Factors and mechanisms of mobility of the high pathogenicity island of Yersinia [Elektronische Ressource] / von Uladzimir Antonenka

Uladzimir Antonenka

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• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Max von Pettenkofer-Institut für Hygiene und M edizinische M ikrobiologie
Ludwig -Maximilians-Universität
München
Direktor: Prof. Dr. Dr. J. Heesemann
Factors and Mechani sms of Mobi lity of the H igh P athogenicity
Island of Y ersinia
Dissertation
zur
Erlangung d es Doktorgrades der
Fakultät für Biologie der
Ludwig-Maximilians-Universität
München
von
Uladzimir Antonenka
aus
Gomel, Belarus
München 2007Dissertation e ingereicht am: 13. Juni 2007
Vorsitzender: Prof. D r. E lizabeth W eiss
Gutachter: Prof. D r. A nton H artmann
Gutachter: Prof. D r. H einrich Jung
Protokoll: Prof. D r. M akoto H ayashi
Sondervotum: Prof. D r. D r. Jürgen H eesemann
Tag de r mündlichen Prüfung: 17. D ezember 2007TABLE OF CONTENTS I
A. INTRODUCTION 1
1. General characteristics of Yersinia species 1
2. The concept of “Pathogenicity Island” (PAI) 2
3. PAIs, as a particular case of “Genomic Islands” (GEIs) 3
4. Evolution of genomic islands 4
5. GEIs strategies for lateral transfer 6
6. Structure and function of the HPI 7
6.1 Yersiniabactin core 8
6.2 Mobility of the Yersinia HPI 10
7. Aims of this research study 13
B. MATERIALS AND METHODS 14
1. Material 14
1.1 Equipment 14
1.2 Other materials 15
1.3 Chemicals and Enzymes 15
2. Bacteria, Plasmids and Primers 15
2.1 Bacterial strains and plasmids 15
2.2 List of primers 17
3. Culture media, Antibiotics, Strain Cultivation and Storage 19
3.1 Culture media 19
3.2 Antibiotics 21
3.3 Cultivation and long term storage of bacteria 21
4. Molecular genetic methods 22
4.1 Isolation of chromosomal DNA with Qiagen Genomic-tip 100/G 22
4.2 Isolation of plasmid DNA 22
4.2.1 Plasmid isolation with QIAprep Spin Miniprep kit (Qiagen) 22
4.2.2 Plasmid isolation with Nucleobond AX100 Kit (Machery-Nagel) 22
4.3 Purification DNA and determination of DNA concentration and purity 22
4.3.1 Phenol extraction and ethanol precipitation of DNA 22
4.3.2 Determination of DNA concentration and purity 23
4.4 Polymerase Chain Reaction 23
4.4.1 Nested PCR screening for genomic islands excision 24
4.4.2 Real Time PCR and quantification of attP -targets 25
4.5 Agarose gel electrophoresis 25TABLE OF CONTENTS II
4.6 Enzymatic modification of DNA 26
4.6.1 Restriction digestion of DNA 26
4.6.2 Dephosphorylation of DNA 27
4.6.3 Ligation of DNA molecules 27
4.7 DNA sequencing 27
4.8 RNA analysis 27
4.8.1 RNA isolation 27
4.8.2 DNase reaction 28
4.8.3 Reverse Transcription 28
4.8.4 Mapping the start of orf1 transcription 29
4.9 Bacterial transformation 29
4.9.1 Production of electrocompetent cells 29
4.9.2 Transformation Procedure 30
4.9.3 Preparation of X-gal/IPTG LB-agar plates for blue-white
screening of recombinants 30
4.10 Conjugation 30
5. Enzyme activity assays 31
5.1 Luciferase assay 31
5.2 Quantification of GFP fluorescence for GFP-reporter
studies with iron-regulated promoters 31
6. In vitro DNA-binding assays 31
6.1 Electrophoretic Mobility Shift Assay (EMSA) 31
6.2 DNase I footprinting assay 32
7. Protein biochemical studies 32
7.1 Sodium-dodecyl-sulphate Polyacrylamide Gel Electrophoresis
(SDS-PAGE) Principle 32
7.2 Cultivation and induction of bacteria 34
7.3 Purification of the 6xHis fusion protein 34
7.4 The Glutathione-S-transferase Gene Fusion System (Pharmacia Biotech) 35
7.4.1 Purification of the GST-fusion protein 35
8. Bioinformatics 35
C. RESULTS 38
1. Characterisation of the HPI integrase, as a DNA-binding trans-acting protein 38 TABLE OF CONTENTS III
1.2 Construction of recombinant integrase expression vectors 38
1.3 Integrase activity assay 39
1.4 Purification of recombinant Int 41HPI
1.5 Integrase/attP electrophoretic mobility shift assay 43
1.6 IHF/attP electrophoretic mobility shift assay 43
2. Recombination Directionality Factor of the HPI 44Yps
2.1 Bioinformatic analysis of AT-rich region of HPI, defining
of the putative excisionase of the HPI 44Yps
2.2 Promoters of orf1 and orf2 46
2.3 Construction of orf 2 mutant 47
2.4 Effect of orf2 on excision of HPI in Y. pseudotuberculosis
YPS06 and YPS06 xis strains 48
2.5 Construction of recombinant excisionase expression vector 50
2.6 Excisionase protein expression and purification 50
2.7 Excisionase-DNA binding experiments 51
3. Evolution of recombination apparatus of GEIs integrated in asn tRNA genes 55
3.1 Comparison of HPI with E. coli Ecoc54N GEI 55
3.2 Recombinase of Ecoc54N island is active and able to promote excision 56
3.3 Xis does not assist the Ecoc54N excision 58HPI
3.4 Xis did not bind to Ecoc54N attP recombination site 58HPI
4. Mechanisms of GEIs dissemination 59
4.1 Construction of the shuttle plasmid 59
4.2 Trapping of the “mini-island” 60
4.3 Horizontal transfer of the whole HPI 62
4.3.1 Introduction of resistance marker in to HPI 62
4.3.2 Conjugative transfer of the HPI 63Yps
−
4.4 Reconstruction of CAS-phenotype in Y. enterocolitica WA-TH strain 65
4.5 Cointegrate instability and HPI reintegration 66
4.6 Efficiency of cointegrate transfer in Y. enterecolitica WA-C wild strain 66
5. Factors reducing the freqency of the lateral gene transfer 67
5.1 Determination of nucleotide sequence of the new restriction-modification (RM)
system YenI. 67
5.2 Bioinformatic analysis of the yenI locus and comparison with
other known restriction-modification systems 68TABLE OF CONTENTS IV
5.3 Construction of YenI expression plasmid 68
5.4 Construction of endonuclease-deficient yenI ORF 69
5.5 Expression of yenI and yenIΔhsrYI 70
5.6 Construction of Yen I endonuclease-deficient mutant 70
5.7 Methylation activity of YenIΔhsrYI protein 72
r
5.8 Efficiency of RP4’asn ::HPI Cm cointegrate transfer in
Y. enterecolitica WA-C and WA-C hsrYI, hsmYI mutant 73
D. DISCUSSION 74
1. Key elements, involved in mobility of Y. pestis HPI 74
2. The model of the HPI dissemination 77Yps
3. Restriction-modification systems as lateral gene transfer reducing factors 80
E. SUMMARY 82
F. REFERENCES 84
G. ABBREVIATIONS 91
PUBLISHED ASPECTS OF THIS WORK 93
CURRICULUM VITAE 94A. INTRODUCTION 1

A. INRODUCTION

1. General characteristics of Yersinia species
The genus Yersinia is composed of Gram-negative coccobacilli belonging to the family of
Enterobacteriaceae. Members of the Yersinia genus are facultative non-sporulating anaerobes
with optimal growth at 27-30 °C. According to biochemical and metabolic characteristics, DNA-
DNA hybridization, and 16S rRNA sequencing results, the genus Yersinia comprises 11 different
species. The G+C content of the DNA of the genus is 46 to 50 mol% (Bercovier and Mollaret,
1984). DNA hybridization studies revealed more than 90% intra- and interspecies relatedness
between Y. pestis and Y. pseudotuberculosis and 20 to 55% between Y. pseudotuberculosis and the
other Yersinia species (Perry and Fetherston, 1997). It was found that the 16S rDNA sequence of
Y. pseudotuberculosis is identical to that of Y. pestis (Trebesius et al., 1998).
Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica are pathogens for humans and other
mammals, birds. Y. ruckeri is known as a fish pathogen (Bottone, 1997). Y. pestis, the bacterial
agent of bubonic plague, has been responsible for devastating epidemics throughout human
history. This pathogen persists among certain wild rodent populations in many parts of the world
(except Australia) and is transmitted by the bite of infected fleas. The blockage of the
proventriculae of fleas by Y. pestis forces infected fleas to bite and subsequently regurgitate the
infected blood meal into the bite site of a new host. The subsequent bacteremia in rodents
completes the rodent-flea-rodent cycle which is essential for Y. pestis spread. The ecology,
pathogenicity, and host range of Y. pseudotuberculosis and Y. enterocolitica differ fundamentally
from those of Y. pestis. Both species are transmitted perorally by contaminated food or drinking
water and subsequently invade Peyer's patches of the small bowel and multiply extracellularly. In
bacteria disseminate to mesenteric lymph nodes and occasionally via the murine infection model
the bloodstream to the spleen, liver, and lungs, causing septicemic plague-like infections.
Normally, infections with Y. enterocolitica or Y. pseudotuberculosis (yersiniosis) are self-limiting
and benign. Y. pseudotuberculosis is widely distributed in nature in aquatic and animal reservoirs
(rodents, cattle, swine, deer, and birds). Although the three pathogenic Yersinia species differ
greatly in their lifestyle, they have evolved common strategies of pathogenesis, e.g., tropism for
lymphatic tissue and extracellular multiplication. Yersiniae carry multiple sets of diverse
pathogenicity- and transmission-related genes localized on the chromosome and on plasmids
(Finlay and Falkow, 1997; Hinnebusch, 1997). There are several genes for cell adhesion (inv, ail, A. INTRODUCTION 2

myfA, psa, and yadA), invasion (invA), evasion of the host immune response (virulence plasmid
pYV, shared by all three Yersinia species), and plague pathogenesis and transmission (hms, pla,
ymt, and caf).
The high-pat