chapter eight: microbial genetics
the hereditary material Griffith 1927 & Avery, et al. 1944 the transforming principle coined by Griffith, identified by Avery
the hereditary material Hershey Chase, 1952
the bacterial chromosome
plasmids F factor (conjugative plasmid) dissimilation plasmids R factors
horizontal & vertical gene transfer
antiparallel replication
vertical gene transfer (VGT): DNA replication synthesis requires primers & the 3 OH
horizontal gene transfer (HGT): gene expression simultaneous transcription & translation
HGT: recombination
RecA & chromosomal recombination
insertion sequences & jumping genes
recombination: transformation
recombination: transduction
recombination: conjugation
genetic transfer Transfer Transformation demo Transduction (specialized) Conjugation F+ cells F- cells Hfr cells Effects naked/free DNA from donor DNA binding proteins on recipient RecA needed for DNA fragments transposons chromosome plasmids self-contained Phage incorporates bacterial donor DNA, delivers to recipient F factor codes for sex pilus, delivers donor DNA Contain F factor (donor cell) Lack F factor (recipient cell) High frequency of recombination (donor cell) F factor integrated into donor chromosome at integration point, donates partial F factor from point of transfer and chromosome portion to recipient cell. Recombined F- cell F+ and F- F+ and F+ Hfr and F- Hfr and recombinant F-
regulating bacterial gene expression: constitutive enzymes operons
regulating gene expression* * decreased levels of cellular glucose create high camp levels which further regulate the expression of lactose catabolizing enzymes- this will not be discussed in this class
inducible operon: effector effects by inhibiting repressor = inducer transcriptional control repressible operon: effector effects by activating repressor = corepressor
quorum sensing & gene regulation B. subtilis sporulation cell density = CSF & ComX ComS competence cell density & CSF = ComS inhibited sporulation Gram negative biofilm formation acylated homoserine lactones (HSLs) in loss of flagella sessile microbes initiate biofilm formation P. aeruginosa virulence high cell density activates virulence genes disease
Chapter Eight Learning Objectives 1. What did the work of Griffith, Avery and Hershey & Chase contribute to the field of biology? 2. How is the bacterial chromosome different from the eukaryotic chromosome? What other molecule contains useful genetic information for prokaryotes? Compare and contrast DNA replication in eukaryotes vs. prokaryotes. 3. Why does the replication of every DNA molecule start with a short segment of RNA? 4. Define: vertical gene transfer, horizontal gene transfer, DNA replication, gene expression, transcription, translation, conjugation, transduction and transformation. 5. How is gene expression in prokaryotes different from eukaryotes, both in the timing of transcription & translation and in how transcription is regulated? 6. How do the RecA protein and transposons enable novel DNA to be integrated and used in the recipient cell? Discuss this for both transformation and transduction. 7. Define F factor, F + cell, F - cell and Hfr cell. Understand what happens when F +, F - & Hfr cells interact during conjugation. 8. Describe the mechanisms of inducible and repressible operons. Include the role of promoters, operators, effectors, inducers, repressors and co-repressors in your answer. 9. Discuss the levels of bacterial control of gene expression, paying particular attention to post-translational and transcriptional control, as discussed in lecture. 10. What is quorum sensing? How does it relate to gene expression, particularly as relates to sporulation, biofilm formation, competence and virulence genes.
chapter nine: biotechnology
biotechnology and recombinant DNA biotechnology: using recombinant DNA (rdna) cells using vectors to produce clones therapeutic applications human enzymes and other proteins subunit vaccines viral DNA vaccines gene therapy disease ID mutant screening!!! natural or mutagen-induced >2000 Abx compounds penicillin 1000 stronger than wild type cloned & expressed recombinant DNA technology
rdna technology
pharmaceutical products
restriction endonucleases in vivo: defense system, cut only non-methylated DNA in vitro: molecular scissors
making RFLPs: restriction endonucleases
making & moving rdna: plasmid vectors
shuttle vectors
finding rdna: blue/white colony selection pbluescript vectors
moving rdna: viral vectors
pathogen detection: PCR (second animation)
E. coli O157:H7 outbreak
chapter eight: microbial genetics
change in the genetic material spontaneous no mutagen 10 9 per bp 10 6 genes mutagens freq. 10 5 10 3 per gene mutation frequency
mutation types base substitution (point mutation) silent 3 rd G to any other base = glycine (redundancy) protein change missense, nonsense, frameshift mutation
mutagens
mutation repair photolyase repair separate thymine dimers nucleotide excision repair various damage repaired UvrA, UvrB, UvrC, UvrD (DNA helicase) SOS reca repair cell cycle arrested DNA repair & mutagenesis induced
replica plating: negative mutant selection wildtype auxotroph mutants die
the Ames test: positive mutant selection & carcinogen identification auxotroph wildtype mutants grow
Chapter Nine & Eight B Learning Objectives CHAPTER 9 1. Define biotechnology & recombinant DNA technology. What applications were discussed in lecture which utilize this technology? 2. Discuss how recombinant DNA molecules are made using restriction enzymes. What are the steps used in making these recombinant molecules? How do both plasmids & viruses play a role in expressing recombinant DNA molecules? 3. There are four essential regions on a shuttle vector. What are they, and what do they do? How do they help to identify in vitro transformed cells? 4. Describe the process of PCR to amplify a DNA template. How can thistechnologies be used to identify a microbial pathogen? CHAPTER 8B 1. Define: silent, missense, nonsense and frameshift mutation. How can these errors be repaired in a cell? 2. How does the term auxotroph relate to mutant selection? 3. Why is replica plating necessary for the indirect selection of mutants? 4. What is the Ames test? How and why does it result in positive mutant selection?