Download Bacterial Handout #3 Genetics 200A September 24, 2012 Genetic

Bacterial Handout #3
Genetic Concepts in Lecture #3:
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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?
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cI (lambda repressor) is the only gene transcribed in a lysogen.
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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
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lexA
sulA
umuDC
din genes
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recA
(low level expression)
RecA
no protease
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lexA
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recA
cA
LexA inactive
Re
active protease
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uvrB
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sulA
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umuDC
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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)
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Uncommitted growth. After lambda infection,
there is a period of uncommitted growth, in which
lambda development proceeds without a decision.
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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.