Live-imaging of Bacillus subtilis spore germination and outgrowth - Front matter

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Live-imaging of Bacillus subtilis spore germination and outgrowth

Pandey, R.

Publication date

2014

Document Version

Final published version

Link to publication

Citation for published version (APA):

Pandey, R. (2014). Live-imaging of Bacillus subtilis spore germination and outgrowth.

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Rachna Pandey

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Live-Imaging Of

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The research described in this thesis was carried out in the Molecular Biology & Microbial Food Safety as well as Van Leeuwenhoek Centre for Advanced

Microscopy, Section of Molecular Cytology, Swammerdam Institute for Life Sciences, University of Amsterdam, Science Park 904, 1098 XH, Amsterdam, The Netherlands. The research was funded by Erasmus Mundas Window 15 Scholar-ship programme (EMECW15), Unilever, STW & University of Amsterdam Typeset by LATEX

Design: LATEX-style adapted from the thesis of Mark P.J. van de Loo

Cover is designed by Rachna Pandey Printed by IPSKAMP

Live-Imaging Of

Bacillus subtilis Spore

Germination And Outgrowth

ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad van doctor

aan de Universiteit van Amsterdam

op gezag van de rector magnificus prof. dr. D.C. van den Boom,

ten overstaan van een door het college voor promoties ingestelde

commissie,in het openbaar te verdedigen in de Agnietenkapel op

vrijdag 17 oktober 2014, te 10:00 uur

door

Rachna Pandey

PROMOTIECOMMISSIE

PROMOTOR: Prof. Dr. S. Brul COPROMOTORES: Dr. E.M.M. Manders

Dr. A.S. Ter Beek OVERIGE LEDEN: Prof. Dr. M. Peck

Prof. Dr. C.G. de Koster Prof. Dr. W.H. Winnok de Vos Dr. R. Kemperman

Om Asato Maa Sad-Gamaya ||

Tamaso Maa Jyotir-Gamaya ||

Mrtyor-Maa Amrtam Gamaya ||

Om Shaantih Shaantih Shaantih ||

Lead me from the asat to the sat ||

Lead me from darkness to light ||

Lead me from death to immortality ||

Om peace, peace, peace ||

Contents

1 General introduction and outline of the thesis 1

1.1 The Bacillus Genus . . . 2

1.2 Sporulation cycle . . . 2

1.3 Endospore structure and its resistance . . . 3

1.4 Spore germination and outgrowth . . . 5

1.5 Bacillus subtilis spores in the Food Industry. . . 8

1.6 Heterogeneity in bacterial (spore) physiology during germination and outgrowth . . . 9

1.7 Thesis outline . . . 12

1.8 References . . . 14

2 SporeTracker: An analysis tool to measure germination and out-growth at single cell resolution 21 2.1 Abstract . . . 22 2.2 Introduction. . . 23 2.2.1 Project Window . . . 24 2.2.2 ObjectJ Results . . . 25 2.2.3 ObjectJ Tools. . . 25 2.2.4 SporeTracker . . . 25 2.2.5 Multichannel-SporeTracker . . . 27 2.3 References . . . 29

3 Live cell imaging of germination and outgrowth of individual Bacillus subtilis spores; the effect of heat stress quantitatively analyzed with the sporeTracker 31 3.1 Abstract . . . 32

x Contents

3.2 Introduction. . . 33

3.3 Materials and Methods. . . 35

3.3.1 Strain and Growth, Sporulation, and Germination Conditions 35 3.3.2 Slide Preparation . . . 35

3.3.3 Time-lapse Microscopy. . . 36

3.3.4 Analysis with SporeTracker . . . 37

3.4 Results. . . 38

3.4.1 Design and Validation of a Novel Closed air-containing Cham-ber for Live Cell Imaging of Exponentially Growing Cells under Aerobic Conditions . . . 38

3.4.2 Live Cell Imaging and Quantitative Analysis of Heteroge-neous Spore Germination and Outgrowth . . . 41

3.4.3 Effect of a Thermal Stress on Spore Germination and Out-growth. . . 42

3.5 Discussion . . . 46

3.6 References . . . 50

4 Quantitative analysis of the effect of specific tea compounds on germination and outgrowth of Bacillus subtilis spores at single cell resolution 53 4.1 Abstract . . . 54

4.2 Introduction. . . 55

4.3 Materials and methods. . . 56

4.3.1 Strain and spore preparation . . . 56

4.3.2 Tea compounds and germination conditions . . . 56

4.3.3 Microscope-slide preparation and time-lapse microscopy . . 57

4.3.4 Image analysis with SporeTracker. . . 57

4.4 Results. . . 58

4.4.1 Effect of different tea compounds on germination, outgrowth, and subsequent vegetative growth of B. subtilis spores at the population level . . . 58

4.4.2 Effect of different tea compounds on germination, outgrowth, and subsequent vegetative growth at single cell resolution . 61 4.5 Discussion . . . 65

4.6 References . . . 69

5 Quantifying the effect of sorbic acid, heat and combination of both on germination and outgrowth of Bacillus subtilis spores at single cell resolution 73 5.1 Abstract . . . 74

Contents xi

5.2 Introduction. . . 75

5.3 Materials and Methods. . . 77

5.3.1 Strain, media, and germination conditions . . . 77

5.3.2 Heat treatment . . . 77

5.3.3 Microscope-slide preparation and time-lapse microscopy . . 78

5.3.4 Data analysis with SporeTracker . . . 78

5.4 Results. . . 79

5.4.1 Quantitative analysis of germination and outgrowth efficiency 81 5.4.2 Germination heterogeneity . . . 82

5.4.3 Outgrowth heterogeneity . . . 84

5.4.4 Generation time heterogeneity . . . 87

5.5 Discussion . . . 87

5.6 References . . . 92

6 Intracellular pH response to weak acid stress in individual Bacil-lus subtilis vegetative cells: Use of IpHluorin for live-imaging 95 6.1 Abstract . . . 96

6.2 Introduction. . . 97

6.3 Materials and Methods. . . 98

6.3.1 Growth conditions . . . 98

6.3.2 Slide preparation and its settings for fluorescent time-lapse imaging . . . 99

6.3.3 Phototoxicity measurements. . . 99

6.3.4 Calibration of pHi . . . 100

6.3.5 pHimeasurements in a microcolony and in single cells within a microcolony . . . 100

6.4 Results. . . 101

6.4.1 Phototoxicity measurements. . . 102

6.4.2 Calibration of pHimeasurements in B. subtilis cells at single cell resolution . . . 102

6.4.3 Effect of sorbic and acetic acids on pHiand growth of grow-ing B. subtilis vegetative cells . . . 104

6.4.4 Discussion. . . 106

6.4.5 Acknowledgments . . . 112

6.4.6 References. . . 113

7 General discussion and future perspectives 117 7.1 Discussion . . . 118

7.2 Future perspectives. . . 120

xii Contents

Summary 125

Samenvatting 127

List of Publications 131