Transcript Microbial Genetics - University of Montana

Microbial Genetics
MICB404, Spring 2008
Lecture #14
Conjugation: Mechanisms of
plasmid-mediated gene transfer
• Summaries, exam and quiz returned today
• New quiz. Due Friday 03-07-08
• Supplemental reading posted on website (URL to be
distributed).
1) Lanka and Wilkins (1995) DNA processing
reactions in bacterial conjugation. Annu. Rev.
Biochem. 64, 141-169.
2) Foley et al., (1998) A short noncoding viral DNA
element showing characteristics of a replication
origin confers bacteriophage resistance to
Streptococcus thermophilus. Virology 250, 377387.
Review
ColE1 plasmids
Mutation of RNA I = Mut of RNA II
Different secondary structure
Altered G + C content or distribution
Foley et al., 1998
Article is, or will be, available on MICB404 website
Plasmids containing phage ori
Acts as an origin of replication driven by phage infection.
Could a plasmid contain no genes?
Conjugation
• One bacterium (the "male" or donor cell)
transfers DNA to another (the "female"
or recipient cell) while the cells are in
physical contact.
• The recipient that has received DNA
from a donor is called a
“transconjugant”
Conjugation
• Lederberg & Tatum, 1947
– started with two strains of E. coli that had
different nutritional deficiencies caused by
mutations in genes that normally synthesize biotin,
cysteine, leucine, phenylalanine, thiamine, and
threonine
• “. . . single nutritional requirements were established as
single mutational steps under the influence of X-ray or
ultra-violet. By successive treatments, strains with
several requirements have been obtained”
– strains were both triple auxotrophic mutants, as
shown below, where the genes are listed
alphabetically:
Conjugation
• Lederberg & Tatum, 1947
Strain Y10
Strain Y24
bio + cys+ leu- phe+ thi- thrbio- cys- leu+ phe- thi+ thr+
– Neither auxotrophic strain grows on
minimal medium
– Mix 2 strains together, plate to minimal
medium
• Isolates recovered with prototrophic phenotype
Conjugation
• Lederberg & Tatum, 1947
Two strains exchanged genetic information; progeny had
characteristics of both parents
Conjugation
• Genetic information shared during
conjugation is plasmid DNA
– Transfer is replicative
– Process similar to rolling-circle replication
• One single DNA strand remains in donor, one
transferred to recipient
• Both ssDNA are replicated
Conjugation
• Plasmids can be
– self-transmissible
• encode all functions required for conjugation
– mobilizable
• encode some functions required for transfer
• rely on self-transmissible plasmid for remainder
Conjugation
• Transfer occurs via physical contact
– Gram-negative bacteria: sex pilus
• produced from genes on self-transmissible
plasmid
• tra genes
– various types of transfer systems
– correlate with Inc group to prevent transfer of
plasmids to recipients already carrying plasmids
incompatible with donor cell plasmid
Conjugation
• tra genes: 11 or
more in this
large selftransmissible
plasmid
– oriT, origin
required for
conjugation
Conjugation
• Transfer between strains of one species
• Transfer between different bacterial
species
– Promiscuous plasmids
• e.g. IncN, IncP, IncW
• IncP: self-transmissible and capable of
mobilization from E. coli to most other Gramnegative bacteria, and to others (g+, plants)
– Evolutionary role: similar genes found in
evolutionarily distant organisms
– Spread of antibiotic resistance
Mechanism of Conjugation
• Overview I
– Contact made between
donor and recipient
• pilus formation
– Plasmid is nicked
– One DNA strand
attached to relaxase
enzyme through ester
bond
• transesterification
• plasmid duplex separated
by strand displacement
Mechanism of Conjugation
• Overview II
– DNA strand attached
to relaxase transferred into recipient
cell
• replication of donor
cell strand from 3’-OH
of transferred strand
– ssDNAs are
recircularized
– complementary strand
synthesized in
recipient
Mechanism of Conjugation
• tra genes
– DNA transfer and replication (Dtr)
component: functions required to prepare
plasmid DNA for transfer
– mating pair formation (Mpf) component:
proteins involved in forming transfer
structure
• Membrane associated
• Sex pilus
Mechanism of Conjugation
• DNA processing (Dtr) component
proteins
– Relaxase: endonuclease, introducing
specific nick in plasmid
• Protein bound to 5’-PO4
– Relaxase is then
transferred into
recipient cell along
with DNA
Mechanism of Conjugation
• Relaxase
– In recipient cell, enzyme recircularizes
plasmid by breaking protein-DNA bond and
ligating 5’-PO4 with 3’-OH
Mechanism of Conjugation
• Relaxosome
– Multi-component complex containing
relaxase
• Accessory proteins (binding)
• Regulatory proteins (coordinating Dtr and Mpf
activities)
• Helicase (strand separation at oriT)
Mechanism of Conjugation
• Primase
– Synthesize RNA primers required for
complementary strand synthesis of
recipient cell strand
– Translated in donor cell, transferred into
recipient cell
Mechanism of Conjugation
• Mpf component proteins
– Cell-cell attachment
– Formation of channel through which
transfer takes place
– Regulate Dtr activity
– Akin to virulence protein secretion systems
& natural transformation factors
Mechanism of Conjugation
• Pilus
– Composed of pilin protein molecules
– Attaches donor to recipient
– DNA transfer occurs
through channel or pore,
not pilus
Mechanism of Conjugation
• Coupling proteins
– Sense contact with recipient cell
– Switch on Dtr activity (signals relaxase)
– Up-regulate what proteins are transferred
to recipient
Mechanism of Conjugation
• tra gene regulation
– Expressed immediately after transfer to
recipient is completed
• Subsequently repressed, with sporadic periods
of expression
– prevents exploitation of pilus as bacteriophage
infection site
• Despite low frequency of tra gene expression in
individual cells, at population level conjugation
leads rapidly to spread of plasmid among
potential recipients
– Transfer is nearly 100% efficient
Mobilizable plasmids
• Plasmids requiring tra gene functions
from self-transmissible plasmids for
cell-cell transfer
– Minimum requirement for mobilizable
plasmid is oriT sequence
– In nature, encode Dtr component functions
• mob genes
• allow mobilizable plasmids to exploit tra
functions without common oriT sequence
• Dtr system of mobilizable plasmid must be able
to respond to coupling protein of coresident
self-transmissible plasmid
Mobilizable plasmids
• Donor cell has mobilizable and selftransmissible plasmids
• Coupling protein of selftransmissible plasmids signals cell
contact made
• mob relaxase initiates transfer of
mobilizable plasmid DNA strand
• Mobilized plasmid replicated in
recipient cell
• Self-transmissible plasmid can be
transferred too
Chromosomal transfer
• During conjugation, transfer of
chromosomal DNA can sometimes occur
– When plasmid has integrated into
chromosome
– When chromosome contain oriT sequence,
e.g. after transposon insertion
– When plasmid contains chromosomal DNA
• Homologous recombination
• transposition
Chromosomal transfer
• Hfr strains (High frequency
recombination)
– Plasmid integrates into chromosome, then
during conjugation, chromosomal DNA
carried along in transfer
– Integration
• recombination
– Insertion Element (IS) transposons provide sequence
homology
• transposition
Chromosomal transfer
• Recombinational integration
– One IS2 site in plasmid, 20
in E. coli chromosome
• 20 different Hfr strains
possible
– Other Insertion Element
sites permit integration
as well
• IS3
• 
Hfr strain DNA transfer
• Expression of tra genes
– Cell-cell contact
– Relaxase expression
• Chromosome nicked at oriT
• Plasmid DNA strand
transferred into recipient
– with replication in donor
Hfr strain DNA transfer
• Plasmid DNA transfer followed by
chromosomal DNA
– Entire chromosome transferred in 100
minutes at 37C
– Usually only fragment of
chromosome transferred
– Fragment can be incorporated into recipient
chromosome by
recombination
• otherwise lost: not all
of plasmid transferred
Hfr strain DNA transfer
• Hfr and recipient of
different genotypes
– Transfer of alleles can
result in recombinant
transconjugant
• Genes close to oriT
transferred at higher
frequency than genes
further away
– gradient of transfer
Hfr gene mapping
• Genetic markers
– Alleles, mutations
– Transposons
– Phenotypic consequences allow position of
marker to be mapped
• in recipient (transconjugant) cell
– Select for recombinant with one marker
• test for other markers
Hfr gene mapping
• Transfer is linear and unidirectional
– So order of markers on chromosome with
respect to oriT can be determined from
frequency of transfer
– By selecting for one marker, then scoring
frequency of recombinants for other
markers, gene positions can be deduced
• Most matings interrupted before entire
chromosome transferred
Hfr gene mapping
• To map rif marker,
cross donor with F- at
42 minutes
– donor is proC and
rif-8 (sensitive to rifampicin)
Hfr gene mapping
F in this
strain
E. coli chromosome genetic map
Hfr gene mapping
• Recipient is hisG1,
argH5, and trpA3
– and rif (resistant to rifampicin)
• Donor & recipient allowed to
mate
• Plated for selection
– No proline or (histidine,
arginine, or tryptophan)
Hfr gene mapping
• Recombinants will be recipients that
have received wildtype hisG, argH, or
trpA from donor
• Replica-plate to tester medium for
unselected markers
– e.g. His+ recombinant: plate to –Arg, -Trp,
and Rif plates
Hfr gene mapping
• Plot frequency of marker recombinants
versus E. coli genetic map
position
– In this case, relative to
hisG
• Frequency of unmapped
marker indicates its
location on chromosome
Prime Factors
• Plasmids that contain
chromosomal genes
– F plasmid with chromosomal genes: F’
– R plasmid with chromosomal genes: R’
• Products of recombination
or transposition
– e.g. from Hfr
– Recombinational excision can
carry chromosomal fragment
with it
Prime Factors
• Complementation testing
– Allelism: are 2 mutations in same or
different gene?
– Dominance/recessivity
– Molecular nature of mutation (point, indel…)
– Function acting in cis or in trans
(cis
– Prime Factors create partial diploids
(merodiploids)
Prime Factors
• Selection
– Early transfer of distal markers
– Replicons
Prime Factors
• Selection
– Early transfer of distal
markers
• Markers that were far from
oriT in chromosome of Hfr
will be close in F’, and
transferred early
• Recipient is merodiploid
and transconjugant, not recombinant
• Capable of conjugation
Prime Factors
• Selection
– Replicons
• Capable of replication
independent of chromosome
• If recipient is defective
in recombination, prime
factor transconjugants can
acquire and transmit selected
marker but no recombinants
from Hfr individuals form
Descriptive Statistics
N
points
Valid N (listwise)
17
17
Minimum
62.50
Maximum
148.13
Mean
99.6691
5
Count
4
3
2
1
80.00
100.00
points
120.00
140.00
Std. Deviation
20.83829
Descriptive Statistics
N
points
Valid N (listwise)
16
16
Minimum
62.50
Maximum
118.13
Mean
96.6406
5
Count
4
3
2
1
70.00
80.00
90.00
points
100.00
110.00
Std. Deviation
17.22987