Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 214 Mechanisms and DNA Specificity in Site-specific Recombination of Integron Cassettes CAROLINA JOHANSSON ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 UPPSALA ISBN 978-91-554-6767-8 2007 urn:nbn:se:uu:diva-7429 ! " # $% $& '( ) * ! ) ) +* * ,- ) ./ 0* 1 2 !*/ # * / $&/ * 34 5 ) 5 6 ) 7 ) 8 ! / 4 / $%/ 9( / / 853 '&:6'6((%69&9&6:/ * / 0* * ; ) * !/ 8 ! 1 * * ) ! ! 6 6 ) 8 8/ 8 8 * ! ! ) * 34 ! ! * < / 5 ) ) ! ! ! / = * * ! ! * * * ! / * 1 * ! ) * ! ) * 1 ;/ 0* 1 ; ! ) * / 0* ) * !" > 0 # 1 * ? / @; * 1 ) / ! * *) * * 1 * 8 8 * ) / ) ! * * )1 * * ) * * * * ) * *!* / 5 ! * 1 A ! / " * * ! 8 8 ! 1 1 * / ! * ! ! 1 ) ) 8 8 * ) ! ) * 634/ 1 1 * * ) * 1 ! ! )) 6 ! * * ) * !/ $% & ! ) 6 ) ! !6 34 * ' ( ) * ( ) + ,-#( ( " .,#/ ( % B # * $& 8553 9(69$9 853 '&:6'6((%69&9&6: 6&%$' ,* CC /;/C D E 6&%$'. To my loving family List of papers This thesis is based on the following papers, which will be referred to in the text by their Roman numerals. I Johansson C, Kamali-Moghaddam M, Sundström L. 2004. Inte- gron integrase binds to bulged hairpin DNA. Nucleic Acids Res 32: 4033-4043. II Hansson K, Johansson C, Sundström L. 2006. Long-range ef- fects of mutations in attC correlate with the formation of an inte- gron hairpin recombination substrate. Manuscript. III Johansson C, Boukharta L, Eriksson J, Åqvist J, Sundström L. 2006. Mutagenesis and homology modelling of the Tn21 inte- gron integrase IntI1. Manuscript. IV Johansson C, Samskog J, Sundström L, Wadensten H, Björkes- ten L, Flensburg J. 2006. Differential expression analysis of Es- cherichia coli proteins using a novel software for relative quanti- tation of LC-MS/MS data. Proteomics 6: 4475-4485. Reprints were made with permission from the publishers. Contents List of papers ..................................................................................................v Introduction.....................................................................................................9 Antibiotics and resistance mechanisms....................................................10 Horizontal gene transfer ...........................................................................11 Recombination .........................................................................................12 A representative selection of proteins ......................................................13 Site-specific recombinases...................................................................14 A mix of proteins directly or indirectly involved in nucleophilic attacks on DNA/RNA......................................................................................19 DNA-binding proteins .........................................................................21 Integrons...................................................................................................22 Function...............................................................................................22 Definition.............................................................................................22 Integron integrases, IntIs .....................................................................26 Gene cassettes......................................................................................27 Recombination sites.............................................................................28 Recombination events..........................................................................32 Origin...................................................................................................33 Mechanisms of strand exchange of integron integrases in comparison with other tyrosine recombinases ........................................................35 Relations to other systems ...................................................................40 Present investigation .....................................................................................42 Aims of this thesis....................................................................................42 Results ......................................................................................................42 Paper I:.................................................................................................42 Paper II: ...............................................................................................43 Paper III ...............................................................................................44 Paper IV:..............................................................................................46 Discussion ................................................................................................46 Concluding remarks .................................................................................50 Acknowledgements.......................................................................................52 References.....................................................................................................55 Abbreviations aadA1 gene for aminoglycoside adenylyl transferase of type A1 attI integron attachment site attC cassette attachment site be base element bp base pair or base pairs CR Common region DR Direct repeat ds double-stranded FIS Factor for inversion stimulation GTTRRRY R is purine (adenine or guanine); Y is pyrimidine (thymine or cytosine) GWTMW (or GNT)W, adenine or thymine; M, adenine or cytosine; N, gua- nine, adenine, thymine or cytosine HTH Helix-turn-helix HU Histone-like heat unstable nucleoid protein ICE Integrative conjugative element IHF Integration host factor intI gene for integron integrase IntI Integron integrase IS Insertion sequence ISCR IS common region LINE Long interspersed repetitive elements MGE Mobile genetic element MRI Multiresistance integron ORF Open reading frame RC Rolling-circle SINE Short interspersed repetitive elements ss single-stranded 2rs secondary site Introduction It is an astounding thought to outline the dimensions of the dark tragedy that never happened thanks to the invention of antibiotics. Antibiotics came as a great relief from the burden of so many painful and often deadly diseases due to bacterial infections. Since the invention of antibiotic therapy in the 1930’s, resistance to almost all useful drugs has appeared in pathogenic or- ganisms (Davies, 1994). This dramatic episode of evolution and gene spread among bacteria is now a major threat to humankind and a concrete manifes- tation of the ability of microorganisms to respond and rapidly adapt to envi- ronmental changes. The rapid and increasing spread of antibiotic resistance is forcing medical care and research to find new solutions to suppress the spread of resistance. New concepts for antibiotic treatment, for example, should include optimization of the therapeutic use and reduction of the the total consumption of antibiotics. If we do not address this serious issue, there may be a worrying future scenario where there will be no antibiotics avail- able to cure bacterial infections. Bacteria gain resistance by spontaneous mutations in either their own housekeeping genes or by acquiring new ge- netic information due to horizontal gene transfer. Research on the dissemina- tion of antibiotic resistance has led to the discovery of many naturally occur- ing mobile elements, vehicles on which resistance genes are usually found. These vehicles of spread include transposons, conjugative plasmids and pathogenicity islands. In the middle of the 1980s it was revealed by analysis of sequence data that many unrelated antibiotic resistance genes in gram- negative pathogens reside in tandem clusters flanked by partially related sequences. It was gradually realized that many plasmids might have a spe- cific mechanism for capture of various genes (Cameron et al., 1986; Hall and Vockler, 1987; Ouellette et al., 1987; Sundström et al., 1987, 1988). When also an adjacent gene of a site-specific recombinase was found (Ouellette and Roy, 1987; Sundström et al., 1988) the contours of a complete gene-packaging system appeared. The term integron was invented (Stokes and Hall, 1989). It was realized early on that integrons probably have a more general role rather than specific dissemination of antibiotic resistance genes (Martinez and de la Cruz, 1988; Stokes et al., 1997; Sundström et al., 1988). The rapid spread of antibiotic resistance genes and the fact that multiresis- tance in clinical isolates correlates with the presence of integrons (Lever- stein-van Hall et al., 2003), strongly encourage research aiming for an im- proved understanding of the recombination mechanisms used by integron 9 systems. The work presented here is focused on analyzing the recognition strategy of integron-borne integrases and the unusual organization of the recombination sites needed for integron-mediated site-specific recombina- tion to occur. Antibiotics and resistance mechanisms Antibiotics are drugs capable of inhibiting bacterial growth and some of them have a lethal