Download Gene movement in bacteria Transformation Bacteria that undergo

Transformation with naked DNA
Gene movement in bacteria
• Griffith mixed dead
pathogenic smooth
Strep. pneumoniae cells
with live rough cells in
mouse infection.
• S.pneumoniae cells
from dead mice were
smooth.
• Avery, MacLeod, &
McCarty showed the
“transforming substance”
from dead smooth
S.pneumoniae cells
was nuclease-sensitive
Phages can mediate
transfer of DNA to new
bacterial cell. Donor for
the DNA dies in lytic
infection. In some
bacteria, naked DNA
from dead donor can be
taken up into recipient
cell.
Fig. 10.11 top
Transformation
Natural transformation (upper
scheme) occurs in variety of
eubacteria and archaea;
uptake of linear DNA
fragments, which must
recombine with host DNA
Molecular biology and cloning
have led to development of
artificial means to make cells
take up autonomously
replicating dsDNAs or
plasmids (lower scheme)
Bacteria that undergo
natural transformation
Gram positive:
Streptococcus
pneumoniae
Bacillus subtilis
Gram negative:
Haemophilus influenza
Neisseria gonorrheae
Acinetobacter
calcoaceticus
Bacteria that undergo
artificial transformation
Escherichia coli
Salmonella typhimurium
Pseudomonas aeruginosa
(Salt + cold temps; dsDNA taken up by cells)
Chemically induced:
Electroporation:
Many species of
eubacteria and archaea
(and some eukaryotes)
(Pulsed electrical fields; dsDNA)
Natural
transformation
Fig. 10.14
Three stages:
a) Acquisition of
competence
b) DNA uptake
c) Recombination
•Stage a varies by species
•Stage b varies between
Gram+ vs. Gram •Stage c is the same in
all Bacteria
Natural transformation : Gram +
Fig. 10.14
DNA uptake:
1) nonspecific
linear dsDNA
(10-20 Kb) binds
to 10-50 sites/cell
2) DNA breaks
to 6-8 Kb pieces
3) nuclease
converts to
ssDNA during
transport across
membrane
(100 nt/sec) Free nucleotides
released in medium
Natural transformation
DNA uptake stage:
•In G–, dsDNA
becomes nuclease
resistant as it passes
through OM into
some protected
compartment via OM
“secretin” protein
•In G+/–, passage
into cytoplasm via
conserved cyto.
membrane channel
Transformation
in Haemophilus influenzae
DNA-binding sites
Cell envelope
Transformasomes
Competence
Uptake
Specific DNA sequences
recognized in transformation
Transformasome
Specific
DNA binding
Seq. specific receptors?
3’
Translocation
Recombination
Specific 9-11 bp sequence in dsDNA required for binding
to competent cell (5’ AAGTGCGGT 3’).
Transformed
genome
Gene movement in bacteria, part III
Conjugation allows DNA movement from live
donor to recipient, dependent on cell-cell contact.
The proteins that enable this movement are
usually encoded by genes on plasmids.
Fig 10.19
• H. influenzae: 5’ AAGTGCGGT 3’ sequence is
present 1465 times in genome-every 4Kb (vs. 8-9
predicted frequency, if random)
• Neisseria: 5’GCCGTCTGAA 3’sequence is present
1910 times in genome (vs. 4 predicted)
• Acinetobacter also seems to prefer to take up specific
DNA, but basis of selectivity is unknown
Specific DNA may allow repair of essential functions or
allow testing of options to ! fitness
No sequence motifs for DNA binding for Gram +
Plasmids
Fundamental characteristics for each plasmid:
• Size/Copy number-from 3 to 200 Kb and
from 1 to 100/cell
• Host range-bacterial strains where the plasmid will
replicate
• Incompatibility group-refers to whether 2 plasmids
can be maintained in same cell; based on
compatibility of replication and segregation
systems.
Representative bacterial plasmids
Plasmid
Size (Kb)
/ CN
100 / 1
Host range
Traits
Narrow
(E. coli)
Conjugal; phage
sensitivity
R100
89 / 1
Enterics
Ti
200 / 1
Agro/Rhizo
Conjugal; phage
sensitivity; drug &
Hg resistance
Causes plant tumor
RP4
60 / 4
ColEI
9/30
Broad:
Conjugal; drug
Gram neg.
resistance; phage
sensitivity
(Ec/Pseud)
Narrow
Mobilizable; colicins
F
F plasmid and R100
• Can mediate their transfer from donor to recipient by
conjugation and are sensitive to particular phages
due to conjugal pili
• Have similar yet distinct Inc functions and are
compatible with each other
Fig 10.18
• Conjugal
ability due to
transfer (tra
genes)
• 99kb plasmid
contains
several IS/Tn
elements
• Rep/Par
functions
Plasmid transfer via F
conjugation
!
!
F plasmid
F (fertility) plasmid
! oriC of
chromosome
R100 (or RP4) plasmid
Low copy number plasmids seem to localize to particular spots during division.
Fig. 10.22a
Mobilizable plasmids
DNA transfer in
F conjugation
Conjugal plasmids encode proteins for an apparatus that
allows DNA movement.
Mobilizable plasmids (like ColEI) can exploit the apparatus
encoded by a co-resident conjugal plasmid. Usually a
moblizable plasmid-specific DNA-processing function (still
using ssDNA transfer process). ColE1 cannot move by
itself!
• oriT is nicked,
5’"3’ ssDNA
transfer
• DNA pol III, etc
replicates
(leading in
donor)
• Religation of
oriT at end
Without a conjugal plasmid,
ColE1 cannot mobilize from here!
Fig. 10.22b
F plasmid can integrate into
E. coli chromosome
Fig. 10.23
= conjugal plasmid
F plasmid and E. coli Hfrs
When plasmids recombine
with chromosome,
conjugation functions
can lead to transfer of
chromosomal DNA (next
to integrated plasmid)
to recipient via conjugative
apparatus.
IS/Tns can allow
plasmids to recombine
with host chromosomes
Process forms
Hfr…
Fig. 10.24
Directionality of process
leads to rare transfer of
tra genes
Conjugal plasmids can mediate
two distinct DNA transfer events
Conjugal or mobilizable
In both cases, ssDNA
transferred
Fig 10.11
DNA must recombine with
recipient; recipient rarely becomes
a donor; 10-100 kb transferred
Gene movement: the bacterium
fights back…
While many mechanisms to move DNA from
one cell to another exist, the bacterial cell is
not necessarily a “passive” recipient. Some
incoming DNA can obviously have negative
impact on cell (Phage infection/sensitivity).
Bacteria have developed one important strategy
to combat the flow of DNA into a cell:
Restriction-Modification (R-M) systems
Outcomes from exchange of genetic info
Gene substitution:
transduction, natural
transformation, or
Hfr conjugation
Gene addition:
phages forming
lysogens, plasmid
conjugation &
mobilization, or
artificial
transformation with
plasmids.
Discovery of R-M systems
Work from several phage groups (50’s-60’s):
# infects both B and K strains of E. coli, but…
• # preps grown on E. coli B strain with 10000x
lower titer on K strain than on B
• # preps grown on E. coli K strain with 10000x
lower titer on B strain than on K
• Discovered that reduction in “efficiency” of
infection due to strain-specific nucleases
• Demonstrated that the R-M enzymes act
indiscrimantly on dsDNA in cell; normal host
DNA is protected due to its modification
Discovery of R-M systems
Grown on B
• Upon entering E. coli K,
DNA from # grown on
B strain could either be
degraded (restricted) or
modified
• If modified, subsequent
infections of phage in K
strains would not be
subject to K-specific
restriction
See Fig. 7.21
K cell
The EcoRI restriction enzyme makes staggered cuts on
both strands of DNA, leaving ss “sticky ends”. The
modifying enzyme adds -CH3 to 1 base of each strand in
recognition sequence and prevents cleavage. R-M systems
widespread in Bacteria and Archaea (rare in euks).
Three types of R-M systems
Type I
EcoB
Type II
More on R-M systems
Type III
TGAN8TGCT
EcoRI
GAATTC
EcoPI
AGACC
ca 1 Kb
away
(distant)
Between G
and A (in
sequence)
24-26 bp on
3’ side
(closeby)
Joint Nuclease/
Methylase?
Yes
No
Yes
ATP-dependent
Yes
No
No
Example
Recognition
site
Cleavage
site
Recognition sequence for
E. coli Type II R-M enzyme
Because they have separate R/M enzymes and they
cleave in recognition sequence, the restriction
endonucleases of Type II systems are useful for
molecular biology.
Restriction enzymes recognizing different sequences
have been isolated from wide variety of bacteria.
Type II systems most common but Type I systems
widely distributed; Type III systems rare
Phages and plasmids have developed ways to
circumvent the protection hosts get from RM systems