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Bacterial Handout #3 Genetic Concepts in Lecture #3: • • • • • • • • • • Promoter/Operator Repressor/Activator Operon Gene regulation Regulon Constitutive Mutation Uninducible Mutant Gain of function allele Immunity Epistasis Genetics 200A September 24, 2012 Now we will consider the LYSOGENIC PROGRAM. First we’ll examine the prophage state. Then we’ll discuss the pathway leading to the prophage state. In the prophage state, the λ geneome is stably associated with the E. coli genome. This occurs via site specific recombination between the phage attachment site (attP) and the bacterial attachment site (attB). Thus, λ integrates into the host genome in a specific locus in the genome, between the gal and bio operons, and replicates passively along with the bacterial chromosome. This prophage has two important new properites: Immunity to λ re-infection The ability to produce 100 λ phages upon treatment with DNA damaging agents. The decision made by λ whether to enter the lytic or lysogenic program is subject to environmental conditions during infection. In particular, low MOI favors lytic growth whereas high MOI favors the lysogenic program. Thus, in the early rounds of infection during plaqe growth, the conditions favor lytic growth. However, as the number of phages increase and the number of bacteria begin to wane, the MOI increases and lysogeny is favored. Thus, plaques formed by λ have a mixture of liberated phage and lysogens. Since the lysogens formed are immune to reinfection, they can now grow within the plaque, giving rise to turbid plaques. There are two phases of the lysogenic program. ESTABLISHMENT of the prophage state involves a critical decision that must be executed with precision (we’ll dive into the decision making process next lecture). MAINTENANCE of the prophage state allows the phage to persist as a prophage and must be robust to ensure the lysogen is not killed under favorable conditions (its survival is linked to survival of the bacterium now) but also sensitive to unfavorable conditions (e.g. host DNA damage) to allow the phage to escape and enter into the lytic program. It is a switch that must be thrown completely in one way or the other. To begin to understand the prophage state, lets see what genes are expressed in the prophage state? • cI (lambda repressor) is the only gene transcribed in a lysogen. • Lambda repressor blocks early promoters PL and PR by binding to OL and OR. How was cI identified? Dale Kaiser and Francois Jacob performed a screen for altered plaque formation and isolated temperature sensitive mutants that were unable to form tubid plaques at the non-permissive temperature. One of the mutants they found was in cI. Importantly, the conditional allele of cI allowed them to test whether cI is also involved in prophage maintenance. cI is required for prophage establishment and maintenance! Important experiments performed in Mark Patashne’s laboratory showed that cI binds to OL and OR and represses transcription from PL and PR. cI is also responsible for immunity, as “excess” cI made by a prophage can bind to the operators of an incoming phage and inhibit transcription. Hybrid phages, where the “immunity region” of lambda has been replaced by the same region from the lambdoid phage 434, are not subject to immunity from a lambda lysogen. However, such a hybrid phage is subject to immunity from a 434 lysogen. Thus, the DNA sequence spanning OL to OR is a master regulatory element that is controlled by binding of cI to the operators. Popsicle Question: Describe how you would isolate λ mutants that can grow in a lambda lysogen. Such mutants were identified (termed λvir) but were extremely rare – 10-10. Knowing what you know about λ so far, give one prediction for the nature of the genetic change(s) that would give rise to λvir. Why were the mutant so rare? Lets turn our attention to prophage induction. Treating a lysogen with UV light (or other DNA damaging agents) induces exit from lysogeny and entry into the lytic pathway. Induction is extremely efficient - nearly 100% of the cells in a culture will induce phage production upon UV treatment. How does λ sense DNA damage in the host? What are the molecular events that allow λ to exit lysogeny and replicate now as a phage? The SOS Response Exposure of E. coli cells to agents that damage DNA, such as UV, results in the induction of a diverse set of physiological responses termed the SOS response. These responses are due to the induction of more than 20 genes (termed damage inducible or din genes), many of which are responsible for the cell to recover from the insult. Much of our knowledge about responses to DNA damage stem from studies of λ induction by UV irradiation: The physiological responses to UV irradiation include: • increased rates of recombination (Radman, 1974) • increased mutagenesis • inhibition of cell septation to form filaments (allows time for repair) Lon inhibits SulA, which inhibits FtsZ (required for cell division). Lon- mutants form long filaments (Buttin, Gottesman & Trisler). SulA- mutants fail to filament (Buttin). The SOS Response uvrB ~~~~~~~~ lexA sulA umuDC din genes ~~~~~~~~ recA (low level expression) RecA no protease ~~~~~~~~ lexA ~~~~~~~~ ~~~~~~~~ ~~~~~~~~ recA cA LexA inactive Re active protease ~~~~~~~~ uvrB ~~~~~~~~ sulA ~~~~~~~~ umuDC ~~~~~~~~ din genes • cI protein, like LexA, is sensitive to RecA coprotease activity and is degraded upon UV treatment. • Decreased levels of cI leads to lower occupancy of OL and OR, relieving repression of PL and PR • Proteins required for phage DNA excision (Int and Xis) are induced and the lytic pathway resumes.. The lysogenic pathway leading to the prophage state: Mutants defective in this process (establishing the prophage state) form clear plaques: (They grow lytically but they are not able to lysogenize.) cI, no lysogeny <105 cII, 0.1% or so: lysogens are stable cIII, 10% or so: lysogens are stable cII and cIII are necessary for establishment not maintenance of lysogenic state. cI is required for both establishment and maintenance of lysogeny. What does cII do? activates transcription of int (from pI) and cI (from pRE) What does cIII do?enhances the amount of cII: we shall see in a little while that it is thought to antagonize a bacterial protease (proteolytic system) that degrades cII protein. There is a lot going on at OR. What regulatory proteins bind to OR immediately after infection, during lysogenic growth, and during lytic growth? cI binds first to OR1-OR2 and only binds to OR3 at high concentrations. Cro binds first to OR3, then OR2 and OR1. The Choice Between Lysis and Lysogeny The activity of cII protein appears to be a key determinant of the decision between lysis and lysogeny. (Good review by Banuett & Herskowitz, 1984) 1. Uncommitted growth. After lambda infection, there is a period of uncommitted growth, in which lambda development proceeds without a decision. 2. Commitment. Then, a decision is made: if cII level high, then lysogeny, if cII level low, then lysis Environmental conditions (multiplicity of infection, nutritional conditions) determine whether lysis or lysogeny is favored: low moi: high moi: starvation: rich medium: lysis>lysogeny lysogeny>lysis lysogeny>lysis lysis>lysogeny The cII protein is unstable; half-life of 3 minutes or so. Host proteins (products of the hfl genes and the catabolite gene activation system [CAP protein and cyclic AMP]) and phage proteins (product of the cIII gene) are thought to govern the level of cII. What are the roles of cAMP and Hfl in E. coli cells? cAMP concentrations are regulated by starvation and carbon source. High glucose levels lead to inactivation of adenylate cyclase (Cya), lowering cAMP concentrations. Likewise, under starvation conditions or growth on unfavorable carbon sources leads to activation of Cya and increased cAMP levels. The concentration of cAMP is read out by the catabolyte activator protein (Cap) which leads to changes in transcription of a subset of genes. Hfl is encoded from two regions of the E. coli genome, hflA and hflB, and is a protease that degrades a wide range of proteins in E. coli. In particular hflB is responsible for the degradation of the heat-shock sigma factor, σ32. σ32 levels are increased during heat stress, for example in a shift from 37˚C to 42˚C. However, when cells are returned to 37˚C, hflB is required for σ32 degradation. cII, in addition to activating PRE and PI, also activates PAQ (Ho & Rosenberg – JBC 260:11838 [1995]) Epistasis - The masking of the phenotypic effect of an allele of one gene by an allele of another gene. A gene is said to be epistatic when its presence suppresses the effect of a gene at another locus. First, let’s examine a classic example of simple epistasis, mouse coat color. In this case, the c phenotype is epistatic to the b phenotype.