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

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

Chemotaxis

Genetic exchange: transformation

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)

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)

Avery Experiment

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

Mechanism of transformation

Transduction

• Generalized— low frequency

• Specialized— high frequency:requires specific integration

Generalized transduction

Specialized transduction

Specialized transduction

Genetic exchange: conjugation

Conjugation: earlyCell-surface structure

Conjugation: middle

Conjugation: late

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

Genomics

• Bioinformatics

• Harvesting genes for biotech (Diversa)– Recent Science article

• Comparing gene families