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
transposon
Stepsin
inthe
thetransfer
Transferofofaaconjugative
Conjugative
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
transposon
Stepsin
inthe
thetransfer
Transferofofaaconjugative
Conjugative
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>
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XerC <41>
XerD <44>
IntDOT <42>
Tn5520 <42>
Tn916 <49>
HP1 <29>
CRE <49>
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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
L
N
K
H372A
COOH
H
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
W186A
H143A K142A
NH2
E
D
C
5
4
3
B
A
2
1
M
L
N
K
COOH
H
C180A
R138A
Y137A
K136A
L135A
T133A
N183A
H179A
T139A
K129A
K131A
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
4 2
5
3
1
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
transposon
Stepsin
inthe
thetransfer
Transferofofaaconjugative
Conjugative
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