Bacillus Cereus Acid Stress Responses

Bacillus cereus acid stress responses Maarten Mols Thesis committee Thesis supervisors Prof. Dr. T. Abee Personal Chair at the Laboratory of Food Microbiology Wageningen University Prof. Dr. Ir. M. H. Zwietering Professor of Food Microbiology Wageningen University Thesis co-supervisor Dr. R. Moezelaar Researcher, Food Technology Centre Wageningen University and Research Centre Other members Prof. Dr. J. van der Oost, Wageningen University Prof. Dr. A.B. Kolstø, University of Oslo Prof. Dr. S. Brul, University of Amsterdam Dr. A.J. Else, PURAC, Gorinchem This research was conducted under the auspices of the graduate school of Voeding, Levensmiddelentechnologie, Agrobiotechnologie en Gezondheid (VLAG) Bacillus cereus acid stress responses Maarten Mols Thesis Submitted in partial fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, In the presence of the Thesis Committee appointed by the Doctorate Board to be defended in public on Wednesday 4 November 2009 at 4 PM in the Aula. Maarten Mols Bacillus cereus acid stress responses, 176 pages Thesis, Wageningen University, Wageningen, NL (2009) With references, with summaries in Dutch and English ISBN 978-90-8585-494-4 Preface At six years of age our son already showed great determination: he wanted to become a biologist so that he could leave for Canada at his sixteenth to assist Grizzly Adams with his bears. Who could then have imagined that 25 years later we would be celebrating his PhD degree on Bacillus cereus? Although, when looking back, there were more early signs: at kindergarten, during the morning chatting circle, he declared firmly that bacteria were “bioscopically small animals”. When strolling through the woods he gathered all that he could find: twigs, pebbles, leaves and so on. Back at home he left all these objects of investigation near the front door and forgot he ever laid them there, very much like a genuine distraught professor. A necessary asset for realising this dream was, however, the ability to read. Teaching him to read could rightfully be called an ambitious project as it took a lot of patience from both his teacher as well as his mom. Not only did he seem to be having difficulties in distinguishing the differences between b, d and p (little drawings had to be made), but in addition he wanted to ensure everything he read had effectively the correct meaning: was the little duck he read about also presented on the picture? When he finally got the hang of reading, his school career was a piece of cake. Favourite subjects were biology and chemistry. Subjects that were followed with a considerable lesser level of enthusiasm were foreign languages. In his view, languages had nothing to do with intelligence, but were just a simple matter of learning by heart. To finish his masters he ventured outside Utrecht University for a traineeship. His mother, who works at Wageningen UR, advised him to look at laboratories of various institutes in Wageningen. He landed a traineeship placement at RIKILT where he performed well and immediately after finishing he was offered to start with the PhD project “Microbial food preservation and safety. Identification of molecular targets for preservation stresses in Bacillus cereus”. Even during this PhD study his family members have proven to be of assistance once again and it turned out to be a huge undertaking for them too. His beloved Brenda nourished and cuddled him, his dad took him fishing to relieve his mind of the stresses and his mom patiently listened to all his frustrations. And now we are proudly attending the conclusion of his achievement, convinced our son was helped by the right surroundings and a tiny bit of help from his family and friends. Mr. and Mrs. Mart and Liesbeth Mols Contents Abstract 9 Chapter 1 General introduction and thesis outline 11 Chapter 2 Metabolic capacity of Bacillus cereus strains ATCC 14579 and ATCC 10987 interlinked with comparative genomics 39 Chapter 3 Role of ureolytic activity in Bacillus cereus nitrogen metabolism and acid survival 61 Chapter 4 Comparative analysis of transcriptional and physiological responses of Bacillus cereus to organic and inorganic acid shocks 79 Chapter 5 Comparative transcriptome and phenotype analysis of acid- stressed Bacillus cereus reveals a major oxidative response and inactivation-associated radical formation 97 Chapter 6 The impact of oxygen availability on stress survival and radical formation of Bacillus cereus 121 Chapter 7 Stress-induced superoxide formation in Bacillus cereus detected using the fluorescent probe MitoSOX 137 Chapter 8 General discussion and concluding remarks 145 Samenvatting 163 Acknowledgements 167 List of publications 169 Curriculum Vitae 171 Overview of completed training activities 173 Abstract Bacillus cereus is a ubiquitous Gram-positive organism, which frequently causes food- borne illnesses. The widespread prevalence of B. cereus makes it a common contaminant in fresh foods where it also can cause spoilage. To prevent food-borne diseases and food spoilage, foods are often processed and/or preserved. In recent years, consumers’ preferences have directed to fresher and tastier foods and this has acted as a driver for food industry to use milder processing and preservation techniques. Examples of hurdles that can be applied to preserve foods are low pH and the addition of organic acids. B. cereus may overcome these adverse conditions by displaying an adaptive stress response. The response of B. cereus upon exposure to these hurdles was investigated using two model strains, ATCC 14579 and ATCC 10987. Comparative analysis revealed numerous strain-specific genes and differences in metabolic capacities, including a urease encoding gene cluster in ATCC 10987 and a nitrate respiration cluster in ATCC 14579. A survey including ATCC 10987 and 48 environmental and outbreak-associated isolates revealed urease activity, i.e., the conversion of urea in ammonia and carbon dioxide, to be present in 10 isolates. However, the activity appeared to be too low to contribute to acid resistance in the strains tested. To search for other acid resistance mechanisms, comparative phenotype and transcriptome analyses of strains ATCC 14579 and ATCC 10987 cells exposed to organic and/or inorganic acid shocks were performed. Upon exposure to low pH with or without the addition of lactic acid or acetic acid, common acid resistance mechanisms and induction of the nitrate reductase cluster in the more acid resistant strain ATCC 14579 were revealed. Furthermore, a major oxidative response was displayed, which included the induction of several oxidative stress related genes and the production of inactivation-associated reactive oxygen species (ROS), such as hydroxyl radicals, peroxynitrite, and superoxide. ROS were detected using fluorescent probes in combination with flow cytometry, including a newly developed method using a specific probe that enables superoxide detection in Gram- positive and Gram-negative bacteria. The formation of ROS was also shown upon exposure to heat and was found to be oxygen dependant. Correspondingly, assessment of B. cereus stress survival capacity revealed increased heat- and acid-resistance with cells grown and exposed to stresses in the absence of oxygen. The excess ROS may originate from stress- induced dysfunction of the aerobic electron transfer chain, which was indicated by the induction of alternative electron transfer chain components upon exposure to organic and inorganic acid shocks. Upon exposure to stress, superoxide is generated through the premature leakage of electrons to oxygen at sites in the electron transfer chain at elevated rates. Subsequently, superoxide may promote the formation of other ROS, which can cause cellular damage leading to cell death. The induction of oxidative stress related genes has been reported in numerous other studies involving a wide range of bacteria exposed to different adverse conditions. However, a clear relation between the formation of ROS and the applied environmental stress was up to now not established. Secondary oxidative 9 responses, including the formation of ROS, are possibly common bacterial responses to severe stresses under aerobic conditions. This thesis describes genomic differences between B. cereus strains and the acid stress response of these strains on transcriptome and phenotype levels, including measurements of intracellular ROS. The findings in this study can contribute to further understanding of bacterial stress responses and secondary oxidative responses. Furthermore, the results obtained may aid to optimize and select (combinations of) stresses to apply in hurdle technology, thus enabling design of safe, milder food processing and preservation techniques. 10 Chapter 1 General introduction and thesis outline Abstract Bacillus cereus is a Gram-positive spore-forming bacterium that is ubiquitously found and can cause two types of food-borne illnesses, emesis and diarrhoea and occasionally it causes serious infections. B. cereus is also an important spoilage bacterium in for example the dairy industry. To prevent food-borne diseases and spoilage, food is often processed and/or food preservatives are added to maintain product quality and extend the shelf-life. Nowadays, food is often preserved by using a series of stresses, called hurdles, to ensure microbial safety and maintain food quality. Bacteria, such as B. cereus, may survive food processing and overcome hurdles

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