Download Chapter 7 - HCC Learning Web

Chapter 7
Microbial Genetics
You are expected to know the basic details of these processes
Genetics
Genetics – the study of heredity
The science of genetics explores:
1.
Transmission of biological traits from parent to offspring
2.
Expression and variation of those traits (phenotype)
3.
Structure and function of genetic material (genotype)
4.
How this material changes
Regulation of Genetic Expression
Most genes are expressed at all times
Other genes transcribed and translated when cells need them
Allows cell to conserve energy
Regulation of polypeptide synthesis:
Typically halts transcription
Can stop translation directly
Regulation of Genetic Expression: Operons
In prokaryotes this regulation is coordinated by operons
An operon consists of a promoter and a series of genes
Controlled by a regulatory element called an operator
Operons are a set of genes all of which are regulated as a single unit
Regulatory gene
3
Operon
Promoter Operator Structural genes
1
2
3
4
5 Template DNA strand
Figure 7.20
Operons
Inducible
operon is turned ON by
substrate
Repressible
operon is turned OFF by the
product synthesized
catabolic operons – made up of
enzymes that metabolize a
nutrient when needed
anabolic operon –made up of
enzymes that stops amino acid
synthesis when they are not
needed
Induced by presence of nutrient
Repressed by presence of amino
acid
Lactose operon
Tryptophan operon
Transcriptional control by regulatory proteins
Regulatory proteins
Repressors
Bind regulatory sequences in the
DNA and prevent transcription of
target genes
Activators
Bind regulatory sequences in the
DNA and stimulate transcription
of target genes
In 1961, Jacques Monod and
François Jacob proposed the
revolutionary idea that
genes could be regulated.
They noticed that, in
Escherichia coli, enzymes
used to metabolize lactose
were inducible, while those
for glucose were constitutive.
Lactose Operon: Inducible Operon
Regulator
Control locus
gene codes for
repressor
Structural locus
coding for enzymes needed
promoter & operator to catabolize lactose
β-galactosidase
hydrolyzes
lactose
permease
brings lactose across cell
membrane
β-galactosidase transacetylase
uncertain function
The lac operon, an inducible operon.
lac operon
Promoter and
regulatory gene
Operator
(blocked)
1
Promoter
3
Transcription
Translation
RNA
2
polymerase
cannot
bind
Repressor
1
Repressor mRNA
2
3
Lactose catabolism genes
5 Template DNA
strand
lac operon repressed
1
3
5
3
Repressor
cannot bind
Transcription
proceeds
2
RNA polymerase
3
5 Template DNA
strand
4
mRNA for
lactose catabolism
5
Repressor
3 Inactivated
repressor
Inducer (allolactose
from lactose)
Figure 7.21
lac operon induced
www.nature.com/scitable
Lactose Operon: Inducible operon
www.nature.com/scitable
The trp operon, a repressible operon.
trp operon with five genes
Regulatory gene
Promoter Operator
3
1
2
3
4
5
5
Template DNA
strand
Transcription
3 mRNA
5
mRNA coding multiple
3 polypeptides
5
Enzymes of tryptophan biosynthetic pathway
Inactive repressor
trp operon active
Tryptophan
Trp
Movement of RNA polymerase ceases
1
3
Inactive
repressor
Trp
Trp
Trp
Trp
Tryptophan
(corepressor)
trp operon repressed
Figure 7.23
Trp
Activated
repressor
Operator
blocked
2
3
4
5
5
Difference between operon types
Type
Inducible operon
Catabolic
Repressible operon
Anabolic
Nutrient/ Amino acid
Inducer
Co-repressor
Operon normally
Off
On
In presence of nutrient/amino acid
On
Off
End result
Nutrient metabolized
Synthesis stopped
Example
Lactose operon
Arginine operon
Regulation of Genetic Expression: Eukaryotes
Control of translation: Regulatory RNAs can regulate translation of
polypeptides
1) microRNAs: Bind complementary mRNA and inhibit its translation
2) Short interference RNA (siRNA): RNA molecule complementary to a
portion of mRNA, tRNA, or DNA that binds and renders the target
inactive by triggering its degradation
Mutations: Changes in the genetic code
A change in phenotype due to a change in genotype (nitrogen base
sequence of DNA) is called a mutation
A natural, nonmutated characteristic is known as a wild type (wild
strain)
An organism that has a mutation is a mutant strain, showing variance
in morphology, nutritional characteristics, genetic control
mechanisms, resistance to chemicals, etc.
Mutations of Genes
Change in the nucleotide base sequence of a genome
Are rare events: Otherwise organisms could not effectively reproduce
Almost always deleterious
Rarely leads to a protein that improves ability of organism to survive
Mutagens increase the mutation rate by a factor of 10 to 1000 times
Causes of mutations
Spontaneous mutations
random change in the DNA
due to errors in replication
that occur without known
cause
Induced mutations
result from exposure to
known mutagens, physical
(primarily radiation) or
chemical agents that
interact with DNA in a
disruptive manner
Categories of mutations
1) Point mutation – addition, deletion, or substitution of a few bases
2) Missense mutation – causes change in a single amino acid
3) Nonsense mutation – changes a normal codon into a stop codon
4) Silent mutation – alters a base but does not change the amino acid
5) Back-mutation – when a mutated gene reverses to its original base
composition
6) Frameshift mutation – when the reading frame of the mRNA is
altered by insertion or deletion of nucleotides
The effects of the various types of point mutations
Figure 7.24
http://www.nature.com/scitable/content/mutations-6656797
I) Wild type
THE BIG BAD DOG ATE THE FAT RED CAT
II) Mutations
A) Substitution mutations
1) Missense:
2) Nonsense:
THE BIG BAD DOG ATE THE FIT RED CAT
THE BIG BAD (stop)
B
B) Frameshift mutations
1) Insertion :
2) Deletion:
THE BIG BAB DDO GAT ETH EFA TRE DCA T
THE BIG BDD OGA TET HEF ATR EDC AT
A
Ultraviolet light
Thymine dimer
G
G
C
C
T
G
A
T =T
G
G
C
A
A
C
C
T
A
A
T
Repair of mutations
Since mutations can be potentially fatal, the cell has several enzymatic
repair mechanisms in place to find and repair damaged DNA
1) DNA polymerase – proofreads nucleotides during DNA replication
2) Mismatch repair – locates and repairs mismatched nitrogen bases
that were not repaired by DNA polymerase
3) Light repair – for UV light damage
4) Excision repair – locates and repairs incorrect sequence by
removing a segment of the DNA and then adding the correct
nucleotides
Identifying Mutants, Mutagens, and Carcinogens
Mutants : Descendants of a cell that does not repair a mutation
Wild types : Cells normally found in nature
Methods to recognize mutants
Positive selection
Negative (indirect) selection
Ames test
Positive selection of mutants
Penicillinresistant cell
Medium with penicillin (only
penicillin-resistant cell grows
into colony)
Penicillinsensitive cells
Medium without
penicillin (both types of
cells form colonies)
Penicillin- resistant
mutants
indistinguishable
from nonmutants
Medium with penicillin
Mutagen
induces
mutations
Medium without
penicillin
Figure 7.29
The use of negative (indirect) selection to isolate a tryptophan
auxotroph.
Auxotroph: An organism that has lost the ability to synthesize certain
substances required for its growth
The Ames Test
Experimental
tube
Suspected
Liver
extract
mutagen
Control
tube
Liver
extract
Culture of his– Salmonella
Medium
lacking
histidine
Incubation
Colony of revertant
(his+) Salmonella
No growth
Genetic Recombination and Transfer
Exchange of nucleotide sequences often occurs between homologous
sequences
Recombinants: Cells with DNA molecules that contain new nucleotide
sequences
Vertical gene transfer: Organisms replicate their genomes and provide
copies to descendants
Horizontal gene transfer: Transfer of genetic material between
organisms of same or different species
Plasmids Replicate Autonomously
Plasmids are much smaller
than chromosomes.
-Found in archaea, bacteria, and
eukaryotic microbes
-Usually circular
-Need host proteins to replicate
Plasmid Properties
Plasmids are advantageous under certain conditions:
Resistance to antibiotics and toxic metals
Pathogenesis
Symbiosis
Plasmids can also be transferred between cells.
R factor/R plasmid
R factor
=
RTF
+
r determinant
R factor is plasmid that can be transferred by conjugation
R factor consists of transfer factor (RTF) and a resistance determinant
(r determinant)
RTF responsible for its own replication and conjugal transfer
r determinant contains genes that code for antibiotic resistance
Horizontal Gene Transfer Among Prokaryotes
Donor cell contributes part of genome to recipient cell
Three types
Transformation
Transduction
Bacterial conjugation
Transformation
Oswald T. Avery
(1877—1955)
Maclyn McCarty
(1911—2005)
Colin M. MacLeod
(1909—1972)
Photo courtesy: Avery & McCarty: The Rockefeller University, Macleod: profiles.nlm.nih.gov
http://www.nature.com/scitable/content/prokaryote-transformation-6656866
Transformation
One of conclusive pieces of proof that DNA is genetic material
Cells that take up DNA are competent
Results from alterations in cell wall and cytoplasmic membrane that
allow DNA to enter cell
Streptococcus pneumoniae
Pneumococcus
Photo courtesy: The Rockefeller University
Pneumococcus colony
.
Transformation of Streptococcus
pneumoniae
Observations of Streptococcus pneumoniae
Griffith's experiment:
Living
strain R
Live cells
Injection
XX
+
Heat-treated
dead cells
of strain S
Mouse dies
Injection
Heat-treated
dead cells of
strain S
DNA fragment
from strain S
Living strain R
Injection
XX
Mouse dies
Mouse lives
Strain R live cells
(no capsule)
XX
DNA broken
into pieces
Capsule
Heat-treated
dead cells of
strain S
In vitro transformation
Culture of
Streptococcus
from dead
mouse
Injection
Living cells
with capsule
(strain S)
Mouse lives
Some cells take
up DNA from the
environment and
incorporate it into
their chromosomes
Transformed cells
acquire ability to
synthesize capsules
Transduction
Generalized transduction
Transducing phage carries random DNA segment from donor to
recipient
Specialized transduction
Only certain donor DNA sequences are transferred
Bacteriophage
Host bacterial cell
(donor cell)
Bacterial
chromosome
Transduction
1 Phage injects its DNA.
2 Phage enzymes
degrade host DNA.
Phage
DNA
Phage with donor DNA
(transducing phage)
3 Cell synthesizes new
phages that incorporate
phage DNA and, mistakenly,
some host DNA.
Transducing phage
Recipient host cell
4 Transducing phage
injects donor DNA.
Transduced cell
Inserted
DNA
5 Donor DNA is incorporated
into recipient’s chromosome
by recombination.
Bacterial conjugation
Pilus
Conjugation involving an Hfr cell.
James Wilson
In: Molecular paradigms of
infectious disease- a bacterial
perspective
Nickerson,C.A. & Schurr,M.
(Eds). 2006
http://www.nature.com/scitable/content/prokaryote-transduction-6656859
Transposons
Special DNA segments that have the capability of moving from one
location in the genome to another – “jumping genes”
http://www.nobelprize.org/nobel_prizes/medicine/laureates/1983/press.html
Transposition
Result is a kind of frameshift insertion (transpositions)
Transposons all contain palindromic sequences at each end
Plasmid with
transposon
Transposon DNA
Jumping transposons.
Transposons move from one
place to another on a DNA
molecule.
Replicating transposons.
Transposons may replicate
while moving, resulting in
more transposons in the cell
Transposons can move onto
plasmids.
Transposons moving onto
plasmids can be transferred to
another cell.
Figure 7.37