1 Molecular genetics of bacteria Emphasis: ways that bacteria differ from eukaryotes DNA structure and function; definitions. DNA replication Transcription and translation Gene regulation and regulation of metabolism Genetic exchange among bacteria Genetic engineering

2 Bacteria tightly regulate their activities Bacteria must respond quickly to changes in the environment. Bacteria are small compared to their environment, have no real capacity for energy storage. Simultaneous transcription and translation allows them to synthesize the proteins they need quickly. Wasteful activities are avoided. If there are sufficient amounts of some metabolite, bacteria will avoid making more AND avoid making the enzymes that make the metabolite. Biosynthesis costs! Biochemical regulation and genetic regulation.

3 Biochemical regulation: allosteric enzymes Allo = other; steric = space. Many enzymes not only have an active site, but an allosteric site. Binding of a molecule there causes a shape change in the enzyme. This affects its function.

4 Feedback inhibition of pathways

5 Genetic regulation Genotype is not phenotype: bacteria possess many genes that they are not using at any particular time. Transcription and translation are expensive; why spend ATP to make an enzyme you don’t need? Example: diauxic growth with sugars.

6 Glucose is the preferred carbon source

7 More on Regulation In biochemical regulation, processes like feedback inhibition prevent wasteful synthesis. To save more energy, bacteria prevent the synthesis of unneeded enzymes by preventing transcription. –In operons, several genes that are physically adjacent are regulated together. Two important patterns of regulation: Induction and repression. –In induction, the genes are off until they are needed. –In repression, the genes normally in use are shut off when no longer needed.

8 Operons and Regulons Nearly 50 years ago, Jacob and Monod proposed the operon model. Many genes in prokaryotes are grouped together in the DNA and are regulated as a unit. Genes are usually for enzymes that function together in the same pathway. At the upstream end are sections of DNA that do not code, but rather are binding sites for proteins involved in regulation (turning genes on and off). The Promoter is the site on DNA recognized by RNA polymerase as place to begin transcription. Operator is location where regulatory proteins bind. Promoter and Operator are defined by function.

9 Our example: the lac operon Lactose is milk sugar, used by a few bacteria like E. coli To use lactose, a couple of proteins are important: the permease which transports the sugar into the cell, and the enzyme beta-galactosidase which breaks the disaccharide lactose into glucose and galactose. To prevent the expense of synthesizing these enzymes if there is no lactose to use, E. coli keeps these genes inactive. But if lactose becomes available, these genes must be turned on quickly so lactose can be taken in and broken down. –Imagine E. coli in your GI tract at breakfast.

10 Structure of the Lac operon KEY: P O are the promoter and operator regions. lac Z is the gene for beta-galactosidase lac Y is the gene for the permease lac A is the gene for a transacetylase lac I, on a different part of the DNA, codes for the lac repressor, the protein which can bind to the operator.

11 Products of the lac operon Each gene codes for a protein that is involved in the use of lactose. Depicted is the mRNA and the proteins that result. Note that P and O are functional regions of DNA but aren’t genes for proteins; no mRNA is made from them.

12 Binding of small molecules to proteins causes them to change shape Characteristic of many DNA-binding proteins Regulation of operons: Inducible operons: Repressor protein comes off DNA Repressible operons: Repressor protein attaches to DNA

13 How the lac operon works When lactose is NOT present, the cell does not need the enzymes. The lac repressor, a protein coded for by the lac I gene, binds to the DNA at the operator, preventing transcription. When lactose is present, and the enzymes for using it are needed, lactose binds to the repressor protein, causing it to change shape and come off the operator, allowing RNA polymerase to find the promoter and transcribe.

14 Lactose is not actually the inducer Low basal levels of beta- galactosidase exist in the cell. This converts some lactose to the related allolactose which binds to the lac repressor protein. Synthetic inducers such as IPTG with a similar structure can take the place of lactose/allolactose for research purposes.

15 Plasmids Plasmids: small, circular, independently replicating pieces of DNA with useful, not essential info Types of plasmids –Fertility, –resistance, –catabolic, –bacteriocin, –virulence, –tumor-inducing, and –cryptic

16 About plasmids-1 Fertility plasmid: genes to make a sex pilus; replicates, and a copy is passed to another cell. Resistance plasmid: genes that make the cell resistant to antibiotics, heavy metals. Catabolic plasmid: example, tol plasmid with genes for breaking down and using toluene, an organic solvent. micr302/transfer.html

17 About plasmids-2 Bacteriocin plasmid: codes for bacteriocins, proteins that kill related bacteria. Virulence plasmid: has genes needed for the bacterium to infect the host. Tumor-inducing plasmid: The Ti plasmid found in Agrobacterium tumefaciens. Codes for plant growth hormones. When the bacterium infects the plant cell, the plasmid is passed to the plant cell and the genes are expressed, causing local overgrowth of plant tissue = gall. Very useful plasmid for cloning genes into plants. Cryptic: who knows?

18 Gene transfer Ways that bacteria can acquire new genetic info –Transformation Taking up of “naked DNA” from solution –Transduction Transfer of DNA one to cell to another by a virus –Conjugation “Mating”: transfer of DNA from one bacterium to another by direct contact.

19 Gene transfer between bacteria Transformation: uptake of “naked” DNA from medium. When Griffith did his experiment combining heat killed, virulent cells with live, harmless mutants, the living cells took up the DNA from solution, changed into capsule-producing, disease-causing bacteria. Next slide

20 Transformation details DNA must be homologous, so transformation only occurs between a few, close relatives.

21 Gene transfer between bacteria-2 Transduction: transfer of DNA via a virus. More common, but still requires close relative.

22 Conjugation: bacterial sex If sex is the exchange of genetic material, this is as close as bacteria get. Conjugation is widespread and does NOT require bacteria to be closely related. Bacteria attach by means of a sex pilus, hold each other close, and DNA is transferred. Plasmids other than F plasmids, such as resistance plasmids, can also be exchanged, leading to antibiotic- resistant bacteria.