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Biofilm Formation Displays Intrinsic Offensive and Defensive Features of 1 Bacillus Cereus 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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Biofilm Formation Displays Intrinsic Offensive and Defensive Features of 1 Bacillus Cereus 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 bioRxiv preprint doi: https://doi.org/10.1101/676403; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Biofilm formation displays intrinsic offensive and defensive features of 2 Bacillus cereus 3 4 Caro-Astorga Joaquin1, Frenzel Elrike2, Perkins James Richard3, de Vicente Antonio1, Ranea 5 Juan A.G.4, Kuipers Oscar P.2, and Romero Diego1* 6 7 1Instituto de Hortofruticultura Subtropical y Mediterránea ‘‘La Mayora’’ –Departamento de 8 Microbiología, Universidad de Málaga, Bulevar Louis Pasteur 31 (Campus Universitario de 9 Teatinos), 29071 Málaga, Spain 10 2Department of Molecular Genetics, Groningen Biomolecular Sciences and Biotechnology 11 Institute, Centre for Synthetic Biology, University of Groningen, Nijenborgh 7, 9747 AG, 12 Groningen, The Netherlands 13 3Research Laboratory, IBIMA, Regional University Hospital of Malaga-UMA, 29009, Málaga, 14 Spain 15 4Department of Molecular Biology and Biochemistry, Universidad de Málaga, Bulevar Louis 16 Pasteur 31 (Campus Universitario de Teatinos), 29071 Málaga, Spain. CIBER de 17 Enfermedades Raras, ISCIII, Madrid. Spain 18 [email protected] 19 [email protected] 20 [email protected] 21 [email protected] 22 [email protected] 23 [email protected] 24 25 * Corresponding author: 26 [email protected] 27 Phone: +34951953056 28 Fax: +34952136645 29 30 Keywords: bacterial multicellularity, metabolism, antimicrobial, toxins, Bacillus cereus 31 biofilm 32 Running title: Offensive-defensive features of B cereus 33 1 bioRxiv preprint doi: https://doi.org/10.1101/676403; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 34 Abstract 35 Background: Biofilm formation is a strategy of many bacterial species to adapt to a 36 variety of stresses and has become a part of infections, contaminations or beneficial 37 interactions. We previously observed that B. cereus ATCC 14579 (CECT148), 38 formed a thick biomass of cells firmly adhered to abiotic surfaces. 39 Results: In this study, we combined two techniques, RNAseq and iTRAQ mass 40 spectrometry, to demonstrate the profound physiological changes that permit Bacillus 41 cereus to switch from a floating to a sessile lifestyle, to undergo further maturation of 42 the biofilm, and to differentiate into offensive or defensive populations. The 43 rearrangement of nucleotides, sugars, amino acids and energy metabolism lead to 44 changes promoting reinforcement of the cell wall, activation of ROS detoxification 45 strategies or secondary metabolite production, all oriented to defend biofilm cells 46 from external aggressions. However, floating cells maintain a fermentative metabolic 47 status along with a higher aggressiveness against hosts, evidenced by the 48 production of toxins and other virulent factors. 49 Conclusions: We show that biofilm-associated cells seem to direct the energy to the 50 individual and global defense against external aggressions and competitors. By 51 contrary, floating cells are more aggressive against hosts. The maintenance of the 52 two distinct subpopulations is an effective strategy to face changeable environmental 53 conditions found in the life cycle of B. cereus. 54 55 56 57 58 59 60 61 62 63 2 bioRxiv preprint doi: https://doi.org/10.1101/676403; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 64 65 Background 66 Bacillus cereus is a widespread bacterium that can colonize a multitude of niches, 67 including soil and seawater, where it survives living as a saprophyte or in transit from 68 other ecological niches. This bacterium can also be found in association with plant 69 tissues, living as a commensal or in symbiosis as a rhizosphere inhabitant [1]. 70 Mammalian and arthropod guts are also a niche for B. cereus, where it can live as a 71 commensal or a pathogen, either opportunistic or not [2, 3]. The versatility shown by 72 these bacteria evidence the resilience ability against a wide range of environmental 73 conditions [4]. B. cereus gives its name to the B. cereus sensu lato group, which 74 includes the phylogenetically similar bacterial species B. thuringiensis and B. 75 anthracis, diverse in their host impact as they affect insects and human health [5]. 76 Some strains of B. cereus provide benefits to plants, as a promoter of growth (PGP) 77 or biocontrol against microbial diseases, while others are proposed as probiotics for 78 cattle and even for humans [6, 7]. By contrast, there are strains responsible for 79 human pathologies mainly caused by food poisoning, contamination of products in 80 the food industry or even food spoilage [8–10]. Biofouling, clogging and corrosive 81 consequences of B. cereus in industrial devices complete the concerns of humans 82 regarding this bacteria species [10]. 83 Regardless of the consequences, most of the scenarios listed above are believed to 84 be related with the organization of bacterial cells in biofilms. The formation of biofilms 85 is considered an important step in the life cycle of most bacterial species, and it is 86 known to be related to outbreaks of diseases, resistance to antimicrobials, or 87 contamination of medical and industrial devices [11]. Approximately 65% of bacterial 88 human diseases are estimated to involve bacterial biofilms, thus these multicellular 89 structures might be considered potential targets to fight against bacterial diseases 90 [12]. Based on the relevance of bacterial biofilms, our research focuses on 91 elucidating the intrinsic factors employed by B. cereus to switch to this sedentary 92 lifestyle. In general, it is known that after encountering an adequate surface, motile 93 bacterial cells switch from a floating or planktonic to a sessile lifestyle followed by the 94 assembly of an extracellular matrix. Studies on biofilm formation in the Gram-positive 95 bacterium Bacillus subtilis have substantially contributed to our understanding of the 96 intricate machinery devoted to efficiently complete this transition [13]. While studies 97 on biofilm formation on specific B. cereus strains indicate that key processes 3 bioRxiv preprint doi: https://doi.org/10.1101/676403; this version posted June 21, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 98 resemble B. subtilis biofilm development, clear differences start to be perceived, 99 representative of the evolutionary distance between the two species [14]: i) the minor 100 role of the exopolysaccharide of B. cereus homologous to the epsA-O of B. subtilis in 101 biofilm formation [15]; ii) the absence of homologues to the accessory protein TapA, 102 necessary for amyloid-like fiber assembly in B. subtilis, iii) the existence of two 103 paralogs of subtilis TasA, i.e., TasA and CalY [16]; iv) the absence of the 104 hydrophobic BlsA protein, which coats the biofilm in B. subtilis and play a role in the 105 biofilm architecture [17]; v) the differences in the regulatory networks of biofilm 106 formation, lacking the regulatory subnetworks II and III that involve SlrA-SlrR-SinR 107 and Abh; and the gain of the pleiotropic regulator PlcR involved in virulence and 108 biofilm formation [14, 18]; vii) the absence in B. cereus of the lipoprotein Med 109 associated with KinD phosphorylation activity that triggers biofilm formation; and viii) 110 the different adhesive properties of the spores of B. cereus [19]. Therefore, novel in- 111 depth studies are required not only to confirm similarities between the two microbes 112 but also to highlight unexplored differences. 113 In a previous work, we reported that the growth of B. cereus ATCC 14579 114 (CECT148) biomass of cells adhered to abiotic surfaces is a process that clearly 115 increases with time [16]. A genomic region containing the two paralogous proteins 116 TasA and CalY, the signal peptidase SipW and the locus BC_1280 were proven 117 essential in the transition from planktonic or floating to sedentary and further growth 118 of the biofilm. The differences found in B. subtilis in this and other reports led us to 119 investigate which are the additional intrinsic genetic features that warrant B. cereus to 120 solve hypothetical environmental situations by the assembly of biofilms. The 121 combination of two techniques, RNA sequencing (RNAseq) and mass spectrometry 122 proteomic (isobaric tags for relative and absolute quantitation – iTRAQ), enabled us 123 to acquire solid evidence of the global changes differentiating floating from biofilm 124 programmed cells and depict how biofilm of B. cereus progresses. We report the 125 reinforcement of the cell wall of biofilm cells, that would prepare cells for further 126 assembly of macromolecules as polysaccharides and other adhesins, and additional 127 protection of cells individually from external aggressions; and the major production of 128 secondary metabolites of biofilm-associated cells to defend against competitors. 129 Additionally, floating cells are maintained in a sustained stationary phase of growth 130 conducive to survival, not in the form of spores, and more aggressive against the 131 human host. Our findings argue in favor of
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