Differentiation of Vegetative Cells into Spores: a Kinetic Model Applied to Bacillus subtilis

The growth-sporulation model describes the progressive transition from vegetative cells to spores with sporulation parameters describing the sporulation potential of each vegetative cell. Consequently, the model constitutes an interesting tool to assess the sporulation potential of a bacterial population over time with accurate parameters such as the time needed to obtain one resistant spore and the probability of sporulation. Further, this model can be used to assess these data under various environmental conditions in order to better identify the conditions favorable for sporulation regarding the time to obtain the first spore and/or the concentrations of spores which could be reached during a food process. ABSTRACT Spore-forming bacteria are natural contaminants of food raw materials, and sporulation can occur in many environments from farm to fork. In order to characterize and to predict spore formation over time, we developed a model that describes both the kinetics of growth and the differentiation of vegetative cells into spores. The model is based on a classical growth model and enables description of the kinetics of sporulation with the addition of three parameters specific to sporulation. Two parameters are related to the probability of each vegetative cell to commit to sporulation and to form a spore, and the last one is related to the time needed to form a spore once the cell is committed to sporulation. The goodness of fit of this growth-sporulation model was assessed using growth-sporulation kinetics at various temperatures in laboratory medium or in whey for Bacillus subtilis, Bacillus cereus, and Bacillus licheniformis. The model accurately describes the kinetics in these different conditions, with a mean error lower than 0.78 log10 CFU/ml for the growth and 1.08 log10 CFU/ml for the sporulation. The biological meaning of the parameters was validated with a derivative strain of Bacillus subtilis 168 which produces green fluorescent protein at the initiation of sporulation. This model provides physiological information on the spore formation and on the temporal abilities of vegetative cells to differentiate into spores and reveals the heterogeneity of spore formation during and after growth. IMPORTANCE The growth-sporulation model describes the progressive transition from vegetative cells to spores with sporulation parameters describing the sporulation potential of each vegetative cell. Consequently, the model constitutes an interesting tool to assess the sporulation potential of a bacterial population over time with accurate parameters such as the time needed to obtain one resistant spore and the probability of sporulation. Further, this model can be used to assess these data under various environmental conditions in order to better identify the conditions favorable for sporulation regarding the time to obtain the first spore and/or the concentrations of spores which could be reached during a food process.

[1]  K. Rohr,et al.  Phenotypic memory in Bacillus subtilis links dormancy entry and exit by a spore quantity-quality tradeoff , 2018, Nature Communications.

[2]  N. Komin,et al.  How to address cellular heterogeneity by distribution biology , 2017, 2109.06691.

[3]  A. Mathot,et al.  Knowledge of the physiology of spore-forming bacteria can explain the origin of spores in the food environment. , 2017, Research in microbiology.

[4]  A. Sant’Ana.  Quantitative Microbiology in Food Processing: Modeling the Microbial Ecology , 2017 .

[5]  Sarah Guiziou,et al.  A part toolbox to tune genetic expression in Bacillus subtilis , 2016, Nucleic acids research.

[6]  Jatin Narula,et al.  Slowdown of growth controls cellular differentiation , 2016, Molecular systems biology.

[7]  M. Wiedmann,et al.  Spore populations among bulk tank raw milk and dairy powders are significantly different. , 2015, Journal of dairy science.

[8]  Jatin Narula,et al.  Chromosomal Arrangement of Phosphorelay Genes Couples Sporulation and DNA Replication , 2015, Cell.

[9]  C. Trunet,et al.  Modeling the behavior of Geobacillus stearothermophilus ATCC 12980 throughout its life cycle as vegetative cells or spores using growth boundaries. , 2015, Food microbiology.

[10]  M. Jobin,et al.  A new chemically defined medium for the growth and sporulation of Bacillus cereus strains in anaerobiosis. , 2014, Journal of microbiological methods.

[11]  M. Jobin,et al.  Absence of oxygen affects the capacity to sporulate and the spore properties of Bacillus cereus. , 2014, Food microbiology.

[12]  T. Benezech,et al.  Sporulation of Bacillus spp. within biofilms: a potential source of contamination in food processing environments. , 2014, Food microbiology.

[13]  S. Al.  Statistical tools for nonlinear regression , 2013 .

[14]  D. Dubnau,et al.  Chance and Necessity in Bacillus subtilis Development. , 2013, Microbiology spectrum.

[15]  O. Igoshin,et al.  Ultrasensitivity of the Bacillus subtilis sporulation decision , 2012, Proceedings of the National Academy of Sciences.

[16]  F. Carlin,et al.  Sporulation boundaries and spore formation kinetics of Bacillus spp. as a function of temperature, pH and a(w). , 2012, Food microbiology.

[17]  M. Hecker,et al.  Cross-talk between the general stress response and sporulation initiation in Bacillus subtilis - the σ(B) promoter of spo0E represents an AND-gate. , 2012, Environmental microbiology.

[18]  B. Ryall,et al.  Culture History and Population Heterogeneity as Determinants of Bacterial Adaptation: the Adaptomics of a Single Environmental Transition , 2012, Microbiology and Molecular Reviews.

[19]  S. Pavan,et al.  Tracking spore-forming bacteria in food: from natural biodiversity to selection by processes. , 2012, International journal of food microbiology.

[20]  Joerg M. Buescher,et al.  Global Network Reorganization During Dynamic Adaptations of Bacillus subtilis Metabolism , 2012, Science.

[21]  B. Schwikowski,et al.  Condition-Dependent Transcriptome Reveals High-Level Regulatory Architecture in Bacillus subtilis , 2012, Science.

[22]  M. Hecker,et al.  Integration of σB Activity into the Decision-Making Process of Sporulation Initiation in Bacillus subtilis , 2011, Journal of bacteriology.

[23]  R. Sen,et al.  Kinetic modeling of sporulation and product formation in stationary phase by Bacillus coagulans RK-02 vis-à-vis other Bacilli. , 2011, Bioresource technology.

[24]  M. Heyndrickx,et al.  The Importance of Endospore-Forming Bacteria Originating from Soil for Contamination of Industrial Food Processing , 2011 .

[25]  Frédéric Carlin,et al.  Origin of bacterial spores contaminating foods. , 2011, Food microbiology.

[26]  F. Carlin,et al.  The wet-heat resistance of Bacillus weihenstephanensis KBAB4 spores produced in a two-step sporulation process depends on sporulation temperature but not on previous cell history. , 2011, International journal of food microbiology.

[27]  A. Durand,et al.  Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure , 2011, Applied Microbiology and Biotechnology.

[28]  Guo-Cheng Yuan,et al.  Broadly heterogeneous activation of the master regulator for sporulation in Bacillus subtilis , 2010, Proceedings of the National Academy of Sciences.

[29]  J. Onuchic,et al.  INAUGURAL ARTICLE by a Recently Elected Academy Member:Deciding fate in adverse times: Sporulation and competence in Bacillus subtilis , 2009 .

[30]  L. Q. Teixeira,et al.  Microbial modeling of thermal resistance of Alicyclobacillus acidoterrestris CRA7152 spores in concentrated orange juice with nisin addition , 2009, Brazilian journal of microbiology : [publication of the Brazilian Society for Microbiology].

[31]  Dong-Hyun Kang,et al.  Effects of minerals on sporulation and heat resistance of Clostridium sporogenes. , 2008, International journal of food microbiology.

[32]  Jeanne-Marie Membré,et al.  Application of predictive modelling techniques in industry: from food design up to risk assessment. , 2008, International journal of food microbiology.

[33]  K. Venkatesh,et al.  Effect of Temperature on the Cannibalistic Behavior of Bacillus subtilis , 2008, Applied and Environmental Microbiology.

[34]  Rajan P Kulkarni,et al.  Tunability and Noise Dependence in Differentiation Dynamics , 2007, Science.

[35]  I. Leguerinel,et al.  Modelling the influence of the sporulation temperature upon the bacterial spore heat resistance, application to heating process calculation. , 2007, International journal of food microbiology.

[36]  Gürol M. Süel,et al.  An excitable gene regulatory circuit induces transient cellular differentiation , 2006, Nature.

[37]  Oscar P Kuipers,et al.  Phosphatases modulate the bistable sporulation gene expression pattern in Bacillus subtilis , 2005, Molecular microbiology.

[38]  Andreas Holzman,et al.  Statistical Tools for Nonlinear Regression , 2004 .

[39]  Wayne L. Nicholson,et al.  Stochastic Processes Influence Stationary-Phase Decisions in Bacillus subtilis , 2004, Journal of bacteriology.

[40]  H. D. Jong,et al.  Qualitative simulation of the initiation of sporulation in Bacillus subtilis , 2004, Bulletin of mathematical biology.

[41]  M. Méndez,et al.  Novel Roles of the Master Transcription Factors Spo0A and σB for Survival and Sporulation of Bacillus subtilis at Low Growth Temperature , 2004, Journal of bacteriology.

[42]  Louis Coroller,et al.  Development and Validation of Experimental Protocols for Use of Cardinal Models for Prediction of Microorganism Growth in Food Products , 2004, Applied and Environmental Microbiology.

[43]  Shane T. Jensen,et al.  The Spo0A regulon of Bacillus subtilis , 2003, Molecular microbiology.

[44]  H. Williams,et al.  Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression. , 2003, Journal of microbiological methods.

[45]  Hanjing Huang,et al.  A segregated model for heterologous amylase production by Bacillus subtilis , 2003 .

[46]  Thomas Ross,et al.  Modeling Microbial Growth Within Food Safety Risk Assessments , 2003, Risk analysis : an official publication of the Society for Risk Analysis.

[47]  A. Sonenshein,et al.  Control of sporulation initiation in Bacillus subtilis. , 2000, Current opinion in microbiology.

[48]  J P Flandrois,et al.  Convenient Model To Describe the Combined Effects of Temperature and pH on Microbial Growth , 1995, Applied and environmental microbiology.

[49]  J. Hoch,et al.  cis‐Unsaturated fatty acids specifically inhibit a signal‐transducing protein kinase required for initiation of sporulation in Bacillus subtilis , 1992, Molecular microbiology.

[50]  K. Franich,et al.  Single, chemically defined sporulation medium for Bacillus subtilis: growth, sporulation, and extracellular protease production , 1984, Journal of bacteriology.

[51]  Larry R. Beuchat,et al.  Food microbiology : fundamentals and frontiers , 2013 .

[52]  J. Dworkin,et al.  Recent progress in Bacillus subtilis sporulation. , 2012, FEMS microbiology reviews.

[53]  John R. King,et al.  Mathematical Modelling of the Sporulation-Initiation Network in Bacillus Subtilis Revealing the Dual Role of the Putative Quorum-Sensing Signal Molecule PhrA , 2011, Bulletin of mathematical biology.

[54]  M. Berlanga.  Food microbiology. Fundamentals and frontiers. 3rd edn. , 2007 .

[55]  T. Campbell.  The Effect of pH on Green Fluorescent Protein: a Brief Review , 2001 .

[56]  S Falkow,et al.  FACS-optimized mutants of the green fluorescent protein (GFP). , 1996, Gene.