ITEM METADATA RECORD
Title: Genomic adaptation of Cupriavidus metallidurans in response to environmental stressors
Other Titles: Genomische adaptatie van Cupriavidus metallidurans aan stressomgevingen
Authors: Vandecraen, Joachim
Issue Date: 2-Jun-2016
Abstract: Bacteria continuously evolve by increasing their genomic variability to survive different environmental challenges. Their genetic adaptation is mostly mediated by horizontal gene transfer with a smaller contribution of point mutations, duplications, insertions and recombination events. The field of evolutionary biology has long been interested in the interplay between generation of genetic diversity and subsequent natural selection favoring the best adapted organism. Epigenetic inheritance and stress-induced mutagenesis have challenged the claim of full independence between mutation and natural selection. One frequently encountered type of mutation results from the hopping of mobile genetic elements (MGEs). The dynamic nature of these elements and the host responses to these elements predict stress-induced events, which will have a significant effect on the genome plasticity and concomitant adaptability of the host cell to environmental challenges. The soil bacterium Cupriavidus metallidurans CH34, which is well-equipped to adapt to and survive harsh and anthropogenic environments, harbours a multitude of MGEs, including: 21 different Insertion Sequence (IS) elements, 11 genomic islands, 5 transposons and 2 plasmids. In this study, we aimed at addressing three challenges. First, the adaptation capabilities of C. metallidurans CH34 to toxic zinc concentrations was scrutinized by performing a laboratory evolution experiment to identify adaptive changes responsible for increased zinc resistance. The mechanisms underlying this increased resistance were identified by whole-genome sequencing and gene expression analysis and were later confirmed by complementing and reconstructing this zinc-resistant genotype. Second, a known IS trap was used to analyze the contribution of IS elements to the adaptation of a zinc-sensitive derivate of C. metallidurans CH34 to toxic zinc concentrations. The interplay between stress and generation of mutations was evaluated. Furthermore, a large quantity of obtained zinc-resistant derivates was screened to identify the involvement of IS elements and the target site specificity and stress induction of these elements were assessed. Third, C. metallidurans CH34 is not only known for its resistance to a wide range of metallic elements but also for its abnormal behaviour when exposed to a temperature shift from 30 °C to 37 °C as CH34 exhibits only temperature-induced mortality and mutagenesis (TIMM) when cultured on rich medium. This phenotype was further scrutinized by comparing different growth media and potential mitigation of this induction was assessed. Furthermore, stable inheritance of TIMM resistance was evaluated and fitness of survivors was examined by competition assays. In addition, mutations responsible for TIMM resistance were identified and validated by complementation assays.
Thus, C. metallidurans strains can readily adapt to fluctuating environmental conditions and this adaptation is often mediated by redistribution of IS elements, highlighting the impact of IS elements on the evolution of their hosts.
Table of Contents: PREFACE I
SAMENVATTING III
SUMMARY VII
LIST OF ABBREVIATIONS XI
TABLE OF CONTENTS XIII
CHAPTER 1 1
GENETIC VARIABILITY IN BACTERIA 1
1.1 Abstract 1
1.2 Introduction 2
1.3 Stress-induced genetic variability in bacteria 3
1.3.1 Global endogenous stress responses promoting genetic variability 4
1.3.2 Environmentally-induced mutagenesis 10
1.3.3 Stress-directed mutagenesis 18
1.4 Insertion Sequences as variability generators 18
1.4.1 Gene inactivation affecting virulence, resistance and metabolism 20
1.4.2 IS elements affecting the expression of neighboring genes 23
1.4.3 IS elements affecting the mobilization of neighboring genes 33
1.4.4 IS elements recognized by the cell's recombination machinery 34
1.4.5 IS elements induced by environmental stimuli 35
1.5 Conclusions 39
CHAPTER 2 41
CUPRIAVIDUS METALLIDURANS AS A MODEL ORGANISM 41
SCOPE OF THE RESEARCH 45
CHAPTER 3 47
ADAPTATION OF CUPRIAVIDUS METALLIDURANS CH34 TO TOXIC ZINC CONCENTRATIONS INVOLVES AN UNCHARACTERIZED ABC-TYPE SUGAR TRANSPORTER 47
3.1 Abstract 47
3.2 Introduction 48
3.3 Material and methods 50
3.3.1 Strains, media and culture conditions 50
3.3.2 Isolation of zinc-resistant mutants 51
3.3.3 Assessment of zinc-resistant phenotype 51
3.3.4 Construction of plasmids 53
3.3.5 Construction of deletion mutant strains 53
3.3.6 Whole-genome gene expression analysis 54
3.3.7 Genome sequencing 55
3.3.8 Fitness analysis 55
3.4 Results 56
3.4.1 Isolation and characterization of zinc-resistant CH34 derivatives 56
3.4.2 Whole-genome expression profile of CH34ZnR 57
3.4.3 Identification of mutations by whole-genome sequencing 59
3.4.4 ABC-type sugar transporter Rmet_2229-2234 is responsible for increased zinc resistance 61
3.4.5 Loss of GlpR function promotes increased zinc resistance 62
3.4.6 Preadaptation to zinc by glycerol-mediated reduced GlpR activity 64
3.5 Discussion 65
CHAPTER 4 71
ZINC-INDUCED TRANSPOSITION OF INSERTION SEQUENCE ELEMENTS CONTRIBUTES TO INCREASED ADAPTABILITY OF CUPRIAVIDUS METALLIDURANS 71
4.1 Abstract 71
4.2 Introduction 72
4.3 Material and methods 74
4.3.1 Strains, media and culture conditions 74
4.3.2 Determination of growth and minimal inhibitory concentration 76
4.3.3 Isolation of zinc-resistant mutants 77
4.3.4 Analysis of IS transposition 77
4.3.5 Whole genome gene expression microarrays 78
4.3.6 cnrH mutant deletion construction 78
4.3.7 Identification of cnrCBAT transcription start site 79
4.3.8 Construction of plasmids 80
4.3.9 Monitoring of transcription 80
4.4 Results 82
4.4.1 Isolation and phenotypic characterization of zinc-resistant AE126 derivatives 82
4.4.2 Genetic characterization of zinc-resistant AE126 derivatives 84
4.4.3 Characterization of IS-independent zinc-resistant AE126 mutants 84
4.4.4 Characterization of IS-dependent zinc-resistant AE126 mutants 86
4.4.5 Characterization of CnrH-independent zinc-resistant AE126 mutants 89
4.4.6 Endogenous promoter activity of IS elements 91
4.4.7 Analysis of upstream flanking genomic sequence on ISRme5 transposase expression 92
4.4.8 Enhanced adaptation potential to other stress challenges 94
4.5 Discussion 94
CHAPTER 5 99
PHENOTYPIC AND GENETIC CHARACTERIZATION OF TEMPERATURE-INDUCED MORTALITY AND MUTAGENESIS IN CUPRIAVIDUS METALLIDURANS 99
5.1 Abstract 99
5.2 Introduction 99
5.3 Material and methods 100
5.3.1 Strains, media and culture conditions 100
5.3.2 TIMM assay and isolation of TIMM-resistant mutants 101
5.3.3 Fitness analysis 101
5.3.4 Construction of plasmids 103
5.3.5 Genome sequencing 103
5.3.6 Whole-genome gene expression analysis 104
5.4 Results 104
5.4.1 The TIMM phenomenon is well-conserved within the C. metallidurans species 104
5.4.2 TIMM-mediated cell mortality after prolonged exposure at 37 °C 105
5.4.3 TIMM-inducing and protective medium 107
5.4.4 Characterization of stably inherited TIMM resistance 110
5.4.5 Analysis of TIMM resistant CH34 and AE104 derivatives 111
5.5 Discussion 117
CHAPTER 6 123
GENERAL DISCUSSION AND PERSPECTIVES 123
6.1 Genome plasticity and heterogeneity 123
6.2 Insertion Sequence elements 125
6.3 Novel zinc resistance mechanism in C. metallidurans 127
6.4 C. metallidurans derivatives thriving in nutrient-rich medium at elevated growth temperature 129
6.5 Conclusions 131
REFERENCES 133
PAGE OF PUBLICATIONS 163
SUPPLEMENTARY DATA 165
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Centre for Food and Microbial Technology

Files in This Item:
File Status SizeFormat
PhDThesisJoachimVandecraen.pdf Published 3033KbAdobe PDFView/Open Request a copy

These files are only available to some KU Leuven Association staff members

 




All items in Lirias are protected by copyright, with all rights reserved.