chapter 08 readings... · chapter 08 lecture ... mutation and horizontal gene transfer 8.1. genetic...

1

Chapter 08

Lecture

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 methicillin-

resistant S. aureus (MRSA)

• Healthcare-associated MRSA (HA-MRSA) resistant to

other antibiotics, including vancomycin

• Vancomycin considered drug of last resort

Antibiotic Resistance

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

8.1. Genetic Change in Bacteria

(a) Mutation

(b) Horizontal gene transfer

Spontaneous

mutation in genome

Vertical gene transfer

Vertical gene transfer

Plasmid transfer

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

8.2. Spontaneous Mutations

Gene X

disrupted

Transposable

element

Transposition

Gene X

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

8.2. Spontaneous 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

8.3. Induced Mutations

Gene X

disrupted

Transposable

element

Transposition

Gene X

Microorganisms commonly acquire genes from

other cells: horizontal gene transfer

• Can demonstrate recombinants

with auxotrophs

• 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

Horizontal Gene Transfer as a Mechanism

of Genetic Change

Mixture

Control: No His+ Trp+

mutants present

Glucose-salts

agar

Control: No Leu+ Thr+

mutants present

Glucose-salts agar Glucose-salts agar

Strain A :

His–

Trp –

Strain B:

Leu –

Thr –

Only phototrophic recombinants

(His+, Trp+, Leu+,Thr+) grow

Genes naturally transferred by three mechanisms

• Transformation: naked DNA uptake by bacteria

• Transduction: bacterial DNA transfer by viruses

• Conjugation: DNA transfer between bacterial cells

Horizontal Gene Transfer as a Mechanism

of Genetic Change

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

+

No effect

Mouse dies

No effect

Mouse dies

Living encapsulated

cells isolated

Living non-encapsulated cells

Heat-killed encapsulated cells

Heat-killed encapsulated cells

Living non-encapsulated cells

Living encapsulated cells

Organisms injected Results

Transformation

• 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 two-

component regulatory system

• Recognizes low nitrogen or carbon

• High concentration of bacteria

(quorum sensing)

• Only a fraction of population

becomes competent

8.6. DNA-Mediated Transformation

1

2

3

4

5

Recipient chromosome

Double-stranded DNA binds to the surface of a competent cell.

Gene

Conferring StrR

Single strand enters the cell; the other strand is degraded.

The strand integrates into the recipient cell’s genome by homologous

recombination. The strand it replaced will be degraded.

Streptomycin-sensitive

daughter cell

Streptomycin-resistant

daughter cell

After replicating the DNA, the cell divides.

Non-transformed cells (StrS) die on streptomycin-containing medium,

whereas transformed cells (StrR) can multiply.

Gene conferring StrS

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

8.7. Transduction

(a) Formation of a transducing particle

Transducing

particle

Phage Transducing particle

A transducing particle

attaches to a specific

receptor on a host cell.

1

The bacterial DNA is

injected in to a cell. 2

The injected bacterial

DNA integrates into the

chromosome by

homologous recombination.

3

Bacteria multiply with new genetic material.

Replaced host DNA is degraded. 4

(b) The process of transduction

Bacterial

DNA

Replaced

host DNA

5

3

4

A bacteriophage attaches to a

specific receptor on a host cell.

1

2 The phage DNA enters the cell.

The empty phage coat remains

on the outside of the bacterium.

Enzymes encoded by the

phage genome cut the bacterial

DNA into small pieces.

Phage nucleic acid is replicated

and coat proteins synthesized.

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.

Conjugation: DNA transfer between bacterial cells

• Requires contact between donor, recipient cells

• Conjugative plasmids direct their own transfer

• Replicons

• F plasmid (fertility)

of E. coli most

studied

8.8. Conjugation

2 µm

F pilus

Conjugation (continued…)

• 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+

8.8. Conjugation Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Making contact

Chromosome F plasmid

F pilus

Donor cell F+ Recipient cell F–

Origin of

transfer

The F pilus contacts the recipient F– cell.

Initiating transfer

The pilus retracts and pulls the donor and recipient cells together.

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.

One strand is cut in

the origin of transfer

Transfer complete

1

2

3

4

F+ cell F+ cell

At the end of the transfer process, both the donor and recipient

cells are F+ and synthesize the F pilus.

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

8.8. Conjugation

Formation of an Hfr cell

The F plasmid sometimes

integrates into the bacterial

chromosome by homologous

recombination, generating

an Hfr cell; the process

is reversible.

Hfr cell

F+ cell

Integrated

F plasmid

F pilus

Formation of an F′ cell

Integrated

F plasmid

Chromosome

Chromosomal

DNA

Hfr cell

F′ cell

F pilus

An incorrect excision of the

integrated F plasmid brings

along a portion of the

chromosome, generating

an F′ cell.

F′ plasmid

F plasmid Chromosome

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

8.8. Conjugation

A

B C

A

A

a ′

b ′ c ′

B A

B

C

A B

C

A A B

C

B c ′

a ′

b ′ c ′

a ′

b ′ c ′

4

3

1

2

Making contact

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.

Origin of

transfer

Origin of

transfer

Recipient cell F– Donor cell Hfr

The F pilus contacts the recipient F–

Transfer ends

Transferring DNA

The donor and recipient cells separate, interrupting DNA transfer.

Integration of transferred DNA

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–.

Chromosome

Origin of

transfer

Chromosomal

genes

Integrated

F plasmid

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

Chloramphenicol-

resistance gene

Pilus-synthesis

genes

Origin of transfer

Origin of

replication

Tetracycline-

resistance

gene

Carbenicillin-resistance

gene


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 non-

homologous recombination

Agrobacterium

tumefacions cell

Agrobacterium cells

inoculated into stem

of plant

Crown gall tumor

develops at site

of inoculation.

Bacteria-free tumor

tissue is hormone

independent and

synthesizes an opine.

Plant tumor cell

contains T-DNA

integrated into the

plant chromosome.

Ti plasmid

Chromosome T-DNA Plant tumor tissue

T-DNA

Plant DNA

Plant cell

nucleus

Chloroplast

Agar

medium

Transposons yielded vancomycin resistant

Staphylococcus aureus strain

• Patient infected with S. aureus

• Susceptible to vancomycin

• Also had vancomycin resistant

strain of Enterococcus faecalis

• Transferred transposon-

containing plasmid to S. aureus

• Transposon jumped to

plasmid in S. aureus


Enterococcus faecalis

plasmid transferred

by conjugation Enterococcus faecalis

resistant to vancomycin

Plasmid


sensitive to vancomycin

Transposon

jumps from

one plasmid

to another.

Plasmid from

Enterococcus

faecalis is

destroyed.

Vancomycin-resistant


Vancomycin-

resistance gene

(encoded on a

transposon

on a plasmid)

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


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