Oxidative Stress Response in Lactobacillus plantarum WCFS: A Functional Genomics Approach L. Mariela Serrano Promotor: Prof. Dr. Willem M. de Vos Hoogleraar Microbiologie Wageningen Universiteit Co-Promotor: Dr. Eddy J. Smid Projectleider NIZO Food Research Leden van de promotiecomissie: Prof. Dr. Tjakko Abee Food Microbiology Laboratory Wageningen University, The Netherlands Dr. Philippe Gaudu INRA, Unité Bactéries Lactiques et pathogènes Jouy en Josas, France Prof. Dr. Jeroen Hugenholtz NIZO Food Research Ede, The Netherlands Prof. Dr. R. Paul Ross Moorepark Food Research Centre Cork, Ireland Dit onderzoek is utigevoerd binnen de onderzoekschool VLAG. Oxidative Stress Response in Lactobacillus plantarum WCFS1: A Functional Genomics Approach L. Mariela Serrano Proefschrift Ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. Dr. M. J. Kropff in het openbaar te verdedigen op vrijdag 18 april 2008 des ochtends om elf uur in de Aula Oxidative Stress Response in Lactobacillus plantarum WCFS1: A Functional Genomics Approach L. Mariela Serrano Ph.D. thesis Wageningen University and Research Centre, The Netherlands, 2008. With summary in dutch ISBN 978-90-8504-920-3 The road not taken Two roads diverged in a yellow wood And sorry I could not travel both And be one traveller, long I stood And looked down one as far as I could To where it bent in the undergrowth; Then took the other, as just as fair, And having perhaps the better claim, Because it was grassy and wanted wear; Though as for that the passing there Had worn them really about the same, And both that morning equally lay In leaves no step had trodden black. Oh, I kept the first for another day! Yet knowing how way leads on to way, I doubted if I should ever come back. I shall be telling this with a sigh Somewhere ages and ages hence: Two roads diverged in a wood, and I- I took the one less travelled by, And that has made all the difference. Robert Frost. Para mis papis Julio y María Sin ellos no habría encontrado mi camino Table of Contents Table of Contents Abstract 9 Chapter 1 11 Introduction and Outline of this Thesis Chapter 2 31 Thioredoxin Reductase is a Key Factor in the Oxidative Stress Response of Lactobacillus plantarum WCFS1 Chapter 3 59 Global Transcriptional Analysis Reveals the Specific Role of Thioredoxin Reductase in Oxidative Stress Response in Lactobacillus plantarum WCFS1 Chapter 4 85 The Thioredoxin System Plays an Important Role in Adaptation of Lactobacillus plantarum WCFS1 to Aerobic and Respiratory Growth. Chapter 5 113 Glutathione Protects Lactobacillus plantarum WCFS1 Against Hydrogen Peroxide Stress Chapter 6 137 Summary, Concluding Remarks, and Future perspectives Appendix 149 Color figures and Supplementary materials Samenvatting 219 Resúmen, comentarios finales, y perspectivas futuras 223 Training and Supervision Plan (VLAG) 233 List of publications 234 About the author 235 Acknowledgements 237 Abstract Abstract Control of activity and functionality of microbial starter and probiotic cultures under indus- trial fermentation conditions is essential in order to provide a tasty, attractive, healthy, and safe product. Oxidative stress is one of the harsh conditions that fermentative microbes have managed to endure during their use in industrial fermentation processes. A widely- used lactic acid bacterium in food fermentations is Lactobacillus plantarum. Hence, un- derstanding oxidative stress response in this micro-organism can be used for engineering robustness towards oxidative stress. There are two systems known to be involved in oxi- dative stress response and redox homeostasis in bacteria: the thioredoxin and glutare- doxin systems. In this study, we constructed a set of L. plantarum WCFS1 strains with alterations in these systems. Using the constructed strains under different oxidative stress conditions (hydrogen peroxide, diamide (thiol-stress), aerobic growth, and respiratory growth), global transcriptome analysis was performed. Subsequently, the functional role of the thioredoxin and glutaredoxin system was analyzed and validated using a number of different techniques including comparative genomics, enzyme assays, and q-PCR. The main results obtained in this study include the role of the thioredoxin system in oxidative stress response in L. plantarum as well as in adaptation to aerobic cultivation; character- ization and overlap of the thioredoxin and glutaredoxin system in this bacterium; insight into transcriptome regulation of oxidative stress response, and unravelling of stress-re- sponse networks present in L. plantarum WCFS1. The comparative genomics and global transcriptome analysis of the oxidative stress response of L. plantarum WCFS1 presented in this thesis can be used for optimization of the performance of lactic acid bacteria in industrial fermentations. 9 1 Introduction and Outline of this Thesis 11 11 12 1 Introduction and Outline of this Thesis Lactobacillus plantarum is a versatile lactic acid bacterium encountered in a variety of niches including industrial food fermentations. The diversity of ecological niches where this bacterium survives and prospers alludes to its flexibility. In an industrial fermentation process the flexibility of L. plantarum is reflected in its capacity to counteract adverse conditions during process including acid, osmotic, temperature, and oxidative stress. Therefore, a major challenge for improving and controlling industrial fermentations is to understand the adaptation mechanisms present in this organism towards stress. A stress may lead to activation of genes required to cope with the altered condition, while repress- ing other genes that do not fulfill an important function in the novel condition. The total set of RNAs, transcriptome, under a stress condition contains information about the biological state of the cell and the genes that play a role under this specific stress condition. Hence, transcriptome analysis is a powerful tool in obtaining new information on gene-regulatory circuits and the effect of stress on the cell. This introduction will provide an overview of the characteristics and applications of L. plantarum WCFS1, the model organism used in this thesis. In addition, oxidative stress in bacteria will be discussed with special focus on the bacteria’s response upon this stress and the antioxidants glutathione and thioredoxin. Furthermore in this introduction the defi- nition and application of global transcriptional analysis will be presented to the reader. 1313 Lactobacillus plantarum zymes that catalyze the synthesis of important Lactic acid bacteria (LAB) are low C+G Gram- fermentation-end-products from pyruvate in- positive, non-sporulating, fermentative organ- cluding flavor and conservation metabolites isms that grow in anaerobic or micro-aerobic (lactate, acetate, acetoine, ethanol, formate, 2, habitats. The growth of these organisms requires 3-butanodiol). The high abundance of transport- high-nutrient containing niches. Their main func- encoding genes (411 proteins; 13.47% of total tion is to convert sugars -present in the raw ma- proteins) reflects the preference of L. plantarum terial- into lactic acid. Lactobacillus plantarum is WCFS1 to reside in nutrient-rich niches. On the a facultative heterofermentative member of the other hand, the capacity of L. plantarum WCFS1 LAB found in a large number of niches including to grow and persist in a range of habitats in- dairy, meat, vegetable fermentations as well as cluding the mammalian and other GI tracts is in the gastro-intestinal (GI) tract (3). Both with reflected by the high number of putative extra- respect to its distribution and its applications, L. cellular proteins (231; 7.47 % of total proteins) plantarum is one of the predominant species of encoded by the genome. A large proportion of the Lactobacillus genus, comprising more than genes encoding enzymes for substrate utilization 80 different species (67). It has been shown that are clustered in a 600-kb region near the origin L. plantarum survives the conditions encountered of replication. This zone was later denominated in the human GI tract (74). In addition, health- the sugar island because most of these genes improving properties have been attributed to are predicted to be involved in sugar metabo- various L. plantarum strains and some are mar- lism and may represent a life-style adaptation is- keted as probiotic (4, 14, 37, 44). land (43). The latter observation was confirmed in a DNA microarray comparison between 20 L. plantarum strains where it was shown that The annotated genome of Lactobacillus the sugar island significantly varied within the plantarum WCFS1 strains suggesting that several genes had been From a single colony isolate of strain L. plan- acquired through interactions with varying envi- tarum NCIMB 8826, originally isolated from ronments, and supports its persistence and sur- human saliva, the complete genome has been vival in diverse ecological niches (43). Moreover, sequenced and annotated (36). The circular 3.3- the genome sequence of L. plantarum WCFS1 Mb chromosome was predicted to contain 3052 reveals the presence of a high number of genes protein-encoding genes of which 70% have a predicted to encode regulatory functions (261 putative function (Table 1). The annotated ge- proteins; 8.58% of total proteins) including three nome confirmed that L. plantarum WCFS1 has sigma-factor encoding genes (rpoD, rpoN, and the capacity for uptake and utilization of many sigH); over