Conjugative Transposons (CTns) - University Of Illinois...– Conjugative transposon (Tn916, CTnDOT) – Tc-stimulated transfer • ermF – Conjugative transposon (CTnDOT family)

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 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

Resistant intestinal bacteria

Swallowed bacteria Other intestinal

bacteria

Fecal-oral transmission

Genes Bacteria

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







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

212 235

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 IntDOT (Brian Swalla)

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

-

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

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







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)






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

Documents

Conjugative Transposons (CTns) - University Of Illinois...– Conjugative transposon (Tn916, CTnDOT) – Tc-stimulated transfer • ermF – Conjugative transposon (CTnDOT family)