bacterial genome plasticity and integrons didier mazel unité plasticité du génome bactérien
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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