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More unA exchange and whaL
bacLerla do abouL lL..
8eadlng: pp 33-34, 309-314
!"#$%&':
() *+,&-./+0,1/&2 4+,0 &'5,16' 0/7'$-
,&7 -'$. 6-) &/&8-'$. 9:;
<) ='-#+%>1/&8?/7%@>,1/& -A-#'0-
B) C=DEF= '$'0'&#-
8ecap: naLural Lransformauon sysLems

WhaL ls Lhe Lransformlng prlnclple, proLeln or unA? (1928/1944)
S. pneumoniae virulence
depends on a capsule:
capsule-deficient rough cells
are avirulent; mixing DNA from
dead smooth virulent cells with
live avirulent bacteria allows
isolation of virulent smooth
bacteria from dead mice.
Work showed that virulence
in a bacterial pathogen is a
genetic characteristic!
!
!
Recovery of !!!!
(Smooth)
Also see llg. 6.1
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Bacteria that undergo
natural transformation

Gram positive: Streptococcus pneumoniae

Bacillus subtilis
Enterococcus faecalis

Gram negative: Haemophilus inuenza
Neisseria gonorrheae
Acinetobacter calcoaceticus
Pseudomonas stutzeri

Transformation
Whlle naLural Lransformauon works dlerenLly
ln dlerenL bacLerlal specles, fundamenLals are
conslsLenL:
# Cells musL be compeLenL" for Lransformauon
# dsunA bound by recepLor on exLernal surface
of cell
# 6-20 kb llnear fragmenLs LransporLed lnLo cell,
wlLh converslon Lo ssunA
# 8ecA medlaLes recomblnauon wlLh reclplenL
chromosome.
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Acquisition of competence
Competence is physiological state that allows cells
to take up DNA and recombine it with genome.
Some strains become at different stages of growth
S. pneumoniae (late exponential phase)
B. subtilis (starvation/stationary)
H. inuenzae (starvation/stationary)
Other strains are always competent at low level
Neisseria gonorrheae, Acinetobacter
Regulation of genes expressing competence
factors will determine when cells are competent
for DNA uptake.
Gram positive vs Gram negative
Fig. 6.2

DNA binds to
specific surface
receptor

DNA uptake into
cells depends
on an ATPase
(ComFA or PilT
type), which
helps to pull
DNA molecule
through a
specific
channel.
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Two Gram negative models
Fig. 6.2B/Fig. 6.5

Transformation
requires
recombination &
homology

DNA
transformation
into these
species depends
on a specific
DNA sequence,
which allows the
DNA to bind to
the receptor.
Specic DNA sequences
recognized in transformation
# H. inuenzae: 5 AAGTGCGGT 3 sequence is present
1465 times in genome-every 4Kb (vs. 8-9 times
predicted frequency in genome, if random)
# Neisseria: 5GCCGTCTGAA 3sequence is present
1910 times in genome (vs. 4 predicted)
# Acinetobacter also seems to prefer to take up specic
DNA, but basis of selectivity is unknown
Specic DNA may allow repair of essential functions or
allow testing of options to ! tness

No sequence motifs for DNA binding for B. subtilis or other

Gram positive species
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Gene movement: the bacterium ghts
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 at least two important
strategies to combat the ow of DNA into a cell:
# Restriction-Modication (R-M) systems
# CRISPR elements
Discovery of R-M systems
Work from several phage groups (50$s-60s):
" infects B, C, 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
# " preps grown on E. coli B, C, or K strains
plate with equal efciency on E. coli C
# " preps grown on E. coli C plate with 10000x
lower titer on E. coli B or K
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Analysis of R-M systems
Work from several phage groups showed that:
# Reduction in efciency of infection due to
strain-specic nucleases that cleaved
(restricted) phage DNA from different host
strain during infection
# Phage DNA was modied by methylases in
host cell in a strain-specic pattern
# Demonstrated that the R-M enzymes act
indiscrimantly on dsDNA in cell; normal host
DNA is protected due to its modication
Discovery of R-M systems
# Upon entering E. coli
K, DNA from " grown
on B strain could either
be degraded
(restricted) or modied
at specic sequences
# If modied, subsequent
infections of phage in K
strains would not be
subject to K-specic
restriction
K cell
Grown on B
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Three main types of R-M systems
Type I Type II Type III
Example EcoB EcoRI EcoPI
Recognition
site
TGAN
8
TGCT
GAATTC AGACC
Cleavage
site
ca 1 Kb
away
(distant)
In sequence
(here between
G and A)
24-26 bp on
3 side
(closeby)
Joint Nuclease/
Methylase?
Yes No Yes
ATP-dependent
Yes No No
Recognition sequence for
E. coli Type II R-M enzyme
The EcoRI restriction enzyme makes staggered cuts on
both strands of DNA, leaving ss sticky ends. The modifying enzyme adds
-CH
3
to 1 base (either A or C) of each strand in recognition sequence and
prevents cleavage. R-M systems widespread in Bacteria and Archaea (rare
in euks).
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Type II R-M enzymes
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 with different recognition
sequences have been isolated from wide
variety of bacteria.
Type II systems most common but Type I
systems widely distributed; Type III systems
rare
Type II R-M enzymes (see Tab 1.3)
Organism Enzyme Recognition
sequence
S. aureus Sau3A GA#TC
*

E. coli EcoRI G#AA

*
TTC

H. inuenzae HindIII A
*
#AGCTT
Nocardia otitidis NotI GC#GGC
*
CGC
Generally, restriction endonucleases with larger recognition
sequences (6-8 bp) are most useful for molecular biology.
Recognition sequences are often palindromic.
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Antagonizing R-M enzymes
Restriction-modication systems widely
distributed

Phages and plasmids have developed ways to

circumvent this protection:
# Modication of T4 DNA allows it to escape the
E. coli R-M system
# Several plasmids have anti-restriction
systems or will reduce frequency of
recognition site in the plasmid sequence.
AnoLher bacLerlal defense sysLem:
C8lS8 elemenLs
8ecenLly, noLed LhaL
bacLerla express anoLher
unlque form of adapuve
lmmunlLy": Lhe ClusLer of
8egularly lnLerspaced
ShorL allndromlc 8epeaLs
(C8lS8s) elemenLs LhaL
funcuon wlLh proLelns
encoded by >,- (C8lS8-
assoclaLed) genes
llg. 7.30C lower panel

Cne Lype of C8lS8 8nA-proLeln
complex LargeLs forelgn" unA,
resulung ln Lhe degradauon of LhaL
unA.

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C8lS8s: defense agalnsL phage/plasmld
# 8acLerlal genome sequences showed dlrecLly repeaLed
ldenucal sequences (21-47 bp) separaLed by ~ 30 bp
spacers LhaL maLched phage/plasmld sequences
# llrsL ldenued ln G)>/$%, buL now known ln 40 of
H,>#'+%,, 90 of ;+>I,',
# Cenerlc arrangemenL shown above (llg. 7.30A): Lhree
maln Lypes, whlch can LargeL elLher unA or 8nA
C8lS8s: defense agalnsL phage/plasmld
# 8acLerlal genome sequences showed dlrecLly
repeaLed ldenucal sequences (21-47 bp) separaLed
by ~ 30 bp spacers
# number of repeaLs varles from a few (3-4) Lo ~400
# Many repeaLs are aL leasL parually pallndromlc

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C8lS8s: defense agalnsL phage/plasmld
# 1ranscrlpuon/processlng of C8lS8 array resulLs ln
many small cr8nAs
# Several assoclaLed >,- genes, whlch encode
nucleases and oLher nuclelc acld-speclc enzymes.
(Cas1 = dsunA nuclease, Cas6 ls endorlbonuclease)

C8lS8 adapLauon
# All C8lS8 locl plck
up new unA
sequences
# Addluons when
elemenL exposed Lo
new geneuc
elemenL
# Addluons occur aL
leader end
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C8lS8 lmmunlLy
# Slngle promoLer
for Lranscrlpuon
of lnlual 8nA
# rocesslng of
8nA ln repeaL
sequences Lo
make cr8nAs
(repeaL + spacer)
# cr8nA + Cas
LargeLs forelgn
unA