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Conjugative Transposons (CTns)
Abigail Salyers, Department of Microbiology, University of Illinois,
Urbana, IL 61801
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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
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Steps in the transfer of a conjugative transposonSteps in the Transfer of a Conjugative Transposon
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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
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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.)
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The Reservoir Hypothesis – Intestinal Bacteria As Reservoirs for Resistance
Genes
Resistant intestinal bacteria
Swallowed bacteria Other intestinal
bacteria
Fecal-oral transmission
Genes Bacteria
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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
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Widespread resistance genes in the human microbiota
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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)
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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
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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
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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
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Steps in the transfer of a conjugative transposonSteps in the Transfer of a Conjugative Transposon
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Excision and Integration of CTnDOT
Rajeev et al, MMBR, 2009
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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)
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Alignment of the Carboxyl Terminal Domain of Some TyrosineRecombinases
212 235
308 311 333 342
259 287
345 348 372 381
Lambda <199> R L A M E L A V V T G Q R . . . . V G D L C E M K W S D I V <9> . . . . K T G V K I A I P T A L H I D A L G I S M K E T L D
XerC <144> R A M L E V M Y G A G L R . . . . L S E L V G L D I K H L D <6> W V M G K G S K E R . . . . R L P I G R N A V A W I E H W L
XerD <136> K A M L E V L Y A T G L R . . . . V S E L V G L T M S D I S <6> R V I G K G N K E R . . . . L V P L G E E A V Y W L E T Y L
IntDOT <238> R D L Y L F C A F T G L S . . . . F S D M R N L T E E N I R <10> I N R Q K T G . . . . . . . . . . . . . . . V V S N I R L L
Tm5520 <237> K N A T H E L V . . . . R D L F V F S V F T G L A Y S D V K <18> T R R K K T N . . . . . . . . . . . . . . . T E S N I R L L
Tn916 <212> Y D E I L I L L K T G L R . . . . I S E F G G L T L P D L D <21> I E T P K T K S . . . G E R Q V P M V E E A Y Q A F K R V L
HP1 <195> G L I V R I C L A T G A R . . . . W S E A E T L T Q S Q V M <4> F T N T K S K K N R . . . . . . . . . . . T V P I S K E L F
CRE <160> L A F L G I A Y N T L L R . . . . I A E I A R I R V K D I S <14> . . . T K T L V S T A G V E K A L S L G V T K L V E R W I S
Lambda <39> F E G D P P T F H E L R . . . S L S A R L Y E K Q I S D K F A Q H L L G H K S D T M A S Q Y R D D R G R E W D K I E I K
XerC <41> N H V H P . . H K L R H . . S F A T H M L E S S G D L R G V Q E L L G H A N L S T T Q I Y T H L D Q H L A S V Y D A A <4>
XerD <44> S E K L S P . . H L V R H . . A F A T H L L N H G A D L R V V Q M L L G H S D L S T T Q I Y T H W A T R L R Q L H Q Q H <8>
IntDOT <42> K I T H W . . . H Q S R H T . A A T T V F L S N G V P I E T V S S M L G H K S I K T T Q I Y A K I T K E K L N Q D M E N <15>
Tn5520 <42> V R L T Y . . . H V A R H T . N A T T V L L S H G V I P E T V S R L L G H T N I K T T Q I Y A K I T A Q K I S Q D M E T <15>
Tn916 <49> D K L P H I T P H S L R H T . . F C T N Y A N A G M N P K A L Q Y I M G H A N I A M T L N Y Y A H A T F D S A M A E M K <11>
HP1 <29> P K G Q L T . . H V L R H T . . F A S H F M M N G G N I L V L K E I L G H S T I E M T M R Y A H F A P S H L E S A V K F <8>
CRE <49> Q R Y L A W S G H S A R V . G A A R D M A R A A G V S I P E I M Q A G G W T N V N I V M N Y I R N L D S E T G A M V R L <3>
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Predicted structure of IntDOT (Brian Swalla)
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B 2
D 4
L
F
J
E 5
A 1
C 3
G
I
NH2
H K
M
N
COOH
WT Recombination Frequency Decrease in activity No Detectable Recombination
Y381F
H372A
R348A
H372A R348A
R247A S259A
Mutations in the CAT Domain of IntDOT and their affect on Recombination
-
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15 Mutations in the CB Domain of IntDOT and their affect on Recombination
H143A
B 2
D 4
L
F
J
E 5
A 1
C 3
G
I
NH2
H K
M
N
COOH
K129A
T194A
K131A
W186A
N183A
C180A
H179A
K142A T139A
R138A
Y137A
K136A
L135A
T133A
WT Recombination Frequency 103-105-fold Decrease No Detectable Recombination
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1 4
3
5
Y137A (no recomb.) Weak ligation
No cleavage
L135A (5x10-8) WT ligation WT cleavage
R138A (10-6) WT ligation WT cleavage
H179A (3x10-6) WT cleavage WT ligation
N183A (no recomb.) WT ligation Weak cleavage
( ) = recombination freqency K142A (6x10-7) Weak ligation WT cleavage
2
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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
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Steps in the transfer of a conjugative transposonSteps in the Transfer of a Conjugative Transposon
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Regulation of excision of CTnDOT
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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)
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Regulation of transfer genes
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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)
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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
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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?
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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)
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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