and sequenced software aligns sequences Bioinformants o Analysis of large data sets of sequencing DNA Annotation of genomes help researchers identify open reading frames allow better determine start and stop points of given gene Genomic analysis of gene expression Genetic libraries 0 Collection of cloned DNA fragments that represents the entire genome of an organism o N In 1 pIn 1 f N number of clone fragments required P probability of generating complete library F average size of each cloned fragmenttotal size of genome 0 Library expressed as mRNA molecules can be formed as complememtary DNA using reverse transcriptase Transcriptomes collection of transcribed mRNA molecues in cell 0 Northern blots separation of RNA fragments by electrophoresis followed by blot transfer and probing with labeled DNA fragment 0 DNA microarrays Method for examining transcriptional activity of all genes in a cell simultaneioulsy Reverse of norhter blods Compares 2 diseases at different temperature Blotted to see what genes are turned on in what type 2 Different colors indicates different expressions 0 RNA seq technology Conversion of mRNA into cDNA by reverse transcriptase DNA sequences using rapid automated methods Successful in studying Saccharomyes cerevisia Proteomes 0 Collection of expressed in a cell 0 Determining 3D shape X ray crystallography 2 X ray shot at crystalized protein 2 Diffraction pattern can be used to discern protein shape Nuclear magnetic resonance 2 Measures distances between atomic nucli to discern protein shape 2 Can measure proteins in solution Comparative genomics Study of evolutionary processes using tools of genomics Diff genes may have arisen from duplication events 0 Paralogs Genes that arose from a duplication event and evolved for other functions within the same species 0 Orthologs Genes that have evolved from same common ancestor with the same function in different species Horizontal gene transfer 0 Genomes tend to have characteristic GC nucleotide content 5072380o genomes with significantly higher or lower are likely areas wehre horizontal transfer has occurred 0 genomic islands DNA segments 10200 kb associated with tRNA genes transposable elements plasmids or bacteriophages Metagenomics Construction and analysis of gene libraries from DNA extracted directly from complex microbial communities Obtain DNA from area of interest sequence it using nextgen seq Analysis 0 Eliminate sequences we already know find new genes and potentially new organisms Chapter 11 Regulation of Gene expression 122814 1113 PM Differential gene expression Constitutive genes need to be constantly on Basic control of gene expression can take place during transcription translation and after translation Enzymes 0 Allosteric inhibition Binding of inhibitor molecule to change shape and substrate can no longer bind Covalent modification can also alter activity 2 Most rapid The operon Transcriptional unit with a series of structurl genes and their transcriptional regulatory elements 0 Lac operon is prime example Operator region where regulatiory proteins bind o System only turned on when lactose is around all glucose is used up diauxic growth Negative control of transcription naked DNA is on o Repressor protein binds to operator blocking RNA polymerase inhibits transcription o Effector molecules can Induce transcription by inhibiting binding of pressor on operatior lac operon Positive control of transcription naked DNA off o Regulartory molecules bind and increase transcription rates o Usually activator molecules increases affinity of RNA polymerase for promoters Laclrepressor o Mutants that constantly express lac operon even when no lactose is present Global Gene regulation Reguons genes that are coordinated to respond to the same regulary systems 0 Catabolite repression Shutdown of several systems that utilize various nutrients when glucose is present o SOS response Multigene system for wide spread DNA repair Cells respond to serious DNA damage 2 Jean Jacques Weigle showed UV exposed induces nonspecific repair mechanisms in bacteria Bacteria pre exposed to UV light then infected reapiredthe phage DNA but produced more mutations Crux white colonies hae genes that respond to DNA damage genes are called din genes damage inducible Important genes and regulators recA and exA Post initiation control of gene expression Regulatory RNAs 0 Small noncoding RNAs sRNA control gene expression at transcription or translation points Botn increase and decrease translation Antisense RNA as a positive factor 2 Makes RpoS not destroyed like it is without Attenuation interaction between translation and transcription processes 0 Occurs after initiation of transcription but prevents it from continuing Control of transcription by mRNA secondary structure 0 Riboswitches regulatory molecules that bind RNA and alter its shape Can prevent continuation of transcription Capable of controlng transcription and translation depending on conformation induced in RNA molecule Quorum sensing Chemical signaling to communicate between microbes Lux prototypical quorum sensing system 0 When grown to high density cells produce AHL Autoinducer stimulates luminescence Low density not enough AHL for light emiison Widespeard occurrence 0 Mechanisms controed biofilm formation 2 component regulatory systems systems can use one protein as sensory and other to control transcription sensory virA reguatory VirG function tumor regulation organism agrobacterium tumefaciens o vir genes on Ti plasmid 0 only expressed under conditions similar to plant wound o virAG required for expression of other virulence genes chemotaxis complex bacterial behavior modulated by shifts in protein activity 0 chemotactic bacteria sense changes in chemical gradients over time differences not felt at different positions study using mutants 0 Che proteins 2 component regulatory systems CheA works as sensor kinase becomes phosphorylated CheA then physophylates CheY RR protein 2 Binds to flagellar motor changing activity 0 Methy accepting chemotaxis proteins MCPS Interacting with CheW proteins autophosphorylation of CheA is modulated 2 Attractants decrease phosphorylation repellants increase Methylation of MCPs regulates attraction during periods of high attractant levels in process of adaptation 2 Remain highly methylated 2 If high levels aren39t maintained CheAB will demethylation of MCP Chapter 12 Microbial Biotechnology 122814 1113 PM Biotechnology Uses of biological processes or organimis for production of goods or services Microbes for biotechnology Sources of microbes 0 Culture collections Preserved speciemds that other scientist can obtain at minimal costs 0 Bioprospectiong Searching for useful new microves to cultivate and add to collections Industrial culture of microbes o Fermentation controlled and regulated culture of microbes to create desired substances 0 Primary metaboite product of metabolic processes required for growth of microbe 0 Secondary metaboite not required for microbial growthoften produced during stationary phase Molecular genetic modification Random mutagenesis by chemicalradiation exposure is followed by screening for desired mutations Mutageneis site directed 0 Specific mutations at specific sites positions might stabilize useful proteins Directed enzyme evolution mutagenesis 0 Progressive rounds of random mutation to direct and select desired traits Used if don39t know enough about enzymes for site directed o 2 methods errorprone PC amplify gene your inserting in a sloppy way leading to random mistakes see what effect the mistakes have on expressed product DNA shuffing take bites you39ve already mutate shea them up and stick them back together randomlyl see what effects thechimeras have on expressed product Production of recombinant proteins 0 Expression vectors can be used to mass produce recombinant proteins 20 or more of total cell 0 Insert eukaryotic DNA sequence for product of interest into plasmid put plasmid into bacterial cells grow and harvest 0 Not easy Eukaryal genes must be reformatted to remove introns Eukaryal proteins often require glycosylation that only a eukaryal system can provide proper levels of disulfide bond formation may require eukaryal system 0 Fusion proteins Combination of elements from 2 or more proteins 2 Affinity tag can be often used to assist in purification Designer organisms synthetic biology 0 Take peives of different organisms with desired traits and put them together Red biotechnology Pharmaceutical industry 0 Microbes are producers of secondary metabolits with therapeutic properties 0 Hosts for production of recombinant human proteins Secondary metabolites as therapeutics o Antibiotics o Statins inhibitors of choloesteral synthesis White biotechnology Industrial biotechnology o Microbial conversion of low cost to products with highervalue and industrial use 0 Microbes act on biomass feedstock and useful materials can be harvested out Biofules Bioplastics 0 Plastic buildup is high 0 Microbes can produce natural polyesters PHB and PHAs PHAs microbial food source in times of starvation 2 Biodegradable plastics Industrial enzymes 0 Laundry proteases lipases anylassea and oxidases for removal of stains 0 Many commercial product Vitamins and amino acids 0 Flavor enhanacement nutritional suppements o Tricking cells to overproducing a compound for human use Lysine Green agricultural Applications of transgenic plants 0 Insect resistance Bt toxin is produced by bacterium Bacillus thuringiensis 2 Highly specific and lethal to certain insect larvae that might feed on crops not naturally in plants
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