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Chapter 08
Lecture
1
Antibiotic Resistance
 Staphylococcus aureus
• Gram-positive coccus; commonly called Staph
• Frequent cause of skin and wound infections
• Since 1970s, treated with penicillin-like antibiotics
• E.g., methicillin
• In 2004, over 60% of S. aureus strains from
hospitalized patients were resistant to methicillin
• ~2.3 million healthy people in U.S. harbor methicillinresistant S. aureus (MRSA)
• Healthcare-associated MRSA (HA-MRSA) resistant to
other antibiotics, including vancomycin
• Vancomycin considered drug of last resort
8.1. Genetic Change in Bacteria
 Organisms adapt to changing environments
• Natural selection favors those with greater fitness
• Bacteria adjust to new circumstances
• Regulation of gene expression (Chapter 7)
• Genetic change (Chapter 8)
• Bacteria excellent system for genetic studies
• Rapid growth, large numbers
• More known about E. coli genetics than any other
• Change in organism’s DNA alters genotype
• Sequence of nucleotides in DNA
• Bacteria are haploid, so only one copy, no backup
• May change observable characteristics, or phenotype
• Also influenced by environmental conditions
8.1. Genetic Change in Bacteria
 Mutation and horizontal gene transfer
Spontaneous
mutation in genome
Vertical gene transfer
(a) Mutation
Plasmid transfer
Vertical gene transfer
(b) Horizontal gene transfer
8.2. Spontaneous Mutations
 Transposons (jumping genes)
• Can move from one location to another
• Process is transposition
• Gene insertionally inactivated
• Function destroyed
• Most transposons have transcriptional terminators
• Blocks expression of downstream genes in operon
Transposition
Transposable
element
Gene X
Gene X
disrupted
8.2. Spontaneous Mutations
 Transposons (jumping genes) (continued…)
• Classic studies carried out by Barbara McClintock
• Observed color variation in corn kernels resulting from
transposons moving into and out of genes controlling
pigment synthesis
8.3. Induced Mutations
 Induced mutations result from outside influence
• Agent that induces change is mutagen
• Geneticists may use mutagens to increase mutation rate
• Two general types: chemical, radiation
8.3. Induced Mutations
 Transposition
• Transposons can be used to generate mutations
• Transposon inserts into cell’s genome
• Generally inactivates gene into which it inserts
Transposition
Transposable
element
Gene X
Gene X
disrupted
Horizontal Gene Transfer as a Mechanism
of Genetic Change
 Microorganisms commonly acquire genes from
other cells: horizontal gene transfer
• Can demonstrate recombinants
with auxotrophs
Strain A :
His–
Trp –
Strain B:
Leu –
Thr –
Mixture
• Combine two strains
– E.g., His–, Trp– with Leu–, Thr–
• Spontaneous mutants unlikely
• Colonies that can grow on
glucose-salts medium most likely
acquired genes from other strain
Glucose-salts agar
Control: No His+ Trp+
mutants present
Glucose-salts agar
Control: No Leu+ Thr+
mutants present
Glucose-salts
agar
Only phototrophic recombinants
(His+, Trp+, Leu+,Thr+) grow
Horizontal Gene Transfer as a Mechanism
of Genetic Change
 Genes naturally transferred by three mechanisms
• Transformation: naked DNA uptake by bacteria
• Transduction: bacterial DNA transfer by viruses
• Conjugation: DNA transfer between bacterial cells
8.6. DNA-Mediated Transformation
• Naked DNA is not within
cell or virus
• Cells release when lysed
• Addition of DNase prevents
transformation
• Demonstration of
transformation in
pneumococci
• Only encapsulated cells
pathogenic
Organisms injected
Results
Mouse dies
Living encapsulated cells
No effect
Living non-encapsulated cells
No effect
Heat-killed encapsulated cells
Mouse dies
Heat-killed encapsulated cells
+
Living non-encapsulated cells
Living encapsulated
cells isolated
8.6. DNA-Mediated Transformation
 Transformation
1
• Recipient cell must be competent
• Most take up regardless of origin
• Some accept only from closely
related bacteria (DNA sequence)
• Process tightly regulated
• Bacillus subtilis has twocomponent regulatory system
• Recognizes low nitrogen or carbon
• High concentration of bacteria
(quorum sensing)
• Only a fraction of population
becomes competent
Gene conferring StrS
Recipient chromosome
Gene
Conferring StrR
Double-stranded DNA binds to the surface of a competent cell.
2
Single strand enters the cell; the other strand is degraded.
3
The strand integrates into the recipient cell’s genome by homologous
recombination. The strand it replaced will be degraded.
4
Streptomycin-sensitive
daughter cell
Streptomycin-resistant
daughter cell
After replicating the DNA, the cell divides.
5
Non-transformed cells (StrS) die on streptomycin-containing medium,
whereas transformed cells (StrR) can multiply.
8.7. Transduction
 Transduction: transfer of genes by bacteriophages
• Specialized transduction: specific genes (Chapter 13)
• Generalized transduction: any genes of donor cell
• Rare error
during phage
assembly
• Transfer of
DNA to new
bacterial host
Transducing particle
Phage
1
A bacteriophage attaches to a
specific receptor on a host cell.
2 The phage DNA enters the cell.
The empty phage coat remains
on the outside of the bacterium.
3
Enzymes encoded by the
phage genome cut the bacterial
DNA into small pieces.
1
Bacterial
DNA
Replaced
host DNA
2
3
A transducing particle
attaches to a specific
receptor on a host cell.
The bacterial DNA is
injected in to a cell.
The injected bacterial
DNA integrates into the
chromosome by
homologous recombination.
4 Phage nucleic acid is replicated
and coat proteins synthesized.
4
Transducing
particle
(a) Formation of a transducing particle
5 During construction of viral
particles, bacterial DNA can
mistakenly enter a protein coat.
this creates a transducing
particle that carries bacterial
DNA instead of phage DNA.
Bacteria multiply with new genetic material.
Replaced host DNA is degraded.
(b) The process of transduction
8.8. Conjugation
 Conjugation: DNA transfer between bacterial cells
• Requires contact between donor, recipient cells
• Conjugative plasmids direct their own transfer
• Replicons
F pilus
• F plasmid (fertility)
of E. coli most
studied
2 µm
8.8. Conjugation
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 Conjugation (continued…)
1
Making contact
Chromosome
F plasmid
F pilus
• F plasmid of E. coli
•
F+
cells have,
F–
do not
• Encodes proteins including
F pilus
•
•
•
•
•
Sex pilus
Brings cells into contact
Enzyme cuts plasmid
Single strand transferred
Complementary strands
synthesized
• Both cells are now F+
Donor cell F+
Origin of
transfer
Recipient cell F–
The F pilus contacts the recipient F– cell.
2 Initiating transfer
One strand is cut in
the origin of transfer
The pilus retracts and pulls the donor and recipient cells together.
3 Transferring DNA
A single strand of the F plasmid is transferred to the recipient cell;
its complement is synthesized as it enters that cell. The strand
transferred by the donor is replaced, using the remaining strand
as a template for DNA synthesis.
4 Transfer complete
F+ cell
F+ cell
At the end of the transfer process, both the donor and recipient
cells are F+ and synthesize the F pilus.
8.8. Conjugation
 Chromosomal DNA transfer
less common
• Involves Hfr cells (high
frequency of recombination)
• F plasmid is integrated into
chromosome via homologous
recombination
• Process is reversible
• F′ plasmid results when small
piece of chromosome is
removed with F plasmid DNA
• F′ is replicon
Formation of an Hfr cell
Chromosome
F plasmid
The F plasmid sometimes
integrates into the bacterial
F pilus
chromosome by homologous
recombination, generating
an Hfr cell; the process
Integrated is reversible.
F plasmid
F+ cell
Hfr cell
Formation of an F′ cell
Integrated
F plasmid
Chromosome
Hfr cell
Chromosomal
DNA
An incorrect excision of the
integrated F plasmid brings
F′ plasmid along a portion of the
chromosome, generating
an F′ cell.
F pilus
F′ cell
8.8. Conjugation
 Chromosomal DNA transfer less
common (continued…)
• Hfr cell produces F pilus
• Transfer begins with genes on one
side of origin of transfer of plasmid
(in chromosome)
• Part of chromosome transferred to
recipient cell
• Chromosome usually breaks before
complete transfer (full transfer
would take ~100 minutes)
• Recipient cell remains F– since
incomplete F plasmid transferred
1
Making contact
Integrated
F plasmid
Chromosomal
genes
Origin of
transfer
Chromosome
a′
C
B
b′
A
c′
Recipient cell F–
Donor cell Hfr
The F pilus contacts the recipient F –
2
Transferring DNA
Origin of
transfer
Origin of
transfer
A
B
C
a′
A
b′
c′
A single strand of the donor chromosome begins to be
transferred, starting at the origin of transfer. Gene A, closest
to the origin, is transferred first. DNA synthesis creates
complementary strands in both cells.
3
Transfer ends
B
A
C
A
a′
B
b′
c′
The donor and recipient cells separate, interrupting DNA transfer.
4
Integration of transferred DNA
B
A
C
A
B
c′
Hfr cell
F– cell
The donor DNA is integrated into the recipient cell’s chromosome
by homologous recombination. Unincorporated DNA is degraded.
The recipient cell is still F–.
8.9. The Mobile Gene Pool
 Genomics reveals surprising variation in gene
pool of even a single species
• Perhaps 75% of E. coli genes found in all strains
• Termed core genome of species
• Remaining make up mobile gene pool
• Plasmids, transposons, genomic islands, phage DNA
8.9. The Mobile Gene Pool
 Resistance plasmids (R plasmids)
• Resistance to antimicrobial medications, heavy metals
(mercury, arsenic)
• Compounds found in hospital environments
• Often two parts
• R genes
• RTF (resistance
transfer factor)
– Codes for conjugation
• Often broad host range
• Normal microbiota can
transfer to pathogens
R Plasmid
Carbenicillin-resistance
gene
Chloramphenicolresistance gene
Tetracyclineresistance
gene
Origin of
replication
Pilus-synthesis
genes
Origin of transfer
8.9. The Mobile Gene Pool
 Bacteria can conjugate with plants
• Natural genetic engineering
• Agrobacterium tumefaciens causes crown gall
• Different properties, produces opine, plant hormones
• Piece of tumor-inducing (Ti) plasmid called T-DNA
(transferred DNA) is transferred to plant
• Incorporated into plant chromosome via nonhomologous recombination
Plant tumor tissue
Chromosome T-DNA
T-DNA
Chloroplast
Plant DNA
Plant cell
nucleus
Ti plasmid
Agrobacterium
tumefacions cell
Agar
medium
Agrobacterium cells Crown gall tumor
inoculated into stem develops at site
of plant
of inoculation.
Bacteria-free tumor
tissue is hormone
independent and
synthesizes an opine.
Plant tumor cell
contains T-DNA
integrated into the
plant chromosome.
8.9. The Mobile Gene Pool
 Transposons yielded vancomycin resistant
Staphylococcus aureus strain
• Patient infected with S. aureus
Vancomycinresistance gene
(encoded on a
transposon
on a plasmid)
Plasmid
• Susceptible to vancomycin
• Also had vancomycin resistant
strain of Enterococcus faecalis
• Transferred transposoncontaining plasmid to S. aureus
• Transposon jumped to
plasmid in S. aureus
Staphylococcus aureus
sensitive to vancomycin
Enterococcus faecalis
resistant to vancomycin
Enterococcus faecalis
plasmid transferred
by conjugation
Transposon
jumps from
one plasmid
to another.
Plasmid from
Enterococcus
faecalis is
destroyed.
Vancomycin-resistant
Staphylococcus aureus
8.9. The Mobile Gene Pool
 Genomic islands: large DNA segments in genome
• Originated in other species
• Nucleobase composition very different from genome
• G-C base pair ratio characteristic for each species
• May provide different characteristics
•
•
•
•
Utilization of energy sources
Acid tolerance
Development of symbiosis
Ability to cause disease
– Pathogenicity islands