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Mechanisms and DNA Specificity in Site-specific Recombination of Integron Cassettes
Uppsala University, Medicinska vetenskapsområdet, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Bacterial resistance to antibiotics has become a serious problem. This is due to the remarkable ability of bacteria to respond and rapidly adapt to environmental changes. Integrons are elements with the capacity for gene capture by an integron-encoded site-specific recombinase called IntI. IntI binds and acts at the recombination sites, attI and attC resulting in excision and integration of short DNA elements called gene cassettes carrying an attC site in the 3’ end. Several families of antibiotic resistance genes are borne on gene cassettes in integrons connected to mobile elements. Other cassettes reside in the larger and ancestral superintegrons located on chromosomes in both pathogenic and environmental bacteria. Due to their close connection with lateral gene transfer systems, it is possible that integrons are functionally dependent on those networks. This work presents arguments for such connections. The attC of the aadA1-qacE cassette junction in Tn21 was characterized in detail. Like other attC sites, it contains two pairs of inverted repeats and is almost palindromic. By using electrophoretic mobility shift assays, this study showed that IntI1 binds only to the bottom strand of attC. Upon folding the strand into a hairpin, a few chiral hairpin distortions define both the strand choice and also the appropriate orientation of the highly symmetrical site. Structural recognition also explains the wide sequence variation among attC sites. We have documented the initial cleavage step in recombination in IntI extracts and integrase levels in extracts were evaluated by a new method. Mutagenesis and homology modelling were performed to find amino acid residues in IntI1 that are important for recognition of attC hairpin-DNA. Comparisons were made with other tyrosine family members to explain how integron integrases differ in site-recognition and also in their mechanism of strand exchange.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2007. , p. 65
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 214
Keywords [en]
Microbiology, lateral gene transfer, site-specific recombination, tyrosine recombinase, integron, single-stranded, DNA hairpin
Keywords [sv]
Mikrobiologi
Research subject
Pharmaceutical Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-7429ISBN: 978-91-554-6767-8 (print)OAI: oai:DiVA.org:uu-7429DiVA, id: diva2:169478
Public defence
2007-01-24, C10:301, BMC, Husargatan 3, Uppsala, 09:15
Opponent
Supervisors
Available from: 2007-01-02 Created: 2007-01-02Bibliographically approved
List of papers
1. Integron integrase binds to bulged hairpin DNA
Open this publication in new window or tab >>Integron integrase binds to bulged hairpin DNA
2004 In: Nucleic Acids Res, ISSN 0305-1048, Vol. 32, no 13, p. 4033-4043Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-95307 (URN)
Available from: 2007-01-02 Created: 2007-01-02Bibliographically approved
2. Long-range effects of mutations in attC correlate with the formation of an integron hairpin recombination substrate
Open this publication in new window or tab >>Long-range effects of mutations in attC correlate with the formation of an integron hairpin recombination substrate
Manuscript (Other academic)
Identifiers
urn:nbn:se:uu:diva-95308 (URN)
Available from: 2007-01-02 Created: 2007-01-02 Last updated: 2010-01-13Bibliographically approved
3. Mutagenesis and homology modelling of the Tn21 integron integrase IntI1
Open this publication in new window or tab >>Mutagenesis and homology modelling of the Tn21 integron integrase IntI1
Show others...
2009 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 8, p. 1743-1753Article in journal (Refereed) Published
Abstract [en]

Horizontal DNA transfer between bacteria is widespread and a major cause of antibiotic resistance. For logistic reasons, single or combined genes are shuttled between vectors such as plasmids and   bacterial chromosomes. Special elements termed integrons operate in such shuttling and are therefore vital for horizontal gene transfer. Shorter elements carrying genes, cassettes, are integrated in the integrons, or excised from them, by virtue of a recombination site, attC, positioned in the 3' end of each unit. It is a remarkable and   possibly restricting elementary feature of attC that it must be single-stranded while the partner target site, attI, may be double-stranded. The integron integrases belong to the tyrosine recombinase family, and this work reports mutations of the integrase IntI1 from transposon Tn21, chosen within a well-conserved region characteristic of the integron integrases. The mutated proteins were  tested for binding to a bottom strand of an attC substrate, by using an electrophoresis mobility shift assay. To aid in interpreting the   results, a homology model was constructed on the basis of the crystal structure of integron integrase VchIntIA from Vibrio cholerae bound to its cognate attC substrate VCRbs. The local stability and hydrogen bonding network of key domains of the modeled structure were further examined using molecular dynamics simulations. The homology model allowed us to interpret the roles of several amino acid residues, four of which were clearly binding assay responsive upon mutagenesis. Notably, we also observed features indicating that IntI1 may be more prone to base-specific contacts with VCRbs than VchIntIA.

National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-95309 (URN)10.1021/bi8020235 (DOI)000263697300009 ()
Available from: 2007-01-02 Created: 2007-01-02 Last updated: 2022-01-28Bibliographically approved
4. Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data
Open this publication in new window or tab >>Differential expression analysis of Escherichia coli proteins using a novel software for relative quantitation of LC-MS/MS data
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2006 (English)In: Proteomics, ISSN 1615-9853, E-ISSN 1615-9861, Vol. 6, no 16, p. 4475-4485Article in journal (Refereed) Published
Abstract [en]

The study of changes in protein levels between samples derived from cells representing different biological conditions is a key to the understanding of cellular function. There are two main methods available that allow both for global scanning for significantly varying proteins and targeted profiling of proteins of interest. One method is based on 2-D gel electrophoresis and image analysis of labelled proteins. The other method is based on LC-MS/MS analysis of either unlabelled peptides or peptides derived from isotopically labelled proteins or peptides. In this study, the non-labelling approach was used involving a new software, DeCydei (TM) S Differential Analysis Software (DeCyder MS) intended for automated detection and relative quantitation of unlabelled peptides in LC-MS/MS data. Total protein extracts of E. coli strains expressing varying levels of dihydrofolate reductase and integron integrase were digested with trypsin and analyzed using a nanoscale liquid chromatography system, Ettan (TM) MDLC, online connected to an LTQ (TM) linear ion-trap mass spectrometer fitted with a nanospray interface. Acquired NIS data were subjected to DeCyder NIS analysis where 2-D representations of the peptide patterns from individual LC-MS/MS analyses were matched and compared. This approach to unlabelled quantitative analysis of the E. coli proteome resulted in relative protein abundances that were in good agreement with results obtained from traditional methods for measuring protein levels.

Keywords
DeCycler MS, DHFR, integrase, label-free quantitation, LC-MS/MS
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-95310 (URN)10.1002/pmic.200500921 (DOI)000240036100005 ()16858737 (PubMedID)
Available from: 2007-01-02 Created: 2007-01-02 Last updated: 2017-12-14Bibliographically approved

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