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Two-Component Signal Transduction in Bacillus Cereus and Closely Related Bacteria

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Two-Component Signal Transduction in Bacillus Cereus and Closely Related Bacteria Two-component signal transduction in Bacillus cereus and closely related bacteria Mark W.H.J. de Been Thesis committee Thesis supervisors Prof. Dr. T. Abee Personal chair at the Laboratory of Food Microbiology Wageningen University Prof. Dr. R.J. Siezen Professor of Bioinformatics of Microorganisms, Centre for Molecular and Biomolecular Informatics, NCMLS UMC St. Radboud, Nijmegen Thesis co-supervisor Dr. C. Francke Senior scientist, TI Food and Nutrition Centre for Molecular and Biomolecular Informatics, NCMLS UMC St. Radboud, Nijmegen Other members Prof. Dr. M. Kleerebezem, Wageningen University Prof. Dr. K.J. Hellingwerf, University of Amsterdam Prof. Dr. M.A. Huynen, UMC St. Radboud, Nijmegen Prof. Dr. M. Ehling-Schulz, University of Veterinary Medicine Vienna, Austria This research was conducted under the auspices of the Graduate School VLAG. Two-component signal transduction in Bacillus cereus and closely related bacteria Mark W.H.J. de Been 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 Monday 14 September 2009 at 1.30 PM in the Aula Mark W.H.J. de Been Two-component signal transduction in Bacillus cereus and closely related bacteria, 176 pages Thesis. Wageningen University, Wageningen, NL (2009) With references, with summaries in Dutch and English ISBN 978-90-8585-435-7 Table of Contents Abstract 7 Chapter 1 Introduction and outline of the thesis 9 Chapter 2 Comparative analysis of two-component signal 25 transduction systems of Bacillus cereus, Bacillus thuringiensis and Bacillus anthracis Chapter 3 The identification of response regulator specific 47 binding sites reveals new roles of two-component systems in Bacillus cereus and closely related low-GC Gram-positives Chapter 4 Integrating phylogenetic footprinting approaches with 69 transcriptome profiling reveals new roles for two Bacillus cereus two-component signal transduction systems Chapter 5 A novel hybrid kinase is essential for activating the 85 σB-mediated stress response of Bacillus cereus Chapter 6 Novel σB activation modules of Gram-positive bacteria 111 involve the use of complex hybrid histidine kinases Chapter 7 Summarising discussion 127 References 139 Samenvatting voor iedereen 159 Dankwoord 165 List of publications 169 Curriculum Vitae 171 VLAG graduate school activities 173 Supplementary material: Chapter 2: http://mic.sgmjournals.org Chapter 3: www.cmbi.ru.nl/RRoperators Chapters 5-6: www.cmbi.ru.nl/~mdebeen/Thesis ABSTRACT Abstract Bacillus cereus is a Gram-positive pathogen that is recognised as an important cause of food-borne disease worldwide. Within the genus Bacillus, B. cereus and its closest relatives form a homogeneous subdivision that has been termed the B. cereus group. This group includes B. anthracis, a pathogen that can cause anthrax in mammals, and B. thuringiensis, an insect pathogen that is used as an insecticide worldwide. Members of the B. cereus group can adapt to a wide range of environmental challenges. In bacteria, these challenges are generally monitored by two-component systems (TCS), which consist of a histidine kinase (HK) and a partner response regulator (RR). Upon sensing a specific environmental stimulus, the HK activates its cognate RR, which in turn controls the expression of genes that are involved in the appropriate response. This thesis describes the functional analysis of TCSs in the B. cereus group. By using in silico techniques, 50-58 HKs and 48-52 RRs were identified in eight different B. cereus group genomes. Biological functions, including the involvement in sporulation, biofilm formation and host-microbe interactions were predicted for these TCS proteins. A phylogenetic footprinting approach was developed and used to identify specific binding sites and target genes for over 50% of the B. cereus group DNA-binding RRs. These predictions allowed relating several RRs to a minimal regulon and thereby to a characteristic transcriptional response. To further support these predictions, the transcriptomes of two B. cereus TCS deletion mutants (ΔyvrHG and ΔyufLM) were analysed and compared with the transcriptome of wild-type B. cereus. This revealed that the minimal regulon predictions were correct for the two respective TCSs. Furthermore, the predicted biological roles for these TCSs, including roles in antibiotic resistance (YvrHG) and fumarate metabolism (YufLM), were supported by phenotypic tests. Besides the many “classical” HKs and RRs detected in the B. cereus group, several a- typical TCS proteins were found. These included five RRs without a DNA-binding output domain and two hybrid HKs (HK-RR fusions). Genome analyses revealed that one of the hybrid HK-encoding genes (BC1008) is located in a conserved gene cluster that also encodes the a-typical RR RsbY. In B. cereus, RsbY is known to activate the key stress-responsive sigma factor σB. As a partner HK for RsbY was still “missing”, the role of BC1008 in the σB-mediated stress response was tested. Indeed, a bc1008 deletion strain appeared incapable of inducing σB and its associated regulon upon stress conditions and appeared impaired in its heat adaptive response. In addition, truncation of the BC1008 fused RR receiver domain indicated that this domain plays a role in fine-tuning BC1008 activity. A comparative genome analysis further indicated that BC1008-type hybrid HKs control σB-like sigma factors in at least several other Gram-positive bacteria, including Geobacillus, Paenibacillus and actinobacteria. In summary, the research described in this thesis contributes to our understanding of B. cereus adaptive responses through TCSs. This knowledge may be applied for the development of novel intervention strategies for an improved control of B. cereus in food production environments. 7 8 Chapter 1 Introduction and outline of the thesis 9 CHAPTER 1 10 INTRODUCTION AND OUTLINE Bacillus cereus Bacillus cereus is a Gram-positive, facultative anaerobic bacterium. It was first described in the United Kingdom in 1887, where it was isolated from air in a cow-shed (Frankland and Frankland, 1887). It was given the name “Bacillus” (small rod) “cereus” (wax-like) because its rod-shaped vegetative cells form easily recognisable, wax-like colonies on agar plates. The first isolated B. cereus strain is now known as the type strain ATCC (American Type Culture Collection) 14579. A phase contrast microscopy image and electron micrograph of the vegetative cells of this strain are shown in Fig. 1.1A and 1.1B, respectively. Most Bacillus species, including B. cereus, are known to produce endospores, which are highly specialised metabolically dormant cell types that are resistant to extreme environmental conditions, including heat, dehydration and other physical stresses (Setlow, 2000) (Fig. 1.1A and 1.1C). Other distinguishing features of B. cereus include its motility, haemolysis, its active production of lecithinase (phospholipase) and its inability to ferment mannitol (Johnson, 1984). Fig. 1.1. Bacillus cereus ATCC 14579 vegetative cells and endospores. (A) Phase contrast microscopy image of B. cereus vegetative cells (lower left) and endospores (upper right). (B) Scanning electron micrograph of B. cereus vegetative cells. (C) Scanning electron micrograph of B. cereus endospores. The scale bars in B and C represent 1 μm. The images were kindly provided by Menno van der Voort (A and C) and Masja Nierop Groot (B). B. cereus is an important food-borne pathogen B. cereus is a human pathogen that can cause severe local or systemic infections, such as endophthalmitis and septicaemia. However, B. cereus is best known for its ability to cause food-poisoning and food spoilage. Many types of food have been associated with B. cereus food-borne disease, including meat, vegetables, puddings, milk, rice, pasta and noodles (Johnson, 1984; Kotiranta et al., 2000). B. cereus can cause two distinct types of food-borne disease: the diarrhoeal and the emetic type. Although both types are generally mild and self- containing, more serious and even lethal cases have been reported (Dierick et 11 CHAPTER 1 al., 2005; Fricker et al., 2007; Lund et al., 2000; Mahler et al., 1997). The diarrhoeal disease is often associated with protein-rich foods, such as meat, vegetables, puddings and milk products. It was first described by Steinar Hauge, who investigated several food-borne outbreaks in Norwegian hospitals between 1947-1949 (Hauge, 1950, 1955). The diarrhoeal disease is thought to be caused by vegetative cells (ingested as viable cells or spores) that produce enterotoxins in the small intestine. Typical symptoms include abdominal pains, watery diarrhoea, nausea and vomiting. The incubation time generally ranges between 8-16 hours after ingestion, while the symptoms normally last for 12-24 hours. However, longer incubation times have been observed and the symptoms can last for up to several days (Kotiranta et al., 2000). The emetic disease is often associated with starch-rich foods, such as fried and cooked rice, pasta and noodles. It was first identified in 1974 when B. cereus was linked to several outbreaks caused by eating cooked rice in the United Kingdom in the early 1970s (Mortimer and McCann, 1974). The emetic disease is caused by the B. cereus emetic toxin cereulide, which is produced in foods before ingestion. Symptoms mainly include nausea and vomiting, which occur between 30 minutes to 6 hours after ingestion and which generally last for 6-24 hours (Ehling-Schulz et al., 2004). B. cereus is recognised as an important cause of food-borne disease worldwide (Granum, 2007). Still, a general consensus about its exact importance as a food-borne pathogen is lacking. This is reflected by the outcome of several surveillance studies, which revealed large differences between the numbers of reported B. cereus outbreaks between different countries. Studies in Europe have shown that especially in The Netherlands and Norway, B. cereus is an important food-borne pathogen. In The Netherlands, between 1999 and 2000, B.
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