Post on 18-Dec-2015
Conjugative Transposons (CTns)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 in the transfer of a conjugative transposonSteps in the Transfer of a Conjugative TransposonSteps in the Transfer of a Conjugative Transposon
Different levels of investigationDifferent levels of investigation
• Ecology: Movement of genes in the colonic Ecology: Movement of genes in the colonic ecosystem by CTnsecosystem 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 Composition of the colonic microbiotamicrobiota
• Numerically predominant groups
– Bacteroides Bacteroides spp.spp.• Polysaccharide fermentationPolysaccharide fermentation• Opportunistic pathogen – resistance to antibioticsOpportunistic pathogen – resistance to antibiotics
– Gram positive anaerobes (e.g. Gram positive anaerobes (e.g. Clostridium Clostridium coccoides, Clostridium leptum, Eubacterium spp.)coccoides, Clostridium leptum, Eubacterium spp.)
The Reservoir Hypothesis – Intestinal Bacteria As Reservoirs for Resistance
Genes
Resistant intestinal bacteria
Swallowed bacteriaSwallowed bacteriaOther intestinal
bacteria
Fecal-oral transmission
Genes Bacteria
QuestionsQuestions
• 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 Widespread resistance genes in the human microbiotamicrobiota
Elements responsible for Elements responsible for transfer eventstransfer events
• tetM, tetQ– Conjugative transposonConjugative transposon (Tn916, CTnDOT)– Tc-stimulated transfer
• ermF– Conjugative transposonConjugative transposon (CTnDOT family)– Self-transmissible plasmid, mobilizable plasmid
• ermG, ermB– Conjugative transposonsConjugative transposons (CTnGERM, CTnBST)
Evidence from genome/metagenome Evidence from genome/metagenome sequencessequences
• 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 CTnDOT – a widespread Bacteroides Bacteroides CTnCTn
• 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 tetracyclineFunctions regulated by tetracycline
Different levels of investigationDifferent levels of investigation
• Ecology: Movement of genes in the colonic ecosystem by CTns
• Mechanisms of CTn integration and excisionMechanisms of CTn integration and excision
• Regulation of CTn functions
• Effects of a CTn on the cell it enters
Steps in the transfer of a conjugative transposonSteps in the Transfer of a Conjugative TransposonSteps in the Transfer of a Conjugative Transposon
Excision and Integration of CTnDOTExcision and Integration of CTnDOT
Rajeev et al, MMBR, 2009
Characterizing the CTnDOT Characterizing the CTnDOT Excision/Integration MechanismExcision/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 Alignment of the Carboxyl Terminal Domain of Some TyrosineRecombinasesDomain 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>
Predicted structure of IntDOTPredicted structure of IntDOT(Brian Swalla)(Brian Swalla)
B
2
B
2
D
4
D
4
LL
FF
JJ
E
5
E
5
A
1
A
1
C
3
C
3
GG
II
NH2
HHKK
MM
NN
COOH
WT Recombination Frequency Decrease in activity No Detectable Recombination
Y381FY381F
H372AH372A
R348AR348A
H372AH372AR348AR348A
R247AR247AS259AS259A
Mutations in the CAT Domain of IntDOT and their affect on Recombination
-
15 Mutations in the CB Domain of IntDOT and their affect on Recombination
H143AH143A
B
2
B
2
D
4
D
4
LL
FF
JJ
E
5
E
5
A
1
A
1
C
3
C
3
GG
II
NH2
HHKK
MM
NN
COOH
K129AK129A
T194AT194A
K131AK131A
W186AW186A
N183AN183A
C180AC180A
H179AH179A
K142AK142AT139AT139A
R138AR138A
Y137AY137A
K136AK136A
L135AL135A
T133AT133A
WT Recombination Frequency 103-105-fold Decrease No Detectable Recombination
14
3
5
Y137A (no recomb.)Weak ligation
No cleavage
L135A (5x10-8)WT ligation
WT cleavage
R138A (10-6)WT ligation
WT cleavageH179A (3x10-6)WT cleavage
WT ligation
N183A (no recomb.)WT ligation
Weak cleavage
( ) = recombination freqency
K142A (6x10-7)Weak ligation
WT cleavage
2
Different levels of investigationDifferent levels of investigation
• Ecology: Movement of genes in the colonic ecosystem by CTns
• Mechanisms of CTn integration and excision
• Regulation of CTn functionsRegulation of CTn functions
• Effects of a CTn on the cell it enters
Steps in the transfer of a conjugative transposonSteps in the Transfer of a Conjugative Transposon
Regulation of excision of CTnDOTRegulation of excision of CTnDOT
How regulation of excision worksHow 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 genesRegulation of transfer genes
How regulationof transfer genes How regulationof transfer genes worksworks
• 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 investigationDifferent levels of investigation
• Ecology: Movement of genes in the colonic ecosystem by CTns
• Mechanisms of CTn integration and excision
• Regulation of CTn functionsRegulation of CTn functions
• Effects of a CTn on the cell it entersEffects of a CTn on the cell it enters
Effect of CTnDOT on a recipient Effect of CTnDOT on a recipient cellcell
• 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 effectglobal regulatory effect?
ApproachApproach
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)
ResultsResults
• 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 CTnscryptic CTns– Labeled with resistance gene to show transferLabeled 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 Conjugative elements with regulatory genes may have broad effects on chromosomal geneshave broad effects on chromosomal genes