Bacterial genome plasticity and integrons

Didier Mazel

Unité Plasticité du Génome Bactérien

http://www.pasteur.fr/recherche/unites/pgb/

- Introduction what we know

How bacteria do evolve:» Mutations» Acquisitions (horizontal gene transfer)

- Amplitude of the phenomenon - HGT Limits

- Mechanisms of mobility

- Mechanisms of gene capture – the exemple of the antibiotic resistance development and the

integrons

– superintegrons

Introduction

• 257 bacterial genome had been sequenced (+24 Archeal genomes)

• 521 were on going (+25 Archaea)

We now know the intimity of bacterial genomes quite well

http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi

in september 2005

• The sequenced bacterial genome* sizes vary from 0.580 Mb (Mycoplasma genitalium)to ≈ 10.0 Mb (cyanobacteria (Lyngbya) and actinobacteria (Streptomuyces-Rhodococcus) )for comparison : S. cerevisiae 12 Mb

• first rule: direct correlation between the adptive capabilities (“versatility”) and the genome size .e.g. intracellular parasite bacteria (constant and rich environnement) have small genomes.

*, genome broad meaning : chromosome(s) + episomes

Plasticity ?

large variation in size and content of bacterial genomes ,

- between different genera and species- but also among strains of the same species

Large variation in the bacterial genome organisation, even between closely related species.

Variation in the bacterial genome organisation:

2nd rule: Variations using 2 mechanisms:Transposition and homologous

Recombinaison

=> repeated sequences play a major role, notably the mobile DNA elements.

The mobilome

Gene position plots of pairs of bacterial and archaeal genomes.

(a) Intracellular symbiotic bacteria (no low-divergence species available).; (b) intracellular pathogenic bacteria; (c) free-living species with a limited number of IS elements; (d) species with a high number of IS elements. Comparisons in the left-hand column have an average sequence similarity for homologous genes higher than 85%, whereas genomes shown on the right-hand column have an average sequence identity lower than 76%. Average non-synonymous substitutions per nucleotide site (Ka values) for the orthologous sequences from thepairwise comparisons are: 0.165 (Buchnera), 0.068 (Rickettsia), 0.213 (Chlamydia), 0.014 (Salmonella), 0.116 (Pyrococcus), 0.125 (Xanthomonas) and 0.247 (Sulfolobus). The origin of the gene position plots correspond to the replication origin unless otherwise stated. ori, origin of replication; ter, terminus of replication.

Mira et al COMB (2002) 5: 506-12

Regression line of the total number of IS elements as a predictor of the number of transposed single genes (TSGs) for each compared bacterial pair. .A TSG was defined as a homologous sequence with over 60% sequence similarity and length that occupy different positions in the two genomes and with no adjacent homologous sequences. The number of TSGs for Xanthomonas was taken from [40]. Datapoints (from left to right) represent comparisons between pairs of Buchnera, Chlamydia, Mycoplasma, Listeria, Rickettsia, Pyrococcus,Helicobacter, Escherichia?x2013;Salmonella, Salmonella, Thermoplasma, Mycobacterium, Bacillus, Escherichia, Neisseria, Streptococcus, Sulfolobus and Xanthomonas. Mira et al COMB (2002) 5: 506-12

Bacterial evolution routes• Mutations …• Duplication and divergent evolution

(paralogues) (Rares ! in général paralogues viennent d’orthologues différents)

• Horizontal gene transfer (up to 15% of the genome)

The three kind of phenomenon co-exist and act in synergy. Therefore it is difficult to measure their respective contribution to complex phenotypes development

=> a well known exemple : the antibiotic resistance

Horizontal gene transfer signatures:

• Comparative Analysis = > differences

• Mobile elements characteristic boundaries:

insertions at highly conserved loci (tRNAs,…)

presence of repeated sequences in direct or inverted orientation, transposase or recombinase genes

• base composition or codon usage deviations

Limits of the exchanges : none ???• based on sequence similarity:

Eucaryotes bactéries

archae• Exprimentaly : idem !

• Natural systems: Ti DNA Agrobacterium -> plantsmigration of the organites genes in the

nucleus

The limit is more a question of sharing the same ecological niche

Mobility mechanims

• Transformation: Limitée à un certain nombre d’espèces intégration par recombinaison homologue

• Transduction:Phages très abondants, spectre d’hôtes restreint// PAI de type prophage

• Conjugaison:Spectre d’hôte variable, transfert de réplicon ou machinerie d’intégration par

recombinaison spécifique de site (affranchi limite)

an example : the antibiotics resistance

• recent phenomenon: 60 years

• Extremely documented

• 80 % of resistances due to exogenous gene acquisitions

the multi-resistance phenomenon

• first apparition in Japon in 1955.

• follows the massive production and use of Ab.

.

1

10

100

0

10

20

30

40

50

60

70

55 60 65

TC

C M

S M

Multi- Resistant Shigella

Year

AbR genes origin

• in most cases : ????

• bacterial producers have had to develop protection mechanisms

=> potential source

vancomycin resistance loci comparison

producers

Tn1546 resistance locus

vanR vanS vanY vanZ

vanH ddl vanX

Tn1546vanA

S. toyocaensis

A. orientalis

54-61%

61-64%

{REQUIRED FOR GLYCOPEPTIDE RESISTANCE

Marshall, C.G et al. (1997) PNAS 94:6480

vanH VanA vanX

resistance genes flow

(Conjugatif) Plasmide

Capture dans Tn

AbR

from the source...

…to the clinical isolate

A crucial question: how do Transposons acquièrent-ils ces gènes

?

the answer has been obtained for two examples:

•CompositeTransposons

•Transposons carrying an integron

composite transposon assembly

AbR

The integrons

• They constitute what can be defined as a natural genetic engineering system:

• Incorporate ORFs

• Express them

Tn21IRtnp

Tn21

tnpA tnpR res merD merA merP merR

tnpM? urf2 merE merC merT

urf2MTn21IRmer

Transposition genes mer genes

attI

IRi IRtintI1

qacEΔ1sul1 orf5 tniBΔ1 tniA

integronIn2…In60

Gene cassettes

aadA1

attC

More than 85 different gene cassettes encoding antibiotic resistance have been found in

integrons.

These cassettes allow to resist to all classes of antibiotics used against human Gram-negative pathogens β-lactams, aminoglycosides, chloramphenicol, trimethoprim, streptothricin, rifampin, erythromycin, …antiseptics).

3 ’ conserved segmentintegrase

intI1sul

qacEΔoxa9cmlA2dfrVIaacA4

inverse core site

54 - 135 nt

:59 base elementsattI site

variable region

core site

RYYYAAC GTTRRRY

Multi-Resistant Integron

intI attI site attC2 siteattC1 site attC3 siteattI site

Excisions occur via attC x attC recombination

Two types of reactions: cassette excision and cassette

integration

Recombination reactions in integrons

Integrations occur through attI x attC recombination

Integron cassette structure and characteristics

Stokes, H. and Hall, R (1989) Mol Micro 3:1669; Collis, C. and Hall, R.(1992) Mol Micro 6:2875; Recchia, G. and Hall (1995) Microbiol 141:3015

attC site

ICS CS (RYYYAAC) (G / TTRRRY)

variable region

•all contain an attC site (59-base element) - integrase target

•the ICS is always complementary to the CS of the circularized cassette; they form imperfect inverted repeats

•most contain a single ORF

•promoterless

Five “classes” of Integrons (MIs)

class 1 Tn21 family (most ubiquitous)class 2* Tn7 familyclass 3 self-transmissible plasmidclass 4 self-transmissible plasmidclass 5 SXT element (constin)

•Share between 45-58% amino acid identity•All are associated with mobile DNA elements•Carry at most 8 resistance cassettes

The Mobile Integrons and the antibiotic Resistance :

What is the origin of the multi-resistance integrons and their

cassettes ?

genomeVibrio cholerae

Integron

rplTrpmIinfC

L20L35IF3 integrase

intI4 orVchintIA

Most are 95% identical

core siteinverse core site

RYYYAAC GTTRRRY121-123 nt

: VCR

126 Kb, 179 cassettes, 3% of genome

Mazel, D et al (1998) Science 280:605; Heidelberg, Jet al. (2000) Nature 406:477; Rowe-Magnus et al. (1999) Res Mic 150:641

There are 3 major differences between MRIs & the SI:

•Size•The function of most SI cassettes are unknown•Homology of the SI attC sites compared to the attC sites of MRIs

sto mrhA

The V. cholerae superintegron

84% of the V. cholerae cassettes

16% of the V. cholerae cassettes carry a Remote attC site

3 ’ conserved segmentintegrase

intI1sul

qacEDoxa9cmlA2dfrVIaacA4

integron Multi-resistant

rplTrpmIinfC

L20L35IF3

intI

Super-integron Chromosomique

integrase

3 differences majeures entre MRIs & SIs:• taille• La fonction de la plupart des cassettes du SI est inconnue

• Homologie des sites attC dans le SI comparé à ceux des MRIs

Are SIs the source of MRIs?

?

intIA

?

?

Vibrio Listonella Alteromonas Photobacterium Moritella

XanthomonasPseudomonas alcaligenes *

mendocina *pseudoalcaligenes

Shewanella oneidensis MR-1 putrefaciens

Treponema denticola (spirochete)Microbulbifer Nitrosomonas europaea (β)Thiobacillus ferrooxidans (β)Geobacter sulfurreducens (δ)

+ 2 new types of MRIs

+ 19 intIs and hundreds of

cassettes from soil extracted DNA

SIs are widespread among the proteobacteria

=> We characterized SIs / Is structures in >30 proteobacterial species (mainly γ)

Rowe-Magnus et al. (2001) PNAS 98:652; Rowe-Magnus and Mazel (2001) Cur. Op. Microbiol. 4:565

The integron is an ancient evolutionary apparatus

0.1

VchIntIAVmiIntIA

VmeIntIALanIntIA

VvuIntIAVpaIntIA

VnaIntIa

VfiIntIAGsuintIA

Tden

Lambdae14

P4Int

P22xis

P2

P22int

EcFimB

EcFimE

SpuIntIB

SonIntIASpuIntIA

NeuIntIA

TfeintIA

I8-2I7-2

I6-2

XspIntIA

XcaIntIAXcaIntIB

PmeIntIAPalcIntIA

LpeIntIALpeIntIA

intIHS

IntI2

IntI9 SXT

IntI1IntI3

0.1

Son

Pal

PprPph

VvuLpe

VhaVpaVme

VorLan

VsaVfi

VmiVch

VhoStyEco

Yen

SpuVmaMma

Ama

Psy

Neu

PflXspXca

Gsu

Tde

Tfe

Pim

Pae

PpsPme

IntI9 SXT

intIHS

IntI2

IntI1IntI3

.

VCRcA

dfrVI

CAR4

XCR7XSR6

SPR6

SPR1SPR4

SPR3SPR5

SPR2catB6

XCRs

XSRs

aadA7aacAaadA1a

aadA6

aadA2

0.1

VFRs

LPRs

VCRcIVPR4

VPR2

VMeRs

0.1

VCRs/VMiRs

XCR9

qacF

aadB

aadA1b

VPRs

SPRs

Les genes intI (capture) et les XXRs (cassette genesis) co-evoluent

Altogether:

• SI functional platforms (intI + attI) are not mobile and co-evolve with host genomes

• Integrases and attC sites co-evolve

• The Vibrio and the Xanthomonas ancestors carried a SI (evolutionary history of > 300 Myrs).

The Gene Cassette Reservoir

To what extent do the different SIs share cassettes?

What kind of functions are found in the cassettes?

Comparison of SI cassette contents

V. Chol179

V. Met(40)

V. Para71

V. Vuln193

V. Fisch(22)

P.alca(33)

V. chol 100 15 0 0 0 0

V. met 100 0 0 0 0

V. para 100 0 0 0

V. vuln 100 0 0

V. fisch 100 0

P.alca 100

•Five SIs gather the equivalent of the Mycoplasma genitalium genome.•If each SI proves to have hundreds of species-specific cassettes, then the cassette reservoir will be immense.

Rowe-Magnus et al. Genome Res (2003) 13 (3): 428-442

84% of the V. cholerae cassettes

16% of the V. cholerae cassettes were likely recruited from other integrons

Comparaison des SI des deux V.vulnificus

intIA

Various demonstrated adaptive functions :

Pathogenicity, Metabolic activities, DNA repair,

Partial inventory of the functions encoded in SI cassettes

But also homologues to several AbR genes (aminoglycoside, chloramphenicol, fosfomycin, phosphinothricine, streptothricine, microcin immunity,...)

… and many plasmid addiction modules

Vaisvila et al. Mol. Microbiol. (2001) 42:587

Paradoxically, despite the fact that most of the hundreds of cassettes have NO

PROMOTER, the SI cassette content is extremely stable !

How does the selection apply to maintain such

large arrays of mobile elements (the cassettes), which, in addition, are silent in

most cases ?

What can explain this ?

Mobile elements, repeated sequences and bacterial genome stability

• Repeated sequences play a primordial role in the overall genome plasticity, especially through recombination.

• It is postulated, and observed, that “useless” or somehow deleterious genes  are lost from bacterial genomes (reduction).

• It is then considered that without a positive selective pressure, a gene will be lost quite rapidly, even faster if this gene is “mobile”.

Recombination between repeated sequences

intI

intI

hom. recomb between repeated cassettes

attC x attC or hom. recomb

intI

1- Does this apparent stability reflect the absence of integrase expression?

How does the selection apply to maintain such

large arrays of mobile elements (the cassettes), which, in addition, are silent in

most cases ?

What can explain this ?

Yes !The intI genes, even the one of class 1 RI, are completely silent in laboratory

conditions !

The integrase expression is tightly controlled

(However, the catalytic properties of the SI integrases are identical to those of the MRI

integrases)

2. The addiction cassettes: an explanation for the SI cassette arrays

stability ?

Paradox:

• each of the characterized SI carries a few repeated cassettes (up to 24X in cholerae)

• Most cassettes are silent

How are such structures maintained?

How are deletions counter selected ?

+

•Each of the characterized SI carries several cassettes encoding a functional genetic system similar to the Post-seggregational killing system

found in plasmids !

The PSK systems :

toxinantidote

stableunstable

Each of the characterized SI carries at least one cassette encoding a functional genetic system similar to the Post-seggregational killing system found in plasmids :

• ccdAB in V. fischeri // F plasmid

• Phd-doc in cholerae and metschnikovii // P1 phage• higAB in cholerae• parDE in cholerae• relBE in parahaemolyticus, vulnificus, cholerae• XbaI RE and methylase in X. campestris badrii

• RE in Pseudomonas, V. metschnikovii,, V. vulnificus

5 different types found in the V. cholerae SI, giving a total of at least 14 psk cassettes!

=> Recombination between repeated cassettes is counter selected and the concomitant loss of large part of the tandem cassette array is avoided.

functional

How adaptative are SIs ?

...but :– A great majority of cassettes bear ORF of

unknown function

– The only known promoter (inside IntI gene) cannot account for expression of the whole set of cassette

– Integration preferently occur at attI (integrase) site, while excision occur between two attC (cassette) site

Pc

How adaptative are SIs ?

IntI

AttI

Adaptative evolution

Stochastic evolution

SELECTION NEUTRAL DRIFT

SIs are evolutionary complex structures

standard molecular evolution

If a cassette’s gene is :

i. Functional & adaptative selection

ii. Not functional/adaptativeiii. Not expressed

neutral drift

Level of genes Level of cassettes

recombination dynamic

Depends on :

i. Mechanistic features ii. The K7 gene function iii. Its effective expression

Ka / Ks : A comparative method for detecting selection

For pairs of sufficiently diverse sequences :

• Ka / Ks = 1 : Neutrality

• Ka / Ks >> 1 : More non-syn. mutations

Diversifying selection• Ka / Ks << 1 : more syn. mutations

Purifying selection

Ka / Ks : Application to Vibrios SIs’ cassettes

• Cassettes extraction : “IRMA”

> ~ 1500 cassettes• Pairing : local blast on the cassettes bank

> ~ 200 groups• Alignment and test for selection

Ka / Ks analysis : Negative selection

• 25 % of the groups show strong features of purifying selection functionality

• Physical link between adaptative cassettes ? promoter identification...

• Lots of TA system

• Proteins involved in stress response

SI are evolutionary complex structures

IntI

Pc

SELECTION

DRIFT

AttI

TA system

Intensity should depends on

- recombination rate

- propency to recombine several cassettes together

STABILIZATION

The genesis of a MRI

3 ’ conserved segmentintegrase

intI1sul

qacEDoxa9cmlA2dfrVIaacA4

Multi-resistant integron

rplTrpmIinfC

L20L35IF3

intI

Chromosomal Super-integron

integrase

There are 3 major differences between MRIs & SIs:•Size

•The function of most SI cassettes are unknown•Homology of the SI attC sites compared to the

attC sites of MRIs

Genesis of a mobile integron

qacEΔsul1intI1

IS

qacEΔsul1intI1

IS IS

qacEΔsul1intI1

IS IS

Composite transposon

Conjugative plasmid

qacEΔsul1intI1

MR integron

Creation of a multi-resistant integron

SI Shewanella sp

SI Vibrio sp

qacEΔsul1 qacEΔsul1

SI Xanthomonas sp

qacEDsul1qacEΔ sul1

SI unknown species

qacEDsul1qacEΔ sul1

In a single environment, a wastewater treatment plant in germany:97 different resistance plasmids, 21 different resistance cassettes

(Tennstedt T., FEMS Microbiol Ecol. (2003) 45:239)

Bacterial resistance evolution by

recruitment of super-integron gene

cassettes

Potential antibiotic resistance cassettes in the V. cholerae N16961 SI

ORF Number on Chromosome IIb

Proposed Function

VCA0300 Chloramphenicol acetyltransferase*

VCA0328, VCA0341, VCA0463 Fosfomycin glutathione-transferase

VCA0387Phosphinothricin acetyltransferase

VCA0473 Streptogramin acetyltransferase

b, the 214 ORFs in the 179 cassettes in the SI of V. cholerae strain N16961are numbered from VCA0292 to VCA0506 (Heidelberg et al., 2000).

*,VCA0300 is 60% identical to CATB6 and 40% identical to SatA

R388

In3

dfrB2 orfA

oriT

IntI1

VCA0xxx

V. cholerae

R388

VCA0xxx orfA [AbR]

oriT

dfrB2

VchintI4

SI

VCA0292 VCA0506

p112

IntI1

IPTG

IntI1

E. coli [NalR]conjugate

Directing promoterless cassettes to the attI site of a class 1 integron

• Sequenced 14 random clones – identified 14 different K7s.

• MRIs randomly recruit SI gene cassettes. Those observed within the MRIs of clinical

isolates are selected according to environmental conditions.

• Selected on media containing different Ab

• Obtained clones that were resistant to Cm

• V. cholerae parent strain is Cms

attI1.1 dfrfus

This is not limited to this unique example.

Recently:

• Two CARB genes have been identified in the SI of environmental Vibrio isolates. These genes show signatures that point them as the ancestors of all the cassette-encoded carbenicillinases found in multi-resistance integrons. (Petroni, AAC (2004) 48:4042-46)

• We have identified two novel dfr cassettes in the SI of two environmental V. splendidus.

Conclusions

• The widely spread multi-resistance integrons and their cassettes derive from the sedentary super-integrons and their cassette pools.

•The stability of the silent SI cassette arrays, hundreds of cassettes long, is due to both a tight control of the integrase expression and the presence of multiple addiction cassettes.

•If we made progress in understanding the recombination reactions, we still do not know the cassette genesis process…

Part 2

Cassette insertion in integrons : a

novel recombination process

involving a folded single stranded

substrate.

intI attI site attC2 siteattC1 site attC3 siteattI site

Excisions occur via attC x attC recombination

Two types of reactions: cassette excision and cassette

integration

Recombination reactions in integrons

Integrations occur through attI x attC recombination

The integron paradox

The attC and attI sites structure differ from the canonical Tyrosine Recombinase

Recombination SitesCre, Flp, XerCD and Intλ : the recombination occurs between two very similar

sites

ArgR PepAInt IHFXis Fis

6 LoxP (Cre)

8 Frt (Flp)

8 Cer (XerCD)

7 attP (Intλ)

RBESimple Site

Potential RBEs in the attI sites ?

CS

attI1 cat--------------------------------------------------------------------------- |||

attI4 catatagttctcactgaatatttaactggttatttgtacagtatttgttggttgtttttatgtcaagaggctatacag

attI1 -----ggcttgttatga-ctg-tttttttgtacagtctatgcctcgggcatccaagcagcaagcgcgtta-cgccg--t || ||| | ||| |||||| | || | ||| | | ||| |||| | | |attI4 acatcagcaatctataagctgagatttttgaatggtgtgatgctcaacatactgatttagaaggttgttatggtagtat

attI1 gggtcga-t-gtttgat-gttatggagcagcaacgatGTTACGCagcagggcagtcgccct-aaaacaaaGTTAGAT | | | | | | | ||| ||| | | | | ||| || || | | | | ||||| |attI4 gcacccagtggtctattaattagatagcggtagcctacctgttggaaaggtaagaagctgtctagaaagcGTTAGTT

attC sites characteristics

Stokes, H. and Hall, R (1989) Mol Micro 3:1669; Collis, C. and Hall, R.(1992) Mol Micro 6:2875; Recchia, G. and Hall (1995) Microbiol 141:3015

attC site

ICS CS (RYYYAAC) (G / TTRRRY)

variable region

• little primary sequence conservation among the attC sites, except their boundaries, ICS and CS

• the ICS is always complementary to the CS of the circularized cassette.

•Sizes vary from 57 to 142 nt

• imperfect palindromic structures

gtcgcgatatgcgGCCTAACaattcGTCCAAGCcgacgcgcttcgcggcgcgGCTTAACtcaggtGTTAGGCcgcatgga

attC structural characteristics conservation

ICS CS

R’’ and R’:AAC/GTT absolutely conserved

L’’ and L’: poorly conserved except for the extra C

R’’ L’’ L’ R’

LH simple site RH simple site

34 to 119 bp

Francia MV, Zabala JC, de la Cruz F, Garcia Lobo JM. J. Bacteriol.(1999) 181:6844-9.

In 1999, the group of Fernando de la Cruz made the following observations in DNA / Integrase binding experiments

Gel mobility shift assays with dsDNA fragments containing aadA1 attC (left) or the attI site (right). Lanes 1, control dsDNA; lanes 2, dsDNA plus E. coli C41 control extract; lanes 3, dsDNA plus pure IntI1-COOH; lanes 4, dsDNA plus purified IntI1. F, free dsDNA; B, DNA-protein complex.

=>IntI does not bind ds attC

+IntI1

+IntI1

Gel retardation assays with ssDNA fragments containing either the top or the bottom strands of the aadA1 attC site (left) or the attI site (right). The substrate DNA used is indicated at the top of the figure. Protein extracts in each lane are as follows: left panel, lane 1, control DNA; lane 2, E. coli C41 control extract; lane 3, IntI1-COOH; lane 4, IntI1; lane 5 E. coli C41 control extract; lane 6, IntI1-COOH; lane 7, IntI1; lane 8, control DNA; right panel, lane 1, DNA alone; lane 2, purified IntI1; lane 3, IntI1-COOH; lane 4, E. coli C41 control extract; lane 5, IntI1; lane 6, IntI1-COOH; lane 7, E. coli C41 control extract; lane 8, control DNA. F, free DNA.

+IntI1

+IntI1

+IntI1

+IntI1

What about ss attC and attI ?

TTATAACAAACGCCTCAAGAGGGACTG -- 79bp -- TCAGCCCCTTAGGCGGGCGTTATAAAATATTGTTTGCGGAGTTCTCCCTGAC -- 79bp -- AGTCGGGGAATCCGCCCGCAATATT3’

5’

R’’ R’L’’ L’

simple site simple site

3’5’

recombination point

R’

AAA TTTATAAC CGCCTAAGGGGCTG CAACGCAATATTG GCGGATTCCCCGAC GTTGCG CGG T

C A A

R’’ L’’

L’

5’

3’

top strand : foldedsingle strand form

R’ L’

GCC ATTATAAC CGCCTAAGGGGCTG CAACGCAATATTG GCGGATTCCCCGAC GTTGCG TTT AG T T

R’’ L’’5’

3’

bottom strand : folded single strand form

5 bpRBE

simple site

RBE

A few examples

* : all are recombinogenic…

A model for the cassette integration at the attI site:

ss attC x ds attI

B

A

Plasmid promiscuity : meeting the challenge of DNA immigration control. B. Wilkins. Env Microbiology, 2002,4:495

We thought that conjugation allowed to test the realism of a such a recombination model involving a folded single strand DNA:

- The transferred strand depends on the transfer origin orientation

=> It is always the same

Indeed conjugation always goes through a single strand transfer

λattB

oriT

oriVR6K

pir

::RP4 (transfer functions)

λattP

IntλX

IHF

PIR

λattB

oriT

oriVR6K

The suicide - conjugation assay

M. Bouvier, G.Demarre and D. Mazel. EMBO J (2005) 24, 4356–4367

==> The recombination freq. after strand 1 transfer equals the recombination freq. after strand 2 transfer

N1

N2

ds x dsN1

=N2 !

+

1,57

E-0

3

1,99

E-0

3

1,02

E-0

5

9,68

E-0

6

1,00

E+

00

1,00

E+

00

1,00E-07

1,00E-06

1,00E-05

1,00E-04

1,00E-03

1,00E-02

1,00E-01

1,00E+00

1,00E-08

bs ts

attI1

oriTRP4+

Recombination site

Strand injected byconjugation

Origin of transfer

bs : bottom strand / ts : top strand

Integrase IntI1

Nature of the Substrate

Inte

gra

tion R

eco

mbin

ati

on F

requenci

es

λattP

orientation 1 orientation 2

oriTRP4+

Intλ

16h of reactionreplicative double

strand substrate

non-replicative singlestrand substrate

16h of conjugation

attI1 x attCaadA7

replicative doublestrand substrate

non-replicative singlestrand substrate

1,75

E-0

8

1,75

E-0

8

1h

1,90

E-0

7

5,68

E-0

8 2h

2,93

E-0

4

1,78

E-0

4

3hof conjugation

1h30 of reaction

λattP x λattB

Nbs

Nts

attCbs x attIds

Nbs >> Nts !

+

1,00E-08

1,00E-07

1,00E-06

1,00E-05

1,00E-04

1,00E-03

1,00E-02

1,00E-01

1,00E+00

Nature of the Substrate

Inte

gra

tion R

eco

mbin

ati

on F

requenci

es

(cata

lyze

d b

y IntI

1)

5,55

E-0

3

1,00

E-0

6

1,46

E-0

2

5,31

E-0

2

bs ts

VCR2/1

oriTRP4+

Recombination site

Strand injected byconjugation

Origin of transfer

bs : bottom strand / ts : top strand

replicative double strand substrate

non-replicative single strand substrate R’ L’

GCC ATTATAAC CGCCTAAGGGGCTG CAACGCAATATTG GCGGATTCCCCGAC GTTGCG TTT AG T T

R’’ L’’

5’

3’

VCR2/1 bs

ATGTCTAACG TT ATTAAGCCGCGCCGCTACAGATTGT AA TAATTCGGCGCGGCG

AG

ATGGT

C GA

3’

5’R’

R’’

L’

L’’

attCaadA7WTattCaadA7 bs

2,02

E-0

2

1,69

E-0

2

attCaadA7

bs

oriTRP4+

3,65

E-0

5

2,80

E-0

22,

80E

-02

bs

3,65

E-0

5

ts

M. Bouvier, G.Demarre and D. Mazel. EMBO J (2005) 24, 4356–4367

1,00E-07

1,00E-06

1,00E-05

1,00E-04

1,00E-03

1,00E-02

1,00E-01

1,00E+001,

57E

-03

9,68

E-0

6

1,02

E-0

5

1,99

E-0

3

bs ts

attI1

oriTRP4+

Recombination site

Strand injected byconjugation

Origin of transfer

bs : bottom strand / ts : top strand

1,00E-08

Integrase IntI1

Nature of the Substrate

replicative double strand substrate

non-replicative single strand substrate

Inte

gra

tion R

eco

mbin

ati

on F

requenci

es

TTATAACAAACGCCTCAAGAGGGACTG -- 79bp -- TCAGCCCCTTAGGCGGGCGTTATAAAATATTGTTTGCGGAGTTCTCCCTGAC -- 79bp -- AGTCGGGGAATCCGCCCGCAATATT3’

5’

R’’ R’L’’ L’

simple site simple site

3’5’

recombination point

R’

AAA TTTATAAC CGCCTAAGGGGCTG CAACGCAATATTG GCGGATTCCCCGAC GTTGCG CGG T

C A A

R’’ L’’

L’

5’

3’

top strand : foldedsingle strand form

R’ L’

GCC ATTATAAC CGCCTAAGGGGCTG CAACGCAATATTG GCGGATTCCCCGAC GTTGCG TTT AG T T

R’’ L’’5’

3’

bottom strand : folded single strand form

G T

sufficient to render the ts recombinogenic (x103)and, inversely, abolish recombination of the complementary strand (former bs)

How do the Integron integrases accomodate and recognize such substrates?

What differentiate these from the other Y recombinases?

<= The additional segment

=> Crystallization of IntIA from the V. cholerae superintegron bound to a substrate mimicking the folded

bs attC site (a VCR).

2 fold symmetry, not 4 X :

-A and C active, bound to G20 in trans-B and D bound to T12 in cis => disorganization of their catalytic domains.

D. MacDonald, G. Demarre, M. Bouvier, D. Mazel and D. N. Gopaul. Nature (2006) 440, 1157-62)

VchIntIA – VCRbs Hairpin tetrameric synapse structure

VchIntIA – VCRbs Hairpin tetrameric synapse structure

94

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Stabilization of the extra-helical base G20”

Stabilization of the T12” extra-helical base

KY(P)

(R)HH

Active sites and catalytic residues