Bacterial genome plasticity and integrons

1. Bacterial genome plasticity and integrons Didier Mazel Unité Plasticité du Génome Bactérien http://www.pasteur.fr/recherche/unites/pgb/

2. 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

3. Introduction We now know the intimity of bacterial genomes quite well • 257 bacterial genome had been sequenced (+24 Archeal genomes) • 521 were on going (+25 Archaea) in september 2005 http://www.ncbi.nlm.nih.gov/genomes/lproks.cgi

4. 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

6. 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.

7. Variation in the bacterial genome organisation: 2nd rule: Variations using 2 mechanisms:Transposition and homologousRecombinaison => repeated sequences play a major role, notably the mobile DNA elements.

8. The mobilome

9. 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

10. 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

11. 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

12. 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

13. Limits of the exchanges : none ??? • based on sequence similarity: Eucaryotes bactéries archae • Exprimentaly : idem ! • Natural systems: Ti DNA Agrobacterium -> plants migration of the organites genes in the nucleus The limit is more a question of sharing the same ecological niche

14. 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)

15. an example : the antibiotics resistance • recent phenomenon: 60 years • Extremely documented • 80 % of resistances due to exogenous gene acquisitions

16. the multi-resistance phenomenon • first apparition in Japon in 1955. • follows the massive production and use of Ab.

17. AbR genes origin • in most cases : ???? • bacterial producers have had to develop protection mechanisms => potential source

18. vancomycin resistance loci comparison producers S . t o y o c a e n s i s 5 4 - 6 1% A . o r i e n t a l i s vanH ddl vanX 6 1 - 6 4% v a n R v a n S v a n Y v a n Z Tn1546 resistance locus T n 1 5 4 6 v a n A { vanH VanA vanX REQUIRED FOR GLYCOPEPTIDERESISTANCE Marshall, C.G et al. (1997) PNAS 94:6480

19. resistance genes flow Capture dans Tn (Conjugatif) Plasmide AbR from the source... …to the clinical isolate

20. A crucial question: how do Transposons acquièrent-ils ces gènes ? the answer has been obtained for two examples: • CompositeTransposons • Transposons carrying an integron

21. composite transposon assembly AbR

22. The integrons • They constitute what can be defined as a natural genetic engineering system: • Incorporate ORFs • Express them

23. Gene cassettes integron In2… In60 aadA1 attC tniBΔ1 tniA qacEΔ1 sul1 orf5 intI1 IRt IRi attI tnpA tnpR res merD merA merP merR tnpM? urf2 merE merC merT Tn21 IRmer urf2M Tn21 Transposition genes mer genes Tn21 IRtnp

24. 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).

25. Multi-Resistant Integron intI1 qacEΔ aacA4 dfrVI cmlA2 oxa9 sul integrase 3 ’ conserved segment :59 base elements attI site 5 4 - 1 3 5 n t RYYYAAC GTTRRRY inverse core site core site variable region

26. attI site intI attI site attC1 site attC2 site attC3 site Recombination reactions in integrons Two types of reactions: cassette excision and cassette integration Excisions occur via attC x attC recombination Integrations occur through attI x attC recombination

27. attC site variable region CS ICS (RYYYAAC) (G / TTRRRY) Integron cassette structure and characteristics • most contain a single ORF • promoterless • 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 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

28. The Mobile Integrons and the antibiotic Resistance : Five “classes” of Integrons (MIs) •Share between 45-58% amino acid identity •All are associated with mobile DNA elements •Carry at most 8 resistance cassettes class 1 Tn21 family (most ubiquitous) class 2* Tn7 family class 3 self-transmissible plasmid class 4 self-transmissible plasmid class 5 SXT element (constin)

29. What is the origin of the multi-resistance integrons and their cassettes ?

30. Integron Vibrio cholerae genome

31. sto mrhA 126 Kb, 179 cassettes, 3% of genome : VCR 121-123 nt RYYYAAC GTTRRRY inverse core site core site Most are 95% identical 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 Mazel, D et al (1998) Science 280:605; Heidelberg, Jet al. (2000) Nature 406:477; Rowe-Magnus et al. (1999) Res Mic 150:641 The V. cholerae superintegron intI4 or VchintIA i n f C r p m I r p l T I F 3 L 3 5 L 2 0 i n t e g r a s e

32. 84% of the V. cholerae cassettes 16% of the V. cholerae cassettes carry a Remote attC site

33. integron Multi-resistant intI1 qacED aacA4 dfrVI cmlA2 oxa9 sul integrase 3 ’ conserved segment Super-integron Chromosomique intI i n f C r p m I r p l T integrase I F 3 L 3 5 L 2 0 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

34. Are SIs the source of MRIs?

35. ? ?

36. SIs are widespread among the proteobacteria => We characterized SIs / Is structures in >30 proteobacterial species (mainly γ) Vibrio Listonella Alteromonas Photobacterium Moritella Xanthomonas Pseudomonas 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

37. IntI1 IntI1 IntI3 IntI3 IntI2 IntI2 0 . 1 IntI9 SXT IntI9 SXT Ppr Vsa Pph Vfi Lpe intIHS intIHS Vvu Vmi Vha Vpa Vch Vho Vme Sty Vor Eco Lan Yen Son Spu Vma Gsu Mma Ama Tfe Pim Tde Xca Pfl Psy Pae Xsp Pme Pal Pps Neu The integron is an ancient evolutionary apparatus TfeintIA SonIntIA SpuIntIA SpuIntIB NeuIntIA XspIntIA I7-2 I8-2 EcFimE I6-2 XcaIntIA XcaIntIB EcFimB PmeIntIA PalcIntIA VchIntIA P22int VmiIntIA VmeIntIA LanIntIA P2 VvuIntIA VpaIntIA LpeIntIA VnaIntIa VfiIntIA P22xis GsuintIA Tden P4Int e14 Lambda 0.1 Rowe-Magnus et al. (2001) PNAS 98:652; Rowe-Magnus and Mazel (2001) Cur. Op. Microbiol. 4:565

38. . Les genes intI (capture) et les XXRs (cassette genesis) co-evoluent VCRs/VMiRs VPR2 VPRs VPR4 VCRcI VFRs dfrVI CAR4 LPRs VCRcA 0.1 VMeRs XSRs catB6 SPR2 aadA1b SPR5 XCR9 SPR3 aadA1a aadB SPRs aacA qacF aadA7 aadA6 SPR4 SPR1 aadA2 SPR6 XSR6 XCR7 XCRs 0.1

39. 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).

40. The Gene Cassette Reservoir To what extent do the different SIs share cassettes? What kind of functions are found in the cassettes?

41. Comparison of SI cassette contents •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

42. 84% of the V. cholerae cassettes 16% of the V. cholerae cassettes were likely recruited from other integrons

43. Comparaison des SI des deux V.vulnificus intIA

44. Partial inventory of the functions encoded in SI cassettes Various demonstrated adaptive functions : Pathogenicity, Metabolic activities, DNA repair, But also homologues to several AbR genes (aminoglycoside, chloramphenicol, fosfomycin, phosphinothricine, streptothricine, microcin immunity,...) … and many plasmid addiction modules

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

46. 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 ?

47. 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”.

48. Recombination between repeated sequences

49. intI hom. recomb between repeated cassettes attC x attC or hom. recomb intI intI

50. 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 ? 1- Does this apparent stability reflect the absence of integrase expression?