University of Groningen Heat resistance of Bacillus spores Berendsen, Erwin Mathijs IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2016 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Berendsen, E. M. (2016). Heat resistance of Bacillus spores: Natural variation and genomic adaptation. Rijksuniversiteit Groningen. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-09-2021 Heat resistance of Bacillus spores Natural variation and genomic adaptation Erwin Mathijs Berendsen The research presented in this thesis was funded by TI Food and Nutrition (Wageningen, the Netherlands), a public-private partnership on pre- competitive research in food and nutrition. The research was conducted at NIZO Food Research BV (Ede, the Netherlands) and was embedded within the Molecular Genetics Group of the Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen (Groningen, the Netherlands). Science (University of Groningen), TIFN, Danone, NIZO, and TNO. Printing of this thesis was financially supported by the Graduate School of Cover Design: Sven Menschel & Erwin Berendsen Layout: Eirlys Pijpers & Erwin Berendsen Printed by: Ipskamp Drukkers B.V. ISBN (print): 978-90-367-8802-1 ISBN (electronic): 978-90-367-8801-4 Copyright © Erwin M. Berendsen, 2016. Heat resistance of Bacillus spores Natural variation and genomic adaptation Proefschrift ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen op gezag van de rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op vrijdag 3 juni 2016 om 16.15 uur door Erwin Mathijs Berendsen geboren op 5 juli 1986 te Kampen Promotor Prof. dr. O.P. Kuipers Copromotor Dr. M.H.J. Wells-Bennik Beoordelingscommissie Prof. dr. J.W. Veening Prof. dr. S. Brul Prof. dr. M. Kleerebezem Contents Chapter 1 7 General introduction Chapter 2 29 Two distinct groups within the Bacillus subtilis different spore heat resistance properties group display significantly Chapter 3 47 A mobile genetic element profoundly increases heat resistance of bacterial spores Chapter 4 81 High-level heat resistance of spores of Bacillus amyloliquefaciens and Bacillus licheniformis results from the presence of a spoVA operon in a Tn1546 transposon Chapter 5 97 Spores of Bacillus thermoamylovorans with very high heat resistances germinate poorly in rich media despite the presence of ger clusters, but efficiently upon non-nutrient Ca-DPA exposure Chapter 6 123 General discussion Addenda 143 Nederlandse samenvatting About the author List of publications Acknowledgements Chapter 1 General introduction Erwin M. Berendsen Partly based on the review published as: Wells-Bennik MHJ, Eijlander RT, den Besten HMW, Berendsen EM, Warda AK, Krawczyk AO, Nierop Groot MN, Xiao Y, Zwietering MH, Kuipers OP, Abee T (2016). Bacterial spores in food: survival, emergence, and outgrowth. Annual Review of Food Science and Technology 7:457-482. Chapter 1 Endospores Bacterial spores are widely present in nature. The resistance properties of spores allow for survival against environmental insults. A very important property of spores is their ability to withstand high temperatures. The level of resistance of spores to heating can vary between different spore forming species, but can also vary between strains. The well understood. Understanding which factors have a major impact on heat resistance mechanisms underlying strain specific variation in heat resistance of spores are not of spores can have important implications for their control in food and in applications to improve health. The focus of this thesis is on establishing variation in heat resistance that underlie this variation. of spores that exists between strains, and on the identification of genomic determinants Spore formers from the Clostridiales and Bacillales to enter sporulation as an adaptive strategy to survive conditions encountered in their orders have the extraordinary ability natural habitat, for instance in soil, aquatic environments or in the gut of insects and cell into a dormant endospore (3, 38, 50, 51, 108), a state in which it can reside for animals (23, 49, 69, 77). This complex regulatory process transforms the bacterial Bacillus sphaericus from 25 to 40 million year old Dominican amber (22), and a Bacillus undefined periods of time. The isolation of spores have been reported for a strain of sp. 2-9-3 from a salt crystal of 250 million years old (118). Although these reports were met with scepticism about potential external contamination, they illustrate the potential extent of longevity of spores. Dormant endospores are resistant to environmental stress low availability of nutrients (71, 77, 103). conditions including heat, salinity, acidity, radiation, oxygen and/or water depletion or The sporulation process is induced by high cell densities and nutrient limitation, and Bacillus subtilis strain 168, which is a gram positive model organism (38, 51). The availability of the genome sequence of B. subtilis 168 and has been extensively studies in the ability to genetically amend this strain greatly facilitated the progress to understand sporulation and spore resistance mechanisms (11, 55). The sporulation process is a and involves differentiation, intercellular signaling and programmed cell death among unique developmental pathway that is fundamentally different from binary fission, others (39). The composition of a spore Bacterial spores have various layered structures (Figure 1) that provide resistance against environmental insults and thus against several food processing conditions. The way in which different structures contribute to spore resistance has been extensively reviewed elsewhere (56, 103) and will be only briefly discussed here. First and foremost, 8 General introduction 1 Coat Coat Outer membrane Outer membrane Cortex ICnonreter xmembrane Inner membrane Core Core 200 nm Figure 1. Transmission electron microscopy (TEM) cross section of a spore of Bacillus subtilis 168 (E.M. Berendsen, unpublished results). The ultra- structure’s of the spore, the coat, cortex, inner membrane, and core are indicated with arrows. the spore core is strongly dehydrated, resulting in proteins present in the spore that are largely rotationally immobilized (107). A specific type of proteins present in spores is α/β-type small acid soluble proteins (SASPs) which protect the genetic material against DNA damage. In addition, the core contains the spore-specific chemical pyridine-2,6- cations (mostly Ca2+) (103). Surrounding the core is the inner membrane that provides carboxylic acid, also known as dipicolinic acid (DPA) which is chelated with divalent protection against chemicals (47, 102) and contains proteins required for germination. Outside the inner membrane lies the germ cell wall, which becomes the cell wall after germination, and the cortex, which consists of spore-specific peptidoglycan, characterized by the muramic-δ-lactam moiety and low peptide cross linking (90). The cortex is required for the development of full resistance toward wet heat (discussed in role in resistance is known [reviewed in (56)]. Finally, the outermost layers make up the more detail below). The cortex is surrounded by an outer membrane for which no clear spore coat, which contains various proteins that provide protection against lysozyme, For certain species, (e.g. Bacillus cereus, Bacillus thuringiensis, Bacillus anthracis, and toxic chemicals, and grazing protozoa, amongst others [extensively reviewed in (71)]. Clostridium in direct contact with the environment and is potentially involved in pathogenicity spp.), the spore coat is further surrounded by an exosporium, which is [reviewed in (48, 85)]. 9 Chapter 1 Sporulation and germination The spore properties, that are at the basis of resistance and dormancy, are largely germination process. Proteins that are required for germination are already produced determined by the sporulation process. To exit the dormant state, spores undergo a during the sporulation process. The sporulation and germination process will be discussed below. The sporulation process The formation of a bacterial endospore is the result of a complex regulatory process process involves a series of steps that ultimately lead to the formation of a dormant that has been extensively reviewed elsewhere (3, 38, 50, 51, 108). The sporulation spore. The point at which a vegetative cell of B. subtilis enters the sporulation process is determined by nutrient limitation and high cell densities. The key sporulation regulator is Spo0A, which requires to be phosphorylated and to form a dimer for initiation of sporulation (19, 59).