2 component regulatory systems

1. 2 component regulatory systems • Maltose=effector, BUT if signal not DIRECTLY involved, but needs to be transmitted and changed = signal transduction • Sensor protein= • kinase, phosphorylates compounds, • membrane associated • Phosphoryl group transmitted to another regulator IN the cell • Often a DNA binding protein involved in transcription • Many examples, N-fixation, sporulation,chemotaxis

3. Chemotaxis • Attractants decrease rate of autophosphorylation • Repellant increased autophosphorylation • CheA-CheW=transducer • CheY controls switch • cheY-P tumbles, CCW-CW • CheB phosporylated by CheA-P, but slower response than CheY-P • CheB involved methylation • Fully methylated = best for repellants • cheB-P demethylates, occurs when attractants High • Degree of methylation regulates attraction/repulsion

4. Chemotaxis

5. Genetic exchange: transformation

6. Genetic exchange: plasmid transfer • Small, usually circular, independently replicating DNA molecules • Generally, G- plasmids replicate as does chromosomal DNA, G+ plasmids by “rolling circle” replication • Genes of replication control, timing initiation on plasmid (ori) • Some plasmids integrate (F+, Hfr) • Most are double-stranded • About 1- 100kb • Code for: • R-factors (R-plasmids) : antibiotic resistance, heavy metal resistance • Virulence plasmids : adhesins, hemolytic factors, toxin, Ti, bacteriocins • Degradation, tol, nah, • Plasmid copy # • Compatibility (inc)

7. Genetic exchange: plasmid transfer • Small, usually circular, independently replicating DNA molecules • Generally, G- plasmids replicate as does chromosomal DNA, G+ plasmids by “rolling circle” replication • Genes of replication control, timing initiation on plasmid (ori) • Some plasmids integrate (F+, Hfr) • Most are double-stranded • About 1- 100kb • Code for: • R-factors (R-plasmids) : antibiotic resistance, heavy metal resistance • Virulence plasmids : adhesins, hemolytic factors, toxin, Ti, bacteriocins • Degradation, tol, nah, • Plasmid copy # • Compatibility (inc)

8. Avery Experiment

9. Transformation • Competence: ability to be transformed • Steps in transformation • DNA binding and uptake (SS or DS, depending on species) • Integration (recA) • Competence may be induced by electroporation, Ca

10. Mechanism of transformation

12. Transduction • Generalized— low frequency • Specialized— high frequency:requires specific integration

13. Generalized transduction

14. Specialized transduction

15. Specialized transduction

16. Genetic exchange: conjugation

17. Conjugation: early Cell-surface structure

18. Conjugation: middle

19. Conjugation: late

20. Genetic Engineering-basics • Basic steps in cloning • Restriction-modification enzymes • “shot gun” or PCR • Ligase • Recombination Plasmids (or phage)= vectors • Expression vectors • Selection of clones • Looking for a clone with a specific gene • Probes (DNA, RNA or antibody) • Wave of the future: DNA chips or “microarrays”, BAC libraries, automated sequencing etc

21. Genomics • Bioinformatics • Harvesting genes for biotech (Diversa) • Recent Science article • Comparing gene families