Major results obtained by the CRAB consortium
Problems addressed.
the principal aim of our project was to explore the mechanisms and process dynamics at work in each of the modules of the chain of AR gene dissemination – integrons, transposons, conjugative plasmids and stability modules – in a concerted approach. We concentrated on the as yet poorly studied aspects of these mechanisms. Our work has been divided in 4 work packages (WP):
WP1: mechanism of integron cassette recombination, signals triggering integrase expression and molecular evolution of gene cassettes.
WP2: Propensities of three major classes of ISs to acquire, stabilize and vehicle AR genes and identification of host factors that affect transposition.
WP3: Identification of host factors that affect conjugation, development of in vitro models for conjugation on cultured mammalian cells.
WP4: Identification of novel stabilization systems and their intracellular targets, and identification of host factors that trigger these systems;
Partnership and practical organization.
CRAB consortium consists of 8 partners from 6 European countries. Industry is represented by the Belgian company Delphi Genetics.
Collaborations between all groups were very successful and in a friendly atmosphere. In depth collaborations have been established through this consortium, as illustrated by the sabbatical year spent by Pr De la Cruz in the Dr Mazel laboratory (from 08/2008 to 07/2009). In addition, during the course of this project, a PhD student from Dr Chandler lab has joined Pr De la Cruz group for a post-doc.
We have had three meetings during the time frame of the project. The last meeting took place in may 2009 in Santander, after 41 months of work and gathered most of the people involved in the different groups. This was the occasion to share all the data collected since the beginning of the project and to discuss these to prepare our final report and future developments from our results. This meeting was a success and confirmed the convergence between several of our projects.
Major Achievements and Prospects.
Concerning the integron study in WP1, we have made progress in the understanding of the recombination reactions involving the cassettes. We have been able to determine which structural features in the attC sites under single stranded form conditioned the strand choice toward the bottom strand exclusive recombination, we have also shown that the recombined nucleotide triplet was not locked for mechanical reasons. Using massive mutagenesis and a powerful genetic screen we have been able to identify the IntI1 integrase residues involved in the attC recognition and we have been able to make a structural modeling of this integrase. In parallel, we have demonstrated that the integrase expression was controlled by the SOS response and we can now draw the integrated picture of the cassette exchange and capture as a function of the environmental threats.
The major objectives of WP2 were to obtain a detailed description of the transposition mechanisms and capacity to sequester and transport AR genes by different paradigmatic ISs of the different catalytic families. We have made considerable progress for all but 2 chosen ISs. In particular, we have demonstrated that IS608 transposition relies on single strand DNA recombination. Together with the IS91 transposition results, the integron recombination data obtained and the knowledge of the conjugation process (which is always a DNA single strand transfer), these data underline the importance of single stranded DNA forms as a key intermediate in the various different recruitment and dissemination mechanisms. We also show that ISs together with conjugative plasmid constitute a powerful combination for horizontal gene transfers.
The WP3 objectives were to analyze the mechanisms of bacterial conjugation, including the characterization of the genetic and environmental factors that affect plasmid conjugative transfer as well as its establishment and stability in the recipient host, and thus affect the acquisition of AR by pathogenic bacteria. The molecular description of the different steps of the relaxosome assembly and activity made considerable progress. We have identified several E. coli host genes that affect the conjugative propensity in the donor context. A model for human catheter –associated urinary tract infection (CAUTI) was developed that enables us to investigate how pathogenic and commensal bacteria colonize and thrive on catheter surfaces. Importantly this approach identifies genes responsible for bacterial motility, growth and the development of polymicrobial biofilms in the catheterized urinary tract. We measured rapid spread of genes that create antibiotic resistance among bacteria in this clinically relevant model, thus conditions controlling genetic exchange can be defined. Our research activities have provided molecular details for the mechanisms underlying gene transfer via bacterial conjugation generally. Elucidation of these mechanisms flows immediately into applications designed to control the proliferation of bacteria as well as restrict transmission of antibiotic resistance in the CAUTI environment.
The toxin-antitoxin systems (TA) studies, targeted by the WP4, were linked to their aptitude to stabilize the mobile genetic elements which disperse AR genes in bacterial populations. These systems are also present in bacterial chromosomes, often in multiple copies. The WP4 goals were (1) to discover new families of TA, (2) to validate experimentally the putative candidates and to analyze the functional TAs and (3) to determine what is the function of TA systems, and (4) to develop new molecular biology tools. We have developed an original bioinformatics approach, which has already led to the identification of tens of potential new toxins and new antitoxins. These are currently being validated by in vivo study. We have also obtained new data which show that TA modules carried on the bacterial chromosome could also play a stabilization role.
To summarize the result gathered within the 42 months of the project, we have succeeded in understanding the basis of several properties shown by mobile genetic elements which disseminate the AR genes. One very important observation we made is the recurrent role of single stranded DNA in the different aspects of the dissemination chain of events, from transposition and recombination to the transfer itself, and finally its role as sensor for the adaptability through the SOS response. Our work has led to a total of more than 75 major articles published or submitted, for the different WPs during this 42 months period.
These publications are:
WP1- The Integrons
MacDonald D, Demarre G, Bouvier M, Mazel D and DN. Gopaul. Structural basis for broad DNA specificity in integron recombination. Nature (2006) 440 : 1157-1162.
Mazel D. Integrons : agents of bacterial evolution. Nature Reviews Microbiology (2006) 4(8):608-20.
Szekeres S, Dauti M, Wilde C, Mazel D, Rowe-Magnus DA. Chromosomal addiction loci can diminish large-scale genome reductions in the absence of selection. Molecular Microbiol. (2007) 63(6),1588-1605.
Cattoir V, Poirel L, Mazel D, Soussy CJ, Nordmann P. Vibrio splendidus as the source of plasmid-mediated QnrS-like quinolone resistance determinants. Antimicrob Agents Chemother. (2007) 51(7) : 2650-2651
Demarre G, Frumerie C, Gopaul D N. and Mazel D. Identification of key structural determinants of the IntI1 integron integrase that influence attC x attI1 recombination efficiency. Nucleic Acids Research (2007) 35(19):6475-89.
Wilde C, Mazel D, Hochhut B, Middendorf B, Le Roux F, Carniel E, Dobrindt U and, Hacker J. Delineation of the recombination sites necessary for integration of pathogenicity islands II and III into the Escherichia coli 536 chromosome. Molecular Microbiol. (2008) 68(1):139-151.
Babic, A. Guérout, A.-M., and D. Mazel. Construction of an improved RP4 (RK2) - based conjugative system. Research in Microbiology (2008) 159(7-8):545-9.
Marcel JP, Alfa M, Baquero F, Etienne J, Goossens H, Harbarth S, Hryniewicz W, Jarvis W, Kaku M, Leclercq R, Levy S, Mazel D, Nercelles P, Perl T, Pittet D, Vandenbroucke-Grauls C, Woodford N, Jarlier V. Healthcare-associated infections: think globally, act locally. Clin Microbiol Infect. (2008) 14(10):895-907.
Cambray G and Mazel D. Synonymous Genes Explore Different Evolutionary Landscapes. PLoS Genetics (2008) 4(11): e1000256.
Guerin E*, Cambray G*, Sanchez-Alberola N*, Campoy S, Erill I, Da Re S, Gonzales-Zorn B, Barbé J, Ploy MC and Mazel D. Recombination of integron cassettes is under control of the SOS response. Science (2009), 324(5930):1034 - (*, co–first authors)
Jacquier H, Zaoui C, Sanson-le Pors M-J, Mazel D and Berçot B. Translation regulation of integrons gene cassette expression by the attC sites. Molecular Microbiology (2009) 72(6): 1475–1486.
Bouvier M, Ducos-Galand M, Loot C, Bikard D and Mazel D. Structural features of single-stranded integron cassette attC sites and their role in strand selection. PLoS Genetics (2009) 5(9):e1000632.
Frumerie C, Ducos-Galand M, Gopaul D N and Mazel D. The relaxed requirements of the integron cleavage site allow predictable changes in integron target specificity. Nucleic Acids Research (2009) in press
WP 2 - Transposons
Siguier, P., Filé, J. and Chandler, M. (2006) Insertion sequences in Bacterial Genomes. Cur. Op. Microbiol. 2006 9(5):526-31.
Gueguen E, Rousseau P, Duval-Valentin G, Chandler M. Truncated forms of IS911 transposase downregulate transposition. Mol Microbiol. (2006) 62(4):1102-16.
Filee J, Siguier P, Chandler M. I am what I eat and I eat what I am: acquisition of bacterial genes by giant viruses. Trends Genet. 2007 Jan;23(1):10-5.
Filee J, Siguier P, Chandler M. Insertion sequence diversity in archaea. Microbiol Mol Biol Rev. (2007) 71:121-57.
Rousseau P, Loot C, Guynet C, Ah-Seng Y, Ton-Hoang B, Chandler M. Control of IS911 target selection: how OrfA may ensure IS dispersion. Mol Microbiol. (2007) 63:1701-9.
Imre A, Olasz, F, Kiss J, and Nagy B (2006): A novel transposon-based method for elimination of large bacterial plasmids. Plasmid. (2006) 55(3):235-41.
Zsolt FP, Noemi N, Olasz, F and Nagy, B: (2006) The role of mobile genetic elements in the horizontal spread of tetracycline resistance and virulence of certain Escherichia coli and Salmonella bacteria. MAGYAR ALLATORVOSOK LAPJA 128 (1): 39-47 2006. In Hungarian
Kiss J, Nagy Z, Toth G, Kiss GB, Jakab J, Chandler M, Olasz F. Transposition and target specificity of the typical IS30 family element IS1655 from Neisseria meningitidis. Mol Microbiol. (2007) 63:1731-47.
Duval-Valentin, G., Marty, B., and Chandler, M. A Window of Opportunity: Co-translational transposase binding to IS911 ends is a new mechanism for a transient protein activity. In prep.
Pasternak, C., Ton-Hoang, B., Coste G, Bailone, A., Chandler, M. and Sommer, S. Irradiation-induced genome fragmentation of Deinoccocus radiodurans triggers extensive excision and reinsertion of a single resident insertion sequence. Submitted
Guynet, C., Achard, A., Ton Hoang B., Barabas, O., Burgess Hickman, A., Dyda, F. and Chandler, M. Resetting the site: Redirecting integration of an insertion sequence in a predictable way. Mol.Cell (2009) 34(5): 612 - 619
Siguier P., Gagnevin L. and Chandler M. The new IS1595 family, its Relation to IS1 and the frontier between insertion sequences and transposons. (2009) Res. Microbiol 160(3):232-41
Rousseau P., Loot C., Turlan C., Nolivos S. and Chandler M. Bias between the left and the right inverted repeats during IS911 targeted insertion. J Bacteriol. (2008) 190: 6111-6118
Filée J., Pouget N. and Chandler M. Phylogenetic evidence for extensive lateral acquisition of cellular genes by Nucleocytoplasmic Large DNA Viruses. BMC Evolutionary Biology (2008) 8: 320
Filée J. and Chandler M. Convergent Mechanisms of Genome Evolution of Large and Giant DNA Viruses. Res Microbiol. (2008) 159(5):325-31
Chandler M., Microbiology: what now? (2008) Res. Microbiol. 120th Anniversary 159(1): 51-8
Barabas O,. Ronning D R., Guynet C., Hickman A B, Ton-Hoang B, Chandler M. and Dyda F. Mechanism of IS200/IS605 Family DNA Transposases: Activation and Transposon-Directed Target Site Selection. Cell (2008) 132: 208-220.
Guynet C., Hickman A B, Barabás O, Dyda F, Chandler. M and Ton-Hoang B. Single-stranded transposition of IS608: in vitro reconstitution of a new transposition mechanism. Mol. Cell (2008) 29: 302-312.
Rousseau P. and Chandler, M. Transposition : Topics in Chemical Biology. Wiley Encyclopaedia of Chemical Biology. Ed. Tadhg Begley (Chair) et al., (2007)
Rousseau P, Loot C, Guynet C, Ah-Seng Y, Ton-Hoang B, and Chandler M. Control of IS911 target selection: how OrfA may ensure IS dispersion. Mol Microbiol. (2007) 63(6):1701-9.
Filee J, Siguier P, Chandler M. Insertion sequence diversity in archaea. Microbiol Mol Biol Rev. (2007) 71(1):121-57.
Kiss, J., Bibó, M., Szabó, M, Olasz F. Stabilty of IS30 mediated cointegrates. In preparation
Imre, A., Olasz, F., Nagy, B. Site-directed (IS30-FljA) transposon mutagenesis system in Salmonella Enteritidis In preparation
Péter Z. Fekete, Elzbieta Brzuszkiewicz, Gabriele Blum-Oehler, Ferenc Olasz, Mónika Szabó, Gerhard Gottschalk, Jörg H. Hacker and Béla Nagy. Sequence analysis and comparative pathogenomics of plasmid pTc conferring virulence and antimicrobial resistance for F18+ porcine enterotoxigenic Escherichia coli. Submitted
Szabó, M., Kiss, J., and Olasz F. Functional compartments in the inverted repeats of IS30. Submitted
Szabó M, Kiss J, Nagy Z, Chandler M*, and Olasz F*. Subterminal sequences modulating IS30 transposition in vivo and in vitro. J. Mol. Biol. (2008) 375: 337-352
Kiss J, Nagy Z, Toth G, Kiss GB, Jakab J, Chandler M, and Olasz F. Transposition and target specificity of the typical IS30 family element IS1655 from Neisseria meningitidis. Mol Microbiol. (2007) 63(6):1731-47.
WP3- Conjugation
Boer, R., Russi, S., Guasch, A., Lucas, M., Blanco, A.G., Perez-Luque, R., Coll, M., and de la Cruz, F. (2006) Unveiling the molecular mechanism of a conjugative relaxase: The structure of TrwC complexed with a 27-mer DNA comprising the recognition hairpin and the cleavage site. J Mol Biol 358: 857-869.
Cesar, C.E., Machon, C., de la Cruz, F., and Llosa, M. (2006) A new domain of conjugative relaxase TrwC responsible for efficient oriT-specific recombination on minimal target sequences. Mol Microbiol 62: 984-996.
Garcillan-Barcia, M.P., Jurado, P., Gonzalez-Perez, B., Moncalian, G., Fernandez, L.A., and de la Cruz, F. (2007) Conjugative transfer can be inhibited by blocking relaxase activity within recipient cells with intrabodies. Mol Microbiol 63: 404-416.
Gonzalez-Perez, B., Lucas, M., Cooke, J.S., Vyle, L.A., de la Cruz, F., and Moncalián, G. (2007) Analysis of DNA processing reactions in bacterial conjugation by using suicide oligonucleotides. EMBO J (in press).
Hormaeche, I., Segura, R.L., Vecino, A.J., Goni, F.M., de la Cruz, F., and Alkorta, I. (2006) The transmembrane domain provides nucleotide binding specificity to the bacterial conjugation protein TrwB. FEBS Lett 580: 3075-3082.
Reisner, A. and E. L. Zechner (2007) Lateral Mobility and Expression of Genes in Microbial Biofilms, in Understanding Biofilms and their importance in industry, environment, and medicine, (J. Anné and W. Streit, eds), Springer, Heidelberg, in press.
Tato, I., Matilla, I., Arechaga, I., Zunzunegui, S., de la Cruz, F. and Cabezon, E. (2007) The ATPase activity of the DNA transporter TrwB is modulated by protein TrwA: implications for a common assembly mechanism of DNA translocating motors. J Biol Chem (in press).
M.P. Garcillán-Barcia, P. Jurado, B. González-Perez, G. Moncalián, L.A. Fernández and F. de la Cruz* “Conjugative transfer can be inhibited by blocking relaxase activity within recipient cells with intrabodies” (2007) Mol. Microbiol. 63: 404–416 (PMID: 17163977).
B. Gonzalez-Perez, M. Lucas, L.A. Cooke, J.S. Vyle, F. de la Cruz* and G. Moncalián “Analysis of DNA processing reactions in bacterial conjugation by using suicide oligonucleotides” (2007) EMBO J. 26: 3847-57 (PMID: 17660746).
I. Tato, I. Matilla, I. Arechaga, S. Zunzunegui, F. de la Cruz* and E. Cabezon* “The ATPase activity of the DNA transporter TrwB is modulated by protein TrwA: implications for a common assembly mechanism of DNA translocating motors”. (2007)J. Biol. Chem. 282: 25569-76 (PMID: 17599913).
C. Revilla, M.P. Garcillán-Barcia, R. Fernández-López, N.R. Thomson, M. Sanders, M. Cheung, C.M. Thomas and F. de la Cruz* “Different pathways to acquiring resistance genes illustrated by the recent evolution of IncW plasmids” (2008) Antimic. Ag. Chemother. 52: 1472-80 (PMID: 18268088).
M.P. Garcillán-Barcia and F. de la Cruz* “Why is entry exclusion an essential feature of conjugative plasmids?” (2008) Plasmid 60: 1-18 (PMID: 18440635).
I. Arechaga*, S. Zunzunegui, A. Peña., M.C. Fernández-Alonso, G. Rivas and F. de la Cruz “ATPase activity and oligomeric state of TrwK, the VirB4 homologue of plasmid R388 type IV secretion system” (2008) J. Bacteriol. 190: 5472-79 (PMID: 18539740).
B. González-Pérez, J. D. Carballeira, G. Moncalián and F. de la Cruz* “Changing the recognition site of a conjugative relaxase by rational design” (2009) J. Biotechnol. 27: 554-557 (PMID: 19039782)
A. Varsaki, G. Moncalián, M.P. Garcillán-Barcia, C. Drainas and F. de la Cruz* “Analysis of ColE1 MbeC unveils an extended ribbon-helix-helix family of nicking-accessory proteins” (2009) J. Bacteriol. 191: 1446-1455 (PMID: 19114496)
D. Pérez-Mendoza and F. de la Cruz* “Escherichia coli genes affecting recipient ability in plasmid conjugation: Are there any?” (2009) BMC Genomics 10:71 (PMID: 19203375).
M.P. Garcillán-Barcia, M.V. Francia and F. de la Cruz* “The diversity and classification of bacterial conjugative transfer systems”. (2009) FEMS Microbiol. Rev. 33: 657–687 (PMID: 19396961)
A. Valverde, R. Cantón, M.P. Garcillán-Barcia, A. Novais, J.C. Galán, A. Alvarado, F. de la Cruz, F. Baquero and T.M. Coque* “Spread of blaCTX-M-14 by IncK and IncHI2 plasmids disseminated among A, B1 and D community Escherichia coli phylogroups in Spain” (2009) Antimicrob. Ag. Chemother. (under revision)
F. de la Cruz, L.S. Frost*, R.J. Meyer and E.L. Zechner “Conjugative DNA Metabolism in Gram-negative Bacteria” (2009) FEMS Microbiol. Rev. (under revision)
M. Lucas, G. Rivas, G. Moncalián and F. de la Cruz “Two different sites in protein TrwC determine ssDNA stable binding and nic-cleavage” (2009) J. Biol. Chem. (under revision)
F. de la Cruz “The role of conjugation in the evolution of bacteria” (2009) pp. xx – xx in “Microbial Evolution 150 Years after The Origin of Species” (S. Maloy & R. Kolter, eds.), ASM Press. (under revision)
R. Fernández-López, I. del Campo and F. de la Cruz* “Architecture and design of a plasmid transcriptional regulatory network” (2009) Plos Genet. (submitted)
S. Mihajlovic, S. Lang, M. Sut, H. Strohmaier, G. Koraimann, G. Moncalián, E. Cabezón, F. de la Cruz and E.L. Zechner* “Regulation at the coupling protein – relaxosome interface: TraD stimulates TraI DNA transesterase activity” (2009) J. Bacteriol. (in press).
S. Mihajlovic, S. Lang, M. Sut, H. Strohmaier, C.J. Gruber, G. Koraimann, G. Moncalián, E. Cabezón, F. de la Cruz and E.L. Zechner “Plasmid R1 conjugative DNA processing is regulated at the coupling protein interface” (2009) J. Bacteriol. in press
Sut, M.V, Mihajlovic, S., Lang, S., Gruber, C. J. and E. L. Zechner “Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1”. (2009) J. Bacteriol. in press
WP4- Stability
Tsilibaris V., Maenhaut-Michel G., Mine N., Van Melderen L. (2007) What is the benefit for E. coli to have multiple toxin-antitoxin systems in their genomes? J. Bacteriol., 189(17):6101-8
Wilbaux M., Mine N., Guerout AM., Mazel D., Van Melderen L. (2007) Functional interactions between coexisting toxin-antitoxin systems of the ccd family in Escherichia coli O157:H7. J. Bacteriol. 189(7):2712-9.
Kędzierska B., Lian LY., Hayes F. (2007) Toxin-antitoxin regulation: bimodal interaction of YefM-YoeB with paired DNA palindromes exerts transcriptional autorepression. Nucleic Acids Res. 35:325-339.
Szekeres S., Dauti M., Wilde C., Mazel D., Rowe-Magnus DA. (2007) Chromosomal toxin-antitoxin loci can diminish large-scale genome reductions in the absence of selection. Mol. Microbiol. 63(6):1588-605.
Bailey S.E.S., Hayes F. (2009) Influence of operator site geometry on transcriptional control by the YefM-YoeB toxin-antitoxin complex. J. Bacteriol. 191:762-772
De Jonge N, Buts L, Vangelooven J, Mine N, Van Melderen L, Wyns L, Loris R. (2007) Purification and crystallization of Vibrio fischeri CcdB and its complexes with fragments of gyrase and CcdA. Acta Crystallograph. Sect F Struct. Biol. Cryst. Commun. 63(Pt 4):356-60
Hayes F, Barillà D (2009) Extrachromosomal components of the nucleoid: recent developments in deciphering the molecular basis of plasmid segregation. In CJ Dorman and RT Dame (ed.), Bacterial Chromatin, Springer Publishing, New York, in press.
Kędzierska B., Lian LY., Hayes F. (2007) Toxin-antitoxin regulation: bimodal interaction of YefM-YoeB with paired DNA palindromes exerts transcriptional autorepression. Nucleic Acids Res. 35:325-339
Mine N., Guglielmini J., Wilbaux M., Van Melderen L. (2009) The Decay of the Chromosomally-encoded ccdO157 Toxin-antitoxin System in the Escherichia coli Species. GENETICS, 181(4):1557-66
Saavedra De Bast M., Mine N., Van Melderen L. (2008) Chromosomal toxin-antitoxin systems may act as anti-addiction modules. J. Bacteriol., 190(13):4603-9
Stieber D., Gabant P., Szpirer C.Y. (2008) The art of selectivekilling: Plasmid toxin/antitoxin systems and their technological applications. Biotechniques 45(3):432-4
Tsilibaris V., Maenhaut-Michel G., Mine N., Van Melderen L. (2007) What is the benefit for E. coli to have multiple toxin-antitoxin systems in their genomes? J. Bacteriol., 189(17):6101-6108
Van Melderen L, Saavedra De Bast M. (2009) Bacterial toxin-antitoxin systems: more than selfish entities? PLoS Genet. 5(3):e1000437
