MB Ch 8 Sp2016 1.pdf Bacterial Genetics Lecture Notes

ANTIBIOTIC RESISTANCE Staphylococcus aureus Gram-positive coccus; commonly called Staph Frequent cause of skin and wound infections Since 1970s, treated with penicillin-like antibiotics E.g., methicillin In 2004, over 60% of S. aureus strains from hospitalized patients w...

ANTIBIOTIC RESISTANCE Staphylococcus aureus Gram-positive coccus; commonly called Staph Frequent cause of skin and wound infections Since 1970s, treated with penicillin-like antibiotics E.g., methicillin In 2004, over 60% of S. aureus strains from hospitalized patients were resistant to methicillin (includes resistance to many common antiobitics) ~2.3 million healthy people in U.S. harbor methicillin-resistant S. aureus (MRSA) found in hospitals = Healthcare-acquired MRSA (HA-MRSA) Vancomycin considered drug of last resort How do multidrug resistant strains arise?  Chapter 8 Bacterial Genetics Section 8.1: Genetic Change in Bacteria Chromosome: circular ds DNA molecule; contains all essential genes Plasmid: smaller, circular ds DNA molecule replicates independently of the chromosome contains nonessential genes Definitions Gene: sequence of nucleotides that specifies a protein (determines a trait) Genome: complete set of genetic information in a cell; all the genes in a organism Genotype: the specific sequence of nucleotides in the DNA of an organism Phenotype: the characteristics displayed by an organism 2 Methods of Genetic Change Vertical gene transfer: mutation arises in single cell of parent and is passed on to all offsprin Horizontal gene transfer: the cell acquires DNA from another organism (lateral move)  SECTION 8.1: GENETIC CHANGE IN BACTERIA Vertical (Mutation) gene change Mutations are caused  Spontaneously Vertical (Mutation) Base substitutions Change Deletion or addition of nucleotides Transposable elements  Induced Chemical mutagens Intentional Transposition Radiation Horizontal gene transfer  Conjugation: horizontal gene transfer  donor cell must physically contact the recipient cell (via sex pili)  DNA-mediated Horizontal gene transfer Transformation: horizontal gene transfer Sections 8.2, 8.3, 8.4: mutation change in nucleotide sequence of DNA alters an organism’s genotype can alter phenotype  Caused by incorporation of incorrect nucleotide during replicati Mutagens agents that increase rate of mutation (chemicals, X ra  spontaneous mutation frequency  occur in natural environment  occur infrequently  random  typically between 10 -4 -- 10—12 for a given gene per cell d  mutagens increase mutation rates 10 to 103 fold most mutations are not beneficial, so are quickly lost from popula about 1/100 mutations are beneficial  SPONTANEOUS AND INDUCED MUTATIONS Base substitutions mutations due to incorporation of incorrect nucleotide during replication most common type of mutation Point mutation = change of a single base pair Wild type = a strain whose properties are similar to the organism first isolated from nature. Geneticists compare mutants to wild type  SPONTANEOUS AND INDUCED MUTATIONS Consequences Base Substitution mutations: 1.Silent mutations – generates a codon that will still specify the correct amino acid 2.Missense mutation – generates a codon that will specify a different amino acid 3.Nonsense mutation – generates a codon that specifies a stop codon  SPONTANEOUS AND INDUCED MUTATIONS, CONT Consequences of Deletion and Addition of nucleotides 1.Shifts the translational reading frame of codons Called frameshift mutation affects @@ downstream from addition or deletion mutations frequently result in premature stop codons @@=amino acids  Spontaneous and Induced Mutations, cont Induced Mutations caused by Chemical mutagens causes base substitutions Causes frameshift mutations 1.Intercalating agents: insert themselves between bases adds a nucleotide, causes frameshifts Ex: Ethidium bromide; a carcinogen Ex: Chloroquine, used to treat malaria 2.Alkylating agents: modify nucleobases alter base-pairing 3. Base analogs: resemble nucleobases alter base-pairing  Spontaneous and Induced Mutations, cont Induced Mutations  caused by Radiation: 2 types 1.Ultraviolet light (UV) Creates covalent bonds between adjacent thymines =thymine dimers this distorts DNA Faulty repair mechanisms/enzymes result in mutations 1.X-rays Cause single & double strand breaks in DNA (ds often lethal)  8.4 Repair of Mismatch repair Damaged DNA DNA Repair Mechanisms (2 types) repair DNA before replication: 1. Proofreading - DNA Polymerase I has editing capability - back ups, excises nucleotide and puts in correct nucleotide 2. Mismatch repair - fixes errors missed by DNA I polymerase - Endonuclease removes short stretch of nt - DNA polymerase fills gap - DNA ligase joins ends Mutations are rare as mistakes generally repaired., Ex: in humans, two breast cancer genes code for DNA repair enzymes - (BRCA 1 & 2) - mutations in either result in 80% probability of breast cancer  Spontaneous and Induced Mutations, cont Transposons  jumping genes or mobile genetic elements - segments of DNA that move spontaneously from gene to gene used to generate mutations able to insert into coding sequence of gene Able to inactivate genes into which it inserts McClintock  Section 8.9: Mobile Gene Pool, cont Transposons as mobile genetic elements: In addition to causing mutations, transposons provide a mechanism for mobilizing genes. transfer genes from chromosome to plasmid and vice versa - Simplest is insertion sequence (IS)  encodes only enzyme transposase & inverte repeats - Composite transposons consist of one or more genes flanked ISs often include antibiotic resistance genes or toxin genes transfer genes from chromosome to plasmid and vice versa In theory, any gene or group of genes can move to another site if the are bounded by IS’s, genes for antibiotic resistance are medically important.  Section 8.9: Mobile Gene Pool, cont Transposons yielded vancomycin resistant Staphylococcus aureus strain :Patient infected with S. aureus (which is susceptible to vancomycin) Also had vancomycin resistant strain of Enterococcus faecalis Enterococcus faecalis plasmid containing vancomycin resistance transferred to S. aureus via conjugation Transposon-containing E. faecalis plasmid cause vancomycin resistant gene to jump to plasmid in S. aureus In the end S. aureus becomes VR  Section 8.5: Mutant selection - how to find mutant cells Major problem induced mutant is rare and difficult to isolate 2 Techniques used to isolate mutants direct & indirect selectio 1.Direct selection – uses selective medium that supports growth of mutant but not parent ex: antibiotic-resistant mutants exposed to antibiotic Selects for antibiotic resistant Bacteria that mutated from antibiotic sensitive parent. Only streptomycin resistant cells (mutan will grow in media containing streptomycin  SECTION 8.5: MUTANT SELECTION - HOW TO FIND MUTANT, CON 2. Indirect selection- Isolates auxotroph from prototrophic parent strain Since prototrophic parents will grow on any media the auxotr can must use replica plating Replica Plating  isolates an organism (mutant) with the inability to synthesize an essential molecule (growth factor) that parent strain can synthesize Ex: Isolating an auxotroph (ex: Trp-) from a prototroph (ex:Trp+) parent is cumbersome Because the parent (Trp+) will grow on every media the mutant (Trp-) will grow on. Replica Plating same orientation of velvet critical  CHAPTER 8: HOROZONTAL GENE TRANSFER AS A MECHANISM OF GENETIC CHANGE (GENETIC RECOMBINATION) Horizontal Gene Transfer microbes acquire genes from other c  Purpose create new combinations of alleles  Requires donor and recipient DNA  Only recipient  is recombinant… (contains new genetic information) Genes are naturally transferred by three ways: 1. Transformation: naked DNA uptake by bacteria 2. Transduction: bacterial DNA transfer by viruses 3. Conjugation: DNA transfer between bacterial Combine two strains His–, Trp– and Leu–, Th Any colonies that grow on glucose-sa medium acquired genes from other s Section 8.6: 1. Transformation DNA-mediated Transformation Transfer of naked, extracellular DNA (from environment to recipient) Recipient bacterial cell must be “competent”  be able to take up DNA from environment Donor doesn't have to be bacterial DNA Donated DNA must be integrated into recipient chromosome Cells rupture during the stationary and death phase; broken donor chromosome bursts thru ruptured cell wall; recipient cell picks up piece of the naked DNA; the naked DNA is integrated onto the recipient chromosome CHAPTER 8: HORIZONTAL GENE TRANSFER AS A MECHANISM OF GENETIC CHANGE (GENETIC RECOMBINATION) DNA replicated only if in a replicon Replicons: Have origin of replication Are plasmids, chromosomes DNA fragments added to (integrated) into chromosome via homologous recombination  DNA-mediated transformation, cont. Electroporation artificial competence transformation assisted by using electric shock to facilitate DNA uptake by recipient cells used in genetic engineering (because not all bacteria become naturally competent) Mix DNA and bacteria + electricity Section 8.7: Transduction transfer of new genetic info by viruses (will be covered in virus section) SECTION 8.8 AND 8.9 : CONJUGATION 2. Conjugation- DNA transfer between cells of same bacterial species much more efficient than transformation requires cell to cell contact Involves a conjugative F plasmid with key properties: » Plasmid is self-replicating (has origin of replication) » Codes for genes necessary for contact (sex pilus) » may contain genes for useful but not essential properties (ex: drug resistance or toxins) Cells must be of opposite mating types: » Donor cells carry a plasmid that codes for “F factor” = F+ cell » Recipient cell does not carry a plasmid F- cell During conjugation, after plasmid is transferred, recipient F- cell becomes F+; donor F+ remains F+  SECTION 8.8 AND 8.9 : CONJUGATION, CONT (E.coli) F plasmid (fertility)most studied:  cell-to-cell contact via sex pilus  plasmid carries gene for sex pilus  if, F factor integrates into bacterial chromosome,cell = Hfr  then, chromosomal genes can then be transferred  Section 8.8 and 8.9 : Conjugation, cont Creation of Hfr cells from F+ cells In some F+ cells, the F factor integrates onto host chromosome  converting F+ to Hfr (Hfr= High frequency of recombinatio  Section 8.8 and 8.9 : Conjugation, cont Hfr cells transfer chromosomal genes Conjugation between Hfr and F- cell Hfr cell produces F pilus Part of chromosome transferred to recipient cell Chromosome usually breaks before complete transfer (full transfer would take to long ~100 minutes) Recipient cell remains F– since incomplete F plasmid transferred F- remains F- F- has new information even though it may not have the F factor gene. Amount transferred is limited by time. Still Hfr Still F-  Section 8.8 and 8.9 : Conjugation, cont Hf F- Selection scheme to r recover recombinants from Hfr transfer Leu + His + Thr + Trp + Selecting prototroph mutants from auxotroph parents Glucose salts medium= minimal medium Colonies able to grow in this media Original single bacteria have no growth factor requirements are + for all amino acids Structure of a typical R plasmid, a conjugative plasmid containing antibiotic-resistance genes Resistance or R plasmids confer resistance to antimicrobial medication and heavy metals, such as mercury and arsenic. Composed of two parts: the R genes which encode the resistance traits. the RTF (resistance transfer factor) which encode conjugation traits. Comparison of Mechanisms of Horizontal Gene Transfer * Dnase is an enzyme that destroys DNA  Type of Characteristics gene transfer Transformati Naked DNA gets transferred on can be any gene cross species transfer-even to animals or plants Conjugation Cell-to-cell contact required conjugative plasmid required only genes attached to plasmids can be transferred Transduction (later…..requires a virus) Transposon can move genes between plasmids and host chromosomes or vice versa