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 Effects

Transformation demo

naked/free DNA from donor DNA binding proteins on recipient

RecA needed for DNA fragments transposons chromosome plasmids self-contained

Transduction (specialized)

Phage incorporates bacterial donor DNA, delivers to recipient

Conjugation F+ cells F- cells Hfr cells

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

transcriptional control repressible operon:

effector effects by activating repressor = corepressor

inducible operon: effector effects by inhibiting repressor

= inducer

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

– 109 per bp

– 106 genes

• mutagens freq.

105 – 103 per gene

mutation frequency

mutation types

• base substitution (point mutation)

– silent

• 3rd G to any other base = glycine (redundancy)

– protein change

• missense, nonsense, frameshift mutation

mutagens

• 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

mutation repair

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?