Download Bacteria Handout #1 Genetics 200A September 17, 2012

Bacteria Handout #1
Genetics 200A
September 17, 2012
Learning Objectives of the Lambda (λ) Lectures
•
•
•
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:
•
•
•
•
•
•
•
How to perform a genetic screen and analyze mutants in bacteria and phage.
How to work with essential genes in bacteria
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:
•
•
•
•
•
•
•
•
•
Gene and Allele
Essential gene/Lethal mutation
Conditional mutation
Genetic screen and selection
Mutagenesis/spontaneous mutation
Complementation
Suppression
Haploid
Recombination
Bacteria:
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 bacteria employ cell-cell communication and employ group behaviors
Despite lacking major membrane bound organelles, bacteria compartmentalize functions withing the cell and
have cytoskeletons, nucleoid packaging, etc.
Bacterial viruses:
“Dark matter of the biological universe” - estimated 1031 phages on earth, ~1025 new infections per sec.
Development in Pr
Some Important Bacterial Experimental Systems
Bacillus/Clostridia
Caulobacter crescentus
Caulobacter crescentus
Bacillus subtilis
Myxobacteria
bobtailed squid
Magnetospirillum magneticum
Vibrio fischeri
Bacteria/Archaea Inhabit Extreme Envi-
Deinococcus radiodurans
Haloquadratum walsbyi
Neisseria gonorrhoeae
E. coli
1.
2.
3.
4.
5.
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 bacterial biology remain mysterious - e.g. host interactions, biofilms, quorum sensing.
1.
2.
3.
4.
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:
1)
2)
3)
4)
5)
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:
•
immunity to λ re-infection
•
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:
1.
2.
3.
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:
•
Begin with an observable phenotype
•
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.
•
Isolate individuals (for genetic screen) and score phenotype
•
Pick mutants with relevant phenotype
Mutant characterization:
•
Complementation tests to determine number of genes
•
Dominance test
•
Mapping (not essential)
•
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:
(1)
Infect nonpermissive wild-type E. coli strain with two different lambda am mutants (each at an moi of 5)
(2)
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.