Genetic Exchange

Sean D. Reid, Ph.D.

Microbiology and Immunology
Rm 8, 402-6287
SeanReid@RossU.edu

Practice questions in eCollege day after lecture

 Learning Objectives

• Demonstrate an understanding of the importance of genetic

exchange to microorganisms and its impact on virulence and
antimicrobial drug resistance
• Demonstrate an understanding of the vocabulary used in
discussing genetic exchange
• Demonstrate knowledge of the different forms of genetic
recombination and their impact
• Demonstrate knowledge of transformation, competence,
bacterial conjugation, and transduction
• Be able to compare and contrast lytic and lysogenic life cycles
• Demonstrate an understanding of transposable elements and
pathogenicity islands
Why is understanding genetic exchange critical
to medicine?
Genetic information can be transmitted by vertical or horizontal transmission

Vertical Bacteria l pathogen with

transmission gene X gene X

Binary fission

gene X gene X

Horizontal
transmission Bacteria lacking
gene X gene X

Gene transfer event

Bacteria acquire
gene X gene X
 Recombination

• Incorporation of
extrachromosomal
(foreign) DNA into the
chromosome
• Homologous
recombination
– Occurs between closely
related DNA sequences and
generally substitutes one
sequence for another
– Requires enzymes (rec
genes)
 Recombination continued

• Nonhomologous (illegitimate) recombination

– Occurs between dissimilar DNA sequences
– Yields insertions of DNA or deletions or both
– Specialized recombination enzymes (sometimes site-specific) such as
those produced by transposons and bacteriophages

Shared endonuclease
site
Case example (MRSA - Lancet, 358:424)

A male infant born with congenital airway obstruction was mechanically

ventilated because of respiratory problems and subsequently developed a
respiratory tract infection. Eventually, his blood cultures grew a methicillin-
susceptible S aureus (MSSA) and he was treated with flucloxacillin. He then
seemed to recover, but amoxicillin and clavulanic acid were initiated on day 32
for 10 more days because his respiratory tract infection recurred. On day 56,
routine cultures of nasal swabs unexpectedly showed a mecA+ MRSA that was
resistant to -lactam antibiotics, but susceptible to other antibiotics. Several
strains of mecA+ coagulase-negative staphylococci were also identified.
Molecular analysis revealed that, except for the mecA DNA, the MSSA and
MRSA isolates were isogeneic — ie, they evolved either by the loss or acquisition
of mecA DNA. The order in which we isolated the strains (mecA− before mecA+)
supports the horizontal gene transfer of mecA DNA.

mecA encodes an altered penicillin binding protein that is proficient for cell wall
synthesis but is not inhibited by -lactam antibiotics or cephalosporins. The
mecA gene allows a bacterium to be resistant to antibiotics such as methicillin,
penicillin and other penicillin-like antibiotics
 Facts on MRSA

• Methicillin introduced in 1959. MRSA appears by

the early 1960s.
• mecA is carried on a mobile genomic element:
Staphylococcal Chromosomal Cassette (SCCmec).
• Encodes an altered penicillin binding protein (PBP
2a).
 MRSA Facts (FYI)

• Four types of SCCmec, each with a varying array of drug

resistance.
• SCCmec II and III are found in many nosocomial MRSA
strains. These chromosomes harbor other antibiotic-
resistance genes conferring resistance to
aminoglycosides, tetracyclines, erythromycin and
clindamycin.
• CA-MRSA carries SCCmec IV which only carries the
mecA gene.
Four major mechanisms of gene transfer (Fig. 13-15)

Transformation: uptake of naked

DNA

Transduction: infection by a
nonlethal virus carrying
bacterial genes

Conjugation: plasmid
mediated exchange
of information between
bacteria in contact

Transposition: exchange of
genetic information via
mobile genetic elements
Relevance of horizontal gene transfer to pathogenesis - example

Transformation: uptake of naked

DNA

Transduction: infection by a
nonlethal virus carrying
bacterial genes

Conjugation: plasmid
mediated exchange
of information between
bacteria in contact

Transposition: exchange of
genetic information via
mobile genetic elements
Clinically relevant example of the impact of Transformation: uptake of naked
transformation on bacterial pathogens DNA

Streptococcus pneumoniae Haemophilus influenzae

•Significant cause of meningitis and otitis media

•Capsule plays an important role in pathogenesis

•Streptococcus pneumoniae and Haemophilus influenzae are two examples of naturally competent
pathogens

•Capsule genes of non capsulated strains acquired by transformation

Relevance of horizontal gene transfer to pathogenesis - example

Transformation: uptake of naked

DNA

Transduction: infection by a
nonlethal virus carrying
bacterial genes

Conjugation: plasmid
mediated exchange
of information between
bacteria in contact

Transposition: exchange of
genetic information via
mobile genetic elements
Phage terminology
Lytic - phage that causes killing of host bacterium
Lysogenic - phage that can survive in the bacterium without killing -
usually integrated in the bacterial chromosome.
Temperate - phage capable of undergoing both lytic or lysogenic
development
Virulent - phage capable of undergoing only lytic development
Lysogen - Bacterium harboring a lysogenic phage

Prophage- Integrated phage genome in a bacterium (lysogen).

 Transduction

DNA transfer mediated by bacteriophages

l E. coli + l close-up

15
Phage Life Cycles –
Lytic and Lysogenic
 Bacteriophage Life Cycle - Lytic

• Goal is to replicate and get out - virulent

• Phage DNA enters cell, replicates, synthesizes phage
heads, assembles, and cell is lysed
 Bacteriophage Life Cycle - Lysogeny

• Goal is long-term – temperate

• Stable integration of a viral genome into bacterial genome; viral genome =
prophage; dormant
• Lysogenic conversion: Prophage alters bacterial cell phenotype;
outcomes e.g. diphtheria, scarlet fever, botulism…
• DNA of phage may encode bacterial virulence factors: e.g. Cholera toxin
• Prophage excises at insertion sequence when stressed (e.g. DNA damage; UV
irradiation)
• Some become lytic, others exit the cell without lysis
 Generalized Transduction
Modern Genetic Analysis.
Griffiths AJF, Gelbart WM, Miller JH, et al.
New York: W. H. Freeman; 1999.

• Lytic lifecycle
• Fragment of host DNA accidentally packaged in
phage instead of phage DNA
• When that phage injects the bacterial DNA into a
new host, bacterial genes are transferred -
homologous recombination
• Phage DNA not present, so recipient cell is
transduced, but not infected

19
1) Prophage exists in host
containing the adjacent gal
gene.
Specialized
2) Phage genome excises, transduction
carrying with it the
adjacent gal gene from
host.
3) Phage matures, cell
lyses, releasing phage
carrying gal gene.

4) Phage infects a cell that

has no gal gene.

5) Along with the

prophage, the bacterial gal
gene integrates into the
new host’s DNA.

6) Lysogenic cell contains

gal gene.
 GENERALIZED SPECIALIZED
TRANSDUCTION TRANSDUCTION
Lytic Cycle: Phage head Lysogenic Cycle: When phage
packages random host excises, it brings flanking host
(bacterial) DNA fragment DNA with it (specific genes =
(‘general’) ‘specialized’)

DNA must be integrated by Phage + host genes integrated

homologous recombination – by phage recombinases and
low rate of success (depending homologous recombination-
on sequence) high rate of success
 Bacteriophage Host Range

• Determined by adhesins (e.g. on tail fibre) and host cell

receptors
• Broad host range
– Relatively rare, but probably very important in DNA
transfer between genera
• Narrow host range
– Much more common, can only infect 1 species, or
even only one or a few strains
Phages play an important role in microbial pathogenesis

•The diphtheria exotoxin of the bacterium Corynebacterium diphtheriae is

produced only when that bacterium is infected with a specific prophage (beta
phage).

•The erythrogenic toxin causing the symptoms of scarlet fever is produced only
when the group A streptococcus is infected with a specific prophage.

•The neurotoxin produced by Clostridium botulinum is encoded by a phage.

•Exfoliatin, an exotoxin that causes scalded skin syndrome, is produced by

Staphylococcus aureus as a result of lysogenic conversion.

•A lysogenic phage encodes an exotoxin produced by Vibrio cholerae.

Relevance of horizontal gene transfer to pathogenesis - example

Transformation: uptake of naked

DNA

Transduction: infection by a
nonlethal virus carrying
bacterial genes

Conjugation: plasmid
mediated exchange
of information between
bacteria in contact

Transposition: exchange of
genetic information via
mobile genetic elements
 F+ x F- Conjugation
• Cell-to-cell transfer of DNA from one bacterium
(male/donor) to another (female/recipient)
• Direct cell-cell contact
• One direction: ♂ → ♀
• ♂ need conjugative F plasmid
– fertility plasmid (F+) encoding
fertility factor
• Transfer occurs via unique
pilus: sex pilus = bridge =
F-pilus
• F-pilus encoded by tra operon
on the F plasmid
• Sex pilus =
Type IV Secretion System 25
http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_3.html
 Bacterial Mating Types
• 1) F- ♀ or 2) F+ ♂ (free plasmid) or 3) Hfr (high frequency
recombination cell
– F plasmid stably crossed into the chromosome = episome
• An HFR strain can transfer chromosomal DNA very
efficiently
– transfer begins at the break in oriT:
• Note: Usually transfer of this large genome/plasmid breaks
off; recipient does not become Hfr or F+ because usually
not all tra genes are transferred (other end of episome)
• Homologous recombination stabilizes the new genes

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter13/animation_quiz_4.html
A typical F+ cell containing an F plasmid.

1) F plasmid integrates into chromosome

by recombination.

2) HFr cells joins an F- cell via a

conjugation pilus.

3) Part of the F plasmid partially moves

into recipient cell trailing a strand of
donor’s DNA.
4) Conjugation ends with pieces of F
plasmid and donor DNA in recipient cell;
cells synthesize complementary DNA
strands.

5) Donor DNA and recipient

DNA recombine, making a recombinant
F-cell (F minus).
 F’ plasmids

• F’ [F prime] plasmids form by incorrect excision of

integrated F plasmid
– Piece of host chromosomal DNA attached to
plasmid.
• Can now horizontally transfer bacterial genes from
host to host
 Conjugative Transfer of Bacterial Genes

• Hfr mating type

• F’ plasmid
• Integration of genes onto F plasmid by another process
(usually transposition, described a bit later)
• Strains permissive for conjugation:
– Have receptor for the specific F pilus
– Can replicate the origin of the plasmid
– Have compatible restriction/modification systems
 Host Range in Conjugation

• Some conjugative plasmids can mediate transfer to many

different species
– broad host range or promiscuous
– related to a strong degree
– Some streptococcal plasmids can be transferred to
Lactococcus spp., Lactobacillus spp., Enterococcus spp.,
Staphylococcus spp., Listeria spp., Pedicoccus spp.
– Even some into eukaryotic cells…
• Some are very specific, known as narrow host range
Relevance of horizontal gene transfer to pathogenesis - example

Transformation: uptake of naked

DNA

Transduction: infection by a
nonlethal virus carrying
bacterial genes

Conjugation: plasmid
mediated exchange
of information between
bacteria in contact

Transposition: exchange of
genetic information via
mobile genetic elements
 Transfer of Bacterial Genes: Transposition

Jumping genes!
DNA that can move itself (non-replicative) or copy itself
(replicative) into new places
Happens within same cell
Plasmid to chromosome
Chromosome site to chromosome site
 Insertion Sequences

• Insertion sequence (IS) is the smallest transposable element (most simple

form of transposon)
• Encodes transposase (tnp) and perhaps regulatory protein(s)
• Flanked by inverted repeats
– Facilitates transposase binding and insertion into new site
• Possesses terminators and stop codons —inactivates the genes into which
it is inserted; cell might die
 Non-composite and composite transposons

• Non-composite transposons are insertion sequences with

genes inserted into the IS
• Composite transposons consist of two IS carrying other genes
between them
– Possible to carry antibiotic resistance genes
– Genes can be added to the element
• Allow transfer of phenotype
 Pathogenicity island
• Clusters of genes, often multiple operons
• Acquired from other places/different G-C content
– Often transported by phages
• Encode numerous virulence factors
• Contain transposable elements or remnants
 Why is all of this so critical?
• Simply put, you are likely to see a patient with
a bacterial infection where the bacterium has
acquired antibiotic resistance and/or
increased virulence properties through one or
more of these mechanisms
 Fig. 13-16 Murray 7th edition

Genetic mechanisms of evolution of methicillin- and vancomycin-resistant Staphylococcus

aureus (MRSA and MVRSA). Vancomycin-resistant enterococcus (VRE) (in red) contains
plasmids with multiple antibiotic resistance and virulence factors. During co-infection, a
MRSA may have acquired the enterococcal resistance plasmid (e-plasmid) by
transformation (after lysis of the enterococcal cell and release of its DNA) or more likely,
by conjugation. A transposon in the e-plasmid containing the vancomycin resistance gene
jumped out and inserted into the multiple antibiotic resistance plasmid of the MRSA. The
new plasmid is readily spread to other S. aureus bacteria by conjugation.
The resulting MVRSA is resistant to :
β-lactams
vancomycin
trimethoprim
Gentamycin/kanamycin/tobramycin
quaternary ammonium disinfectants
 Carbapenem-resitant Enterobacteriaceae

• Also referred to as CRE

• Possesses NDM-1, an enzyme which allows them to cleave
carbapenems, one of the most powerful types of antibiotics
available to doctors.
– New Delhi metallo-ß-lactamase-1
• Nightmare Scenario:
– Gene for NDM-1 can be transferred to a bacterium that is resistant to
all other antibiotics. e.g. VISA, VRSA
– At least one NDM-1 bacterium has been found that is resistant to all
known antibiotic
 There is probably no chemotherapeutic drug to which in

suitable circumstances the bacteria cannot react by in

some way acquiring ‘fastness’ [resistance].”

—Alexander Fleming, 1946

Read more:
http://www.dailymail.co.uk/health/article-1302035/Unbeatable-NDM-1-enzyme-make-
bacterial-diseases-superbugs.html#ixzz1AkZdbx3d
http://www.ncbi.nlm.nih.gov/pubmed/21530894
http://www.ncbi.nlm.nih.gov/pubmed/20920882