Conjugative Transposons (CTns)
Abigail Salyers, Department of
Microbiology, University of Illinois,
Urbana, IL 61801
What is a CTn?
• Integrated DNA segment that excises to form a
circular intermediate which transfers by conjugation
• Also called ICEs (integrated conjugative elements)
• Vary widely in size (18 kbp – 500 kbp)
• Some carry genes not involved in transfer (eg,
antibiotic resistance genes, nitrogen fixation genes)
• Usually able to mobilize plasmids in trans
Steps
Steps in
in the
the transfer
Transferof
ofaaconjugative
Conjugativetransposon
Transposon
Different levels of investigation
• Ecology: Movement of genes in the colonic
ecosystem by CTns
• Mechanisms of CTn integration and excision
• Regulation of CTn functions
• Effects of a CTn on the cell it enters
Composition of the colonic
microbiota
• Numerically predominant groups
– Bacteroides spp.
• Polysaccharide fermentation
• Opportunistic pathogen – resistance to antibiotics
– Gram positive anaerobes (e.g. Clostridium
coccoides, Clostridium leptum, Eubacterium spp.)
The Reservoir Hypothesis – Intestinal
Bacteria As Reservoirs for Resistance
Genes
Other intestinal
bacteria
Swallowed bacteria
Genes
Resistant
intestinal bacteria
Bacteria
Fecal-oral
transmission
Questions
• How much transfer is actually occurring?
How broad is the host range?
– Approach: Find identical or near-identical
resistance genes (>95% DNA sequence identity) in
different species or genera of bacteria
• How is transfer being mediated?
– Approach: Establish genetic linkage between
resistance gene and some mobile element (CTn,
plasmid) using Southern blot or PCR
Widespread resistance genes in the human
microbiota
Elements responsible for
transfer events
• tetM, tetQ
– Conjugative transposon (Tn916, CTnDOT)
– Tc-stimulated transfer
• ermF
– Conjugative transposon (CTnDOT family)
– Self-transmissible plasmid, mobilizable plasmid
• ermG, ermB
– Conjugative transposons (CTnGERM, CTnBST)
Evidence from genome/metagenome
sequences
• Sequences being seen that are
associated with conjugative
transposons (transfer genes, integrase
genes) found in
– Bacteroides group (Bacteroides,
Porphyomonas, Prevotella)
– Gram positives
CTnDOT – a widespread
Bacteroides CTn
• In pre-1970s, found in about 20% of
intestinal Bacteroides strains
• Post 1990, found in over 80% of strains
• Characteristics
– 65 kbp
– tetQ, ermF
– Very stable in the absence of selection
– Functions regulated by tetracycline
Different levels of investigation
• Ecology: Movement of genes in the colonic
ecosystem by CTns
• Mechanisms of CTn integration and excision
• Regulation of CTn functions
• Effects of a CTn on the cell it enters
Steps
Steps in
in the
the transfer
Transferof
ofaaconjugative
Conjugativetransposon
Transposon
Excision and Integration of CTnDOT
Rajeev et al, MMBR, 2009
Characterizing the CTnDOT
Excision/Integration Mechanism
• Construction of a miniature form of CTnDOT
for in vivo assay of integration (suicide
plasmid containing the integrase gene and
the joined ends of the circular form)
• In vitro assays for integration, and steps in
integration process (eg, DNA binding,
cleavage, ligation)
Alignment of the Carboxyl Terminal Domain
of Some TyrosineRecombinases
212
Lambda <199>
XerC <144>
XerD <136>
IntDOT <238>
Tm5520 <237>
Tn916 <212>
HP1 <195>
CRE <160>
R
R
K
R
K
Y
G
L
L
A
A
D
N
D
L
A
A
M
M
L
A
E
I
F
M
L
L
Y
T
I
V
L
E
E
E
L
H
L
R
G
L
V
V
F
E
I
I
I
A
M
L
C
L
L
C
A
V
Y
Y
A
V
L
L
Y
V
G
A
F
.
K
A
N
T
A
T
T
.
T
T
T
G
G
G
G
.
G
G
L
Q
L
L
L
.
L
A
L
R
R
R
S
R
R
R
R
235
.
.
.
.
D
.
.
.
.
.
.
.
L
.
.
.
.
.
.
.
F
.
.
.
.
.
.
.
V
.
.
.
V
L
V
F
F
I
W
I
G
S
S
S
S
S
S
A
D
E
E
D
V
E
E
E
L
L
L
M
F
F
A
I
C
V
V
R
T
G
E
A
E
G
G
N
G
G
T
R
M
L
L
L
L
L
L
I
K
D
T
T
A
T
T
R
W
I
M
E
Y
L
Q
V
S
K
S
E
S
P
S
K
D
H
D
N
D
D
Q
D
I
L
I
I
V
L
V
I
V
D
S
R
K
D
M
S
<9>.
<6>W
<6>R
< 1 0 >I
< 1 8 >T
< 2 1 >I
<4>F
< 1 4 >.
.
V
V
N
R
E
T
.
.
M
I
R
R
T
N
.
.
G
G
Q
K
P
T
T
259
308
Lambda <39>
XerC <41>
XerD <44>
IntDOT <42>
Tn5520 <42>
Tn916 <49>
HP1 <29>
CRE <49>
F
N
S
K
V
D
P
Q
E G
H
E K
I T
R L
K L
K G
R Y
D
V
L
H
T
P
Q
L
P
H
S
W
Y
H
L
A
P
P
P
.
.
I
T
W
T
.
.
.
.
T
.
S
F
.
.
.
.
P
.
G
H
H
H
H
H
H
H
H
345
K
K
K
K
K
K
K
K
L
L
V
S
A
L
L
A
R
R
R
R
R
R
R
R
348
G
S
N
G
N
K
K
L
V
K
K
.
.
S
K
V
K
E
E
.
.
.
N
S
I
R
R
.
.
.
R
T
A
.
.
.
.
.
.
A
I
.
.
.
.
G
.
G
P
.
.
.
.
E
.
V
T
.
.
.
.
R
.
E
333
.
H
H
H
H
H
H
V
A
R
L
.
.
Q
.
K
L
L
V
.
.
V
.
A
H
P
P
.
.
P
.
L
I
I
L
.
.
M
.
S
D
G
G
.
.
V
.
L
A
R
E
.
.
E
.
G
L
N
E
.
.
E
.
V
G
A
A
.
.
A
T
T
I
V
V
V
T
Y
V
K
S
A
Y
V
E
Q
P
L
M
W
W
S
S
A
I
V
K
I
L
N
N
F
S
E
E
E
E
I
I
K
K
R
T
H
T
R
R
R
E
W
L
W
Y
L
L
V
L
I
R
T
T
A
A
Y
A
I
D
H
H
K
K
A
H
R
D
L
W
I
I
H
F
N
R
D
A
T
T
A
A
L
G
Q
T
K
A
T
P
D
R
H
R
E
Q
F
S
S
E
L
L
K
K
D
H
E
W
A
R
L
I
S
L
T
D
S
Q
N
S
A
E
G
K
V
L
Q
Q
M
S
A
I
Y
H
D
D
A
A
M
E
D
Q
M
M
E
V
V
I
A
Q
E
E
M
K
R
K
A
H
N
T
K
F
L
D
L
L
L
L
L
F
S
287
311
E
K
L
Q
V
S
V
S
T
G
G
T
T
T
S
T
.
.
.
T
T
T
T
.
.
.
.
.
.
.
.
G
S
S
A
A
N
.
.
A
L
F
F
A
A
F
F
A
S
A
A
T
T
C
A
R
A
T
T
T
T
T
S
D
R
H
H
V
V
N
H
M
L
M
L
F
L
Y
F
A
Y
L
L
L
L
A
M
R
E
E
N
S
S
N
M
A
K
S
H
N
H
A
N
A
Q
S
G
G
G
G
G
G
I
G
A
V
V
M
G
V
S
D
D
P
I
N
N
S
D
L
L
I
P
P
I
I
K
R
R
E
E
K
L
P
F
G
V
T
T
A
V
E
A
V
V
V
V
L
L
I
Q
Q
Q
S
S
Q
K
M
H
E
M
S
R
Y
E
Q
L
L
L
M
L
I
I
A
L
L
L
L
L
M
L
G
G
G
G
G
G
G
G
G
H
H
H
H
H
H
H
W
372
342
K
A
S
K
T
A
S
T
S
N
D
S
N
N
T
N
D
L
L
I
I
I
I
V
T
S
S
K
K
A
E
N
M
T
T
T
T
M
M
I
A
T
T
T
T
T
T
V
S
Q
Q
Q
Q
L
M
M
Q
I
I
I
I
N
R
N
Y
Y
Y
Y
Y
Y
Y
Y
381
<4>
<8>
<15>
<15>
<11>
<8>
<3>
Predicted structure of IntDOT
(Brian Swalla)
Mutations in the
CAT Domain of
IntDOT and their
affect on
Recombination
H372A
Y381F
R348A
NH2
E
D
C
B
A
5
4
3
2
1
R348A
S259A
R247A
M
H
K
L
N
H372A
COOH
J
G
F
I
WT Recombination Frequency
Decrease in activity
No Detectable
Recombination
15 Mutations in the
CB Domain of
IntDOT and their
T194A
affect on
Recombination
H143A K142A
T139A
NH2
Y137A
E
D
C
B
A
5
4
3
2
1
K136A
W186A
L135A
T133A
N183A
K129A
H179A
M
H
L
N
K131A
C180A
K
COOH
R138A
J
G
F
I
WT Recombination Frequency
103-105-fold Decrease
No Detectable
Recombination
( ) = recombination freqency
K142A (6x10-7)
Weak ligation
WT cleavage
R138A (10-6)
WT ligation
WT cleavage
H179A (3x10-6)
WT cleavage
WT ligation
Y137A (no recomb.)
Weak ligation
No cleavage
1
4 2
5
3
L135A (5x10-8)
WT ligation
WT cleavage
N183A (no recomb.)
WT ligation
Weak cleavage
Different levels of investigation
• Ecology: Movement of genes in the colonic
ecosystem by CTns
• Mechanisms of CTn integration and excision
• Regulation of CTn functions
• Effects of a CTn on the cell it enters
Steps
Steps in
in the
the transfer
Transferof
ofaaconjugative
Conjugativetransposon
Transposon
Regulation of excision of CTnDOT
How regulation of excision works
• Increased translation of TetQ, RteA, RteB proteins due to
translational attenuation (Tc causing stalling of ribosomes on
the leader region of operon)
– Transcriptional fusion constitutive, translation regulated
– Site directed mutations in leader region showed that mRNA
structure involved
• RteA (sensor), RteB (DNA binding protein) is a two component
regulatory system; activates transcription of rteC; RteC protein
activates expression of excision operon (orf2c-orf2d-exc)
– RT-PCR to detect regulated transcription
– RteB and RteC bind DNA in vitro
– Site-directed mutations upstream of promoter region of rteC and
orf2c operon abolished transcription (activator)
Regulation of transfer genes
How regulationof transfer genes
works
• When transfer genes cloned away from rest of CTnDOT,
expression of tra gene was constitutive but within CTnDOT
expression was regulated from nearly zero to high level
expression
• Activation: Excision proteins alone are sufficient to activate tra
gene expression
– Expression of excision operon from heterologous promoter (no
RteB, RteC necessary) caused activation of tra gene operon, but
not repression (protein fusion to start codon of traA, RT-qPCR)
• Repression: Possible small RNA (RteR) causes repression
– Furnishing rteR in trans with tra operon resulted in decreased
expression (no effect on traA fusion, but RT-qPCR showed
reduced transcription of later genes)
– Stop codons in putative start codon had no effect on activity
(probably regulatory RNA)
Different levels of investigation
• Ecology: Movement of genes in the colonic
ecosystem by CTns
• Mechanisms of CTn integration and excision
• Regulation of CTn functions
• Effects of a CTn on the cell it enters
Effect of CTnDOT on a recipient
cell
• What is the effect on a Bacteroides cell
of having CTnDOT enter its
chromosome?
– No evidence for disruption of genes
– Could there be a global regulatory effect?
Approach
Microarrays to compare
No Tc, no CTnDOT
+Tc, +CTnDOT
Generate a “shopping list” of genes to be
checked by qRT-PCR
(Moon and Salyers, Mol. Micro., 2007)
Results
• Expression of nearly 60 chromosomal genes were
up-regulated or down-regulated by more than 7-fold
• Most up-regulated genes were genes of unknown
function
– Some were associated with cryptic CTns
– Labeled with resistance gene to show transfer
• For one of these CTns, RteA and RteB plus Tc were
sufficient. Others required intact CTnDOT
• Conjugative elements with regulatory genes may
have broad effects on chromosomal genes