Download Genetics 200A 2009 Prokaryotes Lecture 1 (Cox)

Prokaryote Handout #1
Genetics 200A
September 14, 2009
Learning Objectives of the Lambda (λ) Lectures
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Understand principles of a genetic approach to answer biological questions.
Learn the molecular interactions that underlie the λ life cycle, a paradigm for developmental processess in higher organisms.
Because we know many of the molecular details of its biology, λ provides an intellectual framework that serves as a useful grid to understand many basic biological processes (e.g. gene regulation, protein-pro
tein interactions, cooperativity, proteolysis, etc.)
Specifically, we will learn:
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How to perform a genetic screen and analyze mutants in bacteria and phage.
How to work with essential genes in prokaryotes
How lambda programs a sequence of events – sequential gene activation
The mechanisms of a decision-making process in which lambda responds to environmental cues
How lambda interacts with its host cell, E. coli
The molecular mechanisms of important regulatory proteins
DNA recombination
Genetic Concepts in Lecture #1:
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Gene and Allele
Essential gene/Lethal mutation
Conditional mutation
Genetic screen and selection
Mutagenesis/spontaneous mutation
Complementation
Suppression
Haploid
Recombination
Prokaryotes:
Diverse in size, shape, metabolism, and environmental niches
Have a high degree of genetic plasticity, can share their genetic material with one another
Some have developmental programs
Likely all prokaryotes employ cell-cell communication and employ group behaviors
Despite lacking major membrane bound organelles, prokaryotes compartmentalize functions withing the cell
and have cytoskeletons, nucleoid packaging, etc.
Prokaryotic viruses:
“Dark matter of the biological universe” - estimated 1031 phages on earth, ~1025 new infections per sec.
Development in Pr
Some Important Prokaryotic Experimental Systems
Bacillus/Clostridia
Caulobacter crescentus
Caulobacter crescentus
Bacillus subtilis
Myxobacteria
bobtailed squid
Magnetospirillum magneticum
Vibrio fischeri
Prokaryotes Inhabit Extreme Environments
Deinococcus radiodurans
Haloquadratum walsbyi
Treponema pallidum
E. coli
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Genome sequenced (haploid) – 4.6 Mb circular chromosome w/ approx. 4500 genes.
Genetically and biochemically tractable
Short doubling time – 20 min under optimal conditions.
Well established model for studying fundamental processes of all cells (transcription, translation, replication, adaptation to environment, intermediate metabolism)
Not just a host for plasmid and protein purification - many aspects of prokarotic biology remain mysterious - e.g. host interactions, biofilms, quorum sensing.
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Genome sequenced in 1983 – 48.5 Kb, 45 genes
“Simple” organism.
Extremely well studied (λ research predominated in the 1960s & 1970s, but continues).
Much of what we know about bacterial cells stems from the study of their associated viruses.
Bacteriophage λ (lambda)
Phage Life Cycle
Many bacteriophages employ a single mode of growth, once they infect their host they replicate and lyse the
bacterium. Thus, these viruses are termed “lytic” bacteriophages. An example is the very well studied phage
T4.
Life cycle of phage infection:
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phage absorption to surface receptors
DNA injection. (Nothing else gets injected)
phage DNA replication
morphogenesis (head/tail assembly, genome packaging into head)
release of phage by bacterial lysis
Infection of E. coli with a single bacteriophage kills the
bacterium and leads to release of approximately 100 viable phages.
Bacteriophage lambda decides between two modes of
growth:
temperate response
host lives
lytic response
host dies
In lytic response, like T4, λ kills its host and releases
100 phage particles.
In temperate response, the host lives with λ genome
stably integrated into the host chromosome. λ is also
referred to as a “temperate phage”. We’ll discuss the
fate of λ DNA in a bit.
This λ lysogen “E. coli (λ)” has new properties:
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immunity to λ re-infection
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can be induced to produce λ (e.g. UV light)
λ senses intracellular environment, then makes a decision.
The λ lectures will be divided into three sections:
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Lytic pathway—production of 100 viral particles, cell death.
• What are the genes required and how is the sequence of events programmed?
Lysogenic pathway—production of viable lysogen (immunity and activation upon UV irradiation)
Choice—After discussing the genes and proteins responsible for this, we shall discuss how lambda makes a choice between (1) and (2).
We will see: (a) what proteins are needed for lambda to grow in these two different modes, (b) how regulatory genes were identified and gain an overview of how their proteins work, (c) how the action of multiple
regulatory proteins is coordinated.
I. LYTIC PATHWAY
Key Concept: There is a temporal order of events once
phage DNA is injected. Phage replication, morphogenesis,
and lysis must be timed appropriately in order for functional phage to be released.
How is this sequence of events programmed by λ?
The answer to this question was elucidated from many elegant genetic and biochemical experiments. In particular,
the genes required for λ growth were first identified from
genetic screens. How was this performed? Before we address this issue, we first will see how to work with phages
experimentally and general strategies to identify mutants.
The plaque assay is the fundamental method to count phages.
Before we move on to the lambda screens, lets quickly review the difference between a genetic screen and
a genetic selection, and the basics of how to identify mutants. Here are a couple of examples:
In a genetic selection, conditions are selected in which the desired mutant can grow but all other non-mutants
cannot. For a screen, there is no selective growth advantage for the desired mutant and thus must be picked out
from all other strains by some observable phenotype.
Mutant isolation:
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Begin with an observable phenotype
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Mutagenize the organism (DNA damaging agents – UV, EMS, etc.) This is not always required due to “background” spontaneous mutagenesis rate due to intrinsic errors in DNA replication and repair.
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Isolate individuals (for genetic screen) and score phenotype
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Pick mutants with relevant phenotype
Mutant characterization:
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Complementation tests to determine number of genes
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Dominance test
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Mapping (not essential)
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Clone the genes
We want to identify all lambda genes necessary for plaque formation but how can we mutate genes that are essential for the organism to grow? The strategy used to identify mutants defective for lytic pathway is a powerful one, the use of conditional lethal mutations. In this way, mutant alleles can be identified that are functional
under one condition (permissive condition) but non-functional under another conditions (restricitve or non-permissive condition). In this way, the mutant can be grown under permissive conditions, and the effects of the
mutated gene can be studied under restrictive conditions.
Two different kinds of of conditional-lethal screens were performed to identify lytic genes: temperature sensitivity and nonsense suppression.
In wild-type E. coli, there are no tRNAs that contain the
anticodon sequence for the three stop (nonsense) codons.
Under normal circumstances, ribosomes and release factors recognize stop codons and terminate translation. If a
λ tail gene has a codon mutated from sense to antisense,
translation will terminate at that codon and will produce
a truncated, likely non-functional protein during infection
of wild-type E. coli. However, tRNA genes can also be
mutated such that the anticodon can now basepair with a
nonsense codon, allowing for translation to read through
individual stop codons. E. coli strains containing such a
mutant tRNA gene allow for the phenotype of nonsense
mutations to be suppressed. Hence, these strains are called
nonsense suppressors.
If you were handed wild-type λ, wild-type E. coli, and E. coli
suII+, how would you isolate λ lytic mutants?
λ wt
λ amber
E. coli wt
E. coli suII+
Results: Alan Campbell isolated 130 mutants: they grow in bacterial strain C600 (suII+) but not in wild-type
bacterial strain such as 594 (su°).
Do the mutations affect different functions/genes? This can be determined by doing pairwise co-infections
with individual mutants. It is important that most of the cells are infected with both phages. If the mutants
have defects in different genes, then the defective gene product from one phage will be helped by the presence of the normal gene product from the other phage, and vice versa. Phage production will occur when
these two mutants are present in the same cell. If the mutants have defects in the same gene, the gene products will not help each other and no viable phages will be produced.
Complementation test: mixed infection of NONPERMISSIVE HOST
The standard complementation test:
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Infect nonpermissive wild-type E. coli strain with two different lambda am mutants (each at an moi of 5)
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Allow infected cells to produce phage (incubate 60 min). This is the test.
(3) Assay total phage produced on permissive strain - this is the assay to determine if the phages complemented each other during infection of the wild-type strain. Compare the number of phages produced per
infected cell, with the number produced by lambda wildtype and with the lambda am mutants grown singly
(that is, not coinfected). Low phage yield indicates failure to complement; high yield indicates complementation.
Campbell: 130 mutants in 18 complementation groups.
Parkinson: 310 mutants; five new genes discovered.
Mapping the 23 essential genes of lambda
Frequency of recombination between two markers is directly proportional to the physical distance between
the markers.
Frequency of recombination = total recombinants/total phage
15% recombination between A and R; ~1% within a gene
The standard cross:
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Infect permissive host (C600) with two different lambda am mutants (each at an moi = 5);
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Allow infected cells to produce phage (incubate 60 min)
(3) Assay phage produced for:
(a) recombinant phages (ability to form plaques on su° host 594)
(b) total phage produced (assayed on suII host, C600)
Gene function: logic of what the genes do. Infect NONPERMISSIVE HOST and see what they do
and what they can’t do. Function of normal gene can be inferred from the behavior of the mutant
LACKING that gene product.
Essential genes were named A-W.
A-F, W -- encode for head proteins
G-M, T-V, Z -- tails
S -- porin permeabilizes inner membrane
R -- lysozyme to degrade peptidoglycan
O,P -- DNA replication
N,Q -- transcriptional activators
Clustering of genes with related function allows for:
1. coordinate regulation
2. genetic modularity, allows for hybrids to
be formed with related “lambdoid” phages
(e.g. 434). Naturally occurring labdoid phages, as well as other kinds of phage, are highly
mosaic.
Before we move on, lets review the phage complementation test and the λ cross. For complementation tests, we are asking whether two amber
mutant phages, which alone can’t grow under
restrictive conditions, can help each other grow.
Thus, the test must be done in wild-type E. coli.
To determine if the phages complemented, we
need to determine how many phages were produced during the test. Therefore, the lysate from
the test infection must be assayed under permissive
conditions (suII+ E. coli). For the cross, we initially want the phages to grow freely and allow the
genomes to recombine. Thus the test is performed
under permissive conditions. After a round of
infection, we now want to determine the frequency
of recombination by enumerating the number of
recombinants. This is performed by determining
the number of wild-type phages produced from
recombination between the two amber alleles by
plating them under restrictive conditions.
In both the complementation test and the cross, it
is important that most of the cells are simultaneously infected with both phages. This is achieved
by having a high multiplicity of infection, MOI.
Lets look at the λ genome. In this figure, there are some new genes that we haven’t discussed yet. Don’t
worry, we’ll get to them soon enough. The point here is to become familiar with what is happening with
the DNA once it gets injected into the host.
In the phage particle, the λ genomic DNA is linear. At both ends of the genome are 12 nucleotide stretches
of complementary, single stranded DNA termed cohesive end sites (cos sites) which can base pair very efficiently. Upon infection, host DNA ligase seals the ends of the λ genome to give rise to a circular genome.
During the lysogenic program, site specific recombination between the phage attachment (attP) site and the
bacterial attachment (attB) site leads to integration of the λ genome into the bacterial chromosome.
As we will soon see, once injected, the DNA is actively transcribed by host RNA polymerase.
Today we will see that the timing of λ functions is reflected by
temporal regulation of λ gene transcription. First, a primer on
transcriptional regualtion in prokaryotes:
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The transcription initiation rate of
any promoter is set by the efficiency with
which RNA polymerase (RNAP) recognizes the promoter sequence elements.
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The sigma subunit plays the major
role in the process of promoter recognition.
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Strong promoters can bind RNAP
w/o help from transcriptional activators but
weak promoters require their function.
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Many transcription activators function by making a direct interaction with
RNAP, although some alter the conformation of promoter DNA.
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Most transcription repressors function by blocking access of RNAP to the
promoter.
The temporal regulation of DNA replication, morphognesis, and lysis during lytic growth is rooted in
the temporal regulation of transcription of λ genes.
Transcription is performed by E. coli RNA polymerase but is controlled by a combination of transacting λ regulatory proteins and cis-acting nucleotide sequences.
There are three phases of λ gene expression: immediate early (N), early (O, P, Q), and late (heads, tails,
S, R)
How is this temporal regulation achieved? Where
are the promoters?
F. Blattner & J. Roberts showed by in vitro transcription that there are three promoters that can be
recognized by RNA polymerase without the help of
any activators (PL, PR, PR΄). However, only N and
cro are transcribed due to terminators TL, TR, and
TR΄.
N function:
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N is a transcriptional activator and works
by inhibiting termination at TL and TR. Thus, N is
termed an antiterminator.
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The mechanism of N function has been determined via both genetics and biochemistry.
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N binds to the nut (N utilization) site present
in the RNA transcript.
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N, in concert with a number of host Nus (Nutilization substance) proteins, modifies the activity
of RNA polymerase such that now it fails to terminate at TL and TR, leading to transcription of downstream genes.
Therefore, N is a cool transcriptional activator. It
doesn’t affect transcription initiation but rather transcription elongation.
Transcription of early genes:
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N accumulation leads to transcription of
O, P, and Q from PR
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N accumulation also leads to transcription of genes downstream of N in the nonessential region of lambda (including exo, bet, gam)
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Increased expression of O and P leads to
DNA replication
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Increased synthesis of Q leads to late
gene expression.
Q function:
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Like N, Q is an antiterminator that inhibits termination at TR΄.
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Unlike N, Q binds to a DNA sequence (termed
the qut site) in the promoter at PR΄.
Transcription of late genes:
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Antitermination at TR΄ leads to transcription of all late genes, including tails, heads, and the lysins
S and R.
Therefore, temporal expression is achieved through a sequence of regulatory proteins that
modify the activity of RNA polymerase:
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First, N is made.
N leads to O, P, and Q synthesis
O and P lead to DNA replication
Q leads to late gene expression.