Pan-Genome Portrait of Bacillus mycoides Provides Insights into the Species Ecology and Evolution

Bacillus mycoides is poorly known despite its frequent occurrence in a wide variety of environments. To provide direct insight into its ecology and evolutionary history, a comparative investigation of the species pan-genome and the functional gene categorization of 35 isolates obtained from soil samples from northeastern Poland was performed. The pan-genome of these isolates is composed of 20,175 genes and is characterized by a strong predominance of adaptive genes (∼83%), a significant amount of plasmid genes (∼37%), and a great contribution of prophages and insertion sequences. The pan-genome structure and phylodynamic studies had suggested a wide genomic diversity among the isolates, but no correlation between lineages and the bacillus origin was found. Nevertheless, the two B. mycoides populations, one from Białowieża National Park, the last European natural primeval forest with soil classified as organic, and the second from mineral soil samples taken in a farm in Jasienówka, a place with strong anthropogenic pressure, differ significantly in the frequency of genes encoding proteins enabling bacillus adaptation to specific stress conditions and production of a set of compounds, thus facilitating their colonization of various ecological niches. Furthermore, differences in the prevalence of essential stress sigma factors might be an important trail of this process. Due to these numerous adaptive genes, B. mycoides is able to quickly adapt to changing environmental conditions.

Izabela Święcicka: [email protected]

Krzysztof Fiedoruk - Department of Microbiology, Medical University of Bialystok, Bialystok, Poland

Justyna M. Drewnowska - Department of Microbiology, Faculty of Biology, University of Bialystok, Bialystok, Poland

Jacques Mahillon - Laboratory of Food and Environmental Microbiology, Earth and Life Institute, Université Catholique de Louvain, Louvain-la-Neuve, Belgium

Monika Zambrzycka - Department of Microbiology, Faculty of Biology, University of Bialystok, Bialystok, Poland

Izabela Święcicka - Department of Microbiology, Faculty of Biology, University of Bialystok, Bialystok, Poland; Laboratory of Applied Microbiology, Faculty of Biology, University of Bialystok, Bialystok, Poland

Swiecicka I, De Vos P. 2003. Properties of Bacillus thuringiensis isolated from bank voles. J Appl Microbiol 94:60–64. https://doi.org/10.1046/j.1365-2672.2003.01790.x.

Ceuppens S, Boon N, Uyttendaele M. 2013. Diversity of Bacillus cereus group strains is reflected in their broad range of pathogenicity and diverse ecological lifestyles. FEMS Microbiol Ecol 84:433–450. https://doi.org/10.1111/1574-6941.12110.

Vidic J, Chaix C, Manzano M, Heyndrickx M. 2020. Food sensing: detection of Bacillus cereus spores in dairy products. Biosensors 10:15. https://doi.org/10.3390/bios10030015.

Swiecicka I. 2008. Natural occurrence of Bacillus thuringiensis and Bacillus cereus in eukaryotic organisms: a case for symbiosis. Biocontrol Sci Technol 18:221–239. https://doi.org/10.1080/09583150801942334.

Mock M, Fouet A. 2001. Anthrax. Annu Rev Microbiol 55:647–671. https://doi.org/10.1146/annurev.micro.55.1.647.

Stenfors Arnesen LP, Fagerlund A, Granum PE. 2008. From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev 32:579–606. https://doi.org/10.1111/j.1574-6976.2008.00112.x.

Drewnowska JM, Stefanska N, Czerniecka M, Zambrowski G, Swiecicka I.2020. Potential enterotoxicity of phylogenetically diverse Bacillus cereus sensu lato soil isolates from different geographical locations. Appl Environ Microbiol 86:e03032-19. https://doi.org/10.1128/AEM.03032-19.

Swiecicka I, Bideshi DK, Federici BA. 2008. Novel isolate of Bacillus thuringiensis subsp. thuringiensis that produces a quasicuboidal crystal of Cry1Ab21 toxic to larvae of Trichoplusia ni. Appl Environ Microbiol 74:923–930. https://doi.org/10.1128/AEM.01955-07.

Jiménez G, Blanch AR, Tamames J, Rosselló-Mora R. 2013. Complete genome sequence of Bacillus toyonensis BCT-7112T, the active ingredient of the feed additive preparation Toyocerin. Genome Announc 1:e01080-13. https://doi.org/10.1128/genomeA.01080-13.

Liu Y, Lai Q, Shao Z. 2018. Genome analysis-based reclassification of Bacillus weihenstephanensis as a later heterotypic synonym of Bacillus mycoides. Int J Syst Evol Microbiol 68:106–112. https://doi.org/10.1099/ijsem.0.002466.

Lechner S, Mayr R, Francis K, Prüss BM, Kaplan T, Wiessner-Gunkel E, Stewart GS, Scherer S. 1998. Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group. Int J Syst Bacteriol 48:1373–1382. https://doi.org/10.1099/00207713-48-4-1373.

Liu Y, Lai Q, Göker M, Meier-Kolthoff JP, Wang M, Sun Y, Wang L, Shao Z. 2015. Genomic insights into the taxonomic status of the Bacillus cereus group. Sci Rep 5:14082. https://doi.org/10.1038/srep14082.

Drewnowska JM, Swiecicka I. 2013. Eco-genetic structure of Bacillus cereus sensu lato populations from different environments in Northeastern Poland. PLoS One 8:e80175. https://doi.org/10.1371/journal.pone.0080175.

Soufiane B, Côté J-C. 2013. Bacillus weihenstephanensis characteristics are present in Bacillus cereus and Bacillus mycoides strains. FEMS Microbiol Lett 341:127–137. https://doi.org/10.1111/1574-6968.12106.

Guinebretière M-H, Thompson FL, Sorokin A, Normand P, Dawyndt P, Ehling-Schulz M, Svensson B, Sanchis V, Nguyen-The C, Heyndrickx M, De Vos P. 2008. Ecological diversification in the Bacillus cereus group. Environ Microbiol 10:851–865. https://doi.org/10.1111/j.1462-2920.2007.01495.x.

Swiecicka I, Bartoszewicz M, Kasulyte-Creasey D, Drewnowska JM, Murawska E, Yernazarova A, Lukaszuk E, Mahillon J. 2013. Diversity of thermal ecotypes and potential pathotypes of Bacillus thuringiensis soil isolates. FEMS Microbiol Ecol 85:262–272. https://doi.org/10.1111/1574-6941.12116.

Fiedoruk K, Drewnowska JM, Daniluk T, Leszczynska K, Iwaniuk P, Swiecicka I. 2017. Ribosomal background of the Bacillus cereus group thermotypes. Sci Rep 7:46430. https://doi.org/10.1038/srep46430.

Drewnowska JM, Fiodor A, Barboza-Corona JE, Swiecicka I. 2020. Chitinolytic activity of phylogenetically diverse Bacillus cereus sensu lato from natural environments. Syst Appl Microbiol 43:126075. https://doi.org/10.1016/j.syapm.2020.126075.

Drewnowska JM, Zambrzycka M, Kalska-Szostko B, Fiedoruk K, Swiecicka I. 2015. Melanin-like pigment synthesis by soil Bacillus weihenstephanensis isolates from Northeastern Poland. PLoS One 10:e0125428. https://doi.org/10.1371/journal.pone.0125428.

Makart L, Commans F, Gillis A, Mahillon J. 2017. Horizontal transfer of chromosomal markers mediated by the large conjugative plasmid pXO16 from Bacillus thuringiensis serovar israelensis. Plasmid 91:76–81. https://doi.org/10.1016/j.plasmid.2017.04.001.

Hu X, Huang D, Ogalo J, Geng P, Yuan Z, Xiong H, Wan X, Sun J. 2020. Application of Bacillus thuringiensis strains with conjugal and mobilizing capability drives gene transmissibility within Bacillus cereus group populations in confined habitats. BMC Microbiol 20:363. https://doi.org/10.1186/s12866-020-02047-4.

Swiecicka I, Mahillon J. 2005. The clonal structure of Bacillus thuringiensis isolates from north-east Poland does not correlate with their cry gene diversity. Environ Microbiol 7:34–39. https://doi.org/10.1111/j.1462-2920.2004.00662.x

Van der Auwera G, Mahillon J. 2008. Transcriptional analysis of the conjugative plasmid pAW63 from Bacillus thuringiensis. Plasmid 60:190–199. https://doi.org/10.1016/j.plasmid.2008.07.003.

Fiedoruk K, Daniluk T, Mahillon J, Leszczynska K, Swiecicka I. 2017. Genetic environment of cry1 genes indicates their common origin. Genome Biol Evol 9:2265–2275. https://doi.org/10.1093/gbe/evx165.

Fayad N, Kallassy Awad M, Mahillon J. 2019. Diversity of Bacillus cereus sensu lato mobilome. BMC Genomics 20:436. https://doi.org/10.1186/s12864-019-5764-4.

Gillis A, Fayad N, Makart L, Bolotin A, Sorokin A, Kallassy M, Mahillon J. 2018. Role of plasmid plasticity and mobile genetic elements in the entomopathogen Bacillus thuringiensis serovar isrealensis. FEMS Microbiol Rev 42:829–856. https://doi.org/10.1093/femsre/fuy034.

Medini D, Donati C, Tettelin H, Masignani V, Rappuoli R. 2005. The microbial pan-genome. Curr Opin Genet Dev 15:589–594. https://doi.org/10.1016/j.gde.2005.09.006.

Yu J, Zhao J, Song Y, Zhang J, Yu Z, Zhang H, Sun Z. 2018. Comparative genomics of the herbivore gut symbiont Lactobacillus reuteri reveals genetic diversity and lifestyle adaptation. Front Microbiol 9:1151. https://doi.org/10.3389/fmicb.2018.01151.

Azarian T, Huang I-T, Hanage WP. 2020. Structure and dynamics of bacterial populations: pangenome ecology, p 115–128. In Tettelin H, Medini D (ed), The pangenome: diversity, dynamics and evolution of genomes. Springer International Publishing, Cham, Switzerland.

Laing CR, Whiteside MD, Gannon VPJ. 2017. Pan-genome analyses of the species Salmonella enterica, and identification of genomic markers predictive for species, subspecies, and serovar. Front Microbiol 8:1345. https://doi.org/10.3389/fmicb.2017.01345.

Zhang X, Liu Z, Wei G, Yang F, Liu X. 2018. In silico genome-wide analysis reveals the potential links between core genome of Acidithiobacillus thiooxidans and its autotrophic lifestyle. Front Microbiol 9:1255. https://doi.org/10.3389/fmicb.2018.01255.

Romaniuk K, Golec P, Dziewit L. 2018. Insight into the diversity and possible role of plasmids in the adaptation of psychrotolerant and metalotolerant Arthrobacter spp. to extreme Antarctic environments. Front Microbiol 9:3144. https://doi.org/10.3389/fmicb.2018.03144.

Koskella B, Vos M. 2015. Adaptation in natural microbial populations. Annu Rev Ecol Evol Syst 46:503–522. https://doi.org/10.1146/annurev-ecolsys-112414-054458.

Inglin RC, Meile L, Stevens MJA. 2018. Clustering of pan- and core-genome of Lactobacillus provides novel evolutionary insights for differentiation. BMC Genomics 19:284. https://doi.org/10.1186/s12864-018-4601-5.

Bazinet AL. 2017. Pan-genome and phylogeny of Bacillus cereus sensu lato. BMC Evol Biol 17:176. https://doi.org/10.1186/s12862-017-1020-1.

Nourdin-Galindo G, Sánchez P, Molina CF, Espinoza-Rojas DA, Oliver C, Ruiz P, Vargas-Chacoff L, Cárcamo JG, Figueroa JE, Mancilla M, MaracajaCoutinho V, Yañez AJ. 2017. Comparative pan-genome analysis of Piscirickettsia salmonis reveals genomic divergences within genogroups. Front Cell Infect Microbiol 7:459. https://doi.org/10.3389/fcimb.2017.00459.

Collingro A, Tischler P, Weinmaier T, Penz T, Heinz E, Brunham RC, Read TD, Bavoil PM, Sachse K, Kahane S, Friedman MG, Rattei T, Myers GSA, Horn M. 2011. Unity in variety–the pan-genome of the Chlamydiae. Mol Biol Evol 28:3253–3270. https://doi.org/10.1093/molbev/msr161.

Kim Y, Koh I, Young Lim M, Chung W-H, Rho M. 2017. Pan-genome analysis of Bacillus for microbiome profiling. Sci Rep 7:10984. https://doi.org/10.1038/s41598-017-11385-9.

Hoton FM, Andrup L, Swiecicka I, Mahillon J. 2005. The cereulide genetic determinants of emetic Bacillus cereus are plasmid-borne. Microbiology (Reading) 151:2121–2124. https://doi.org/10.1099/mic.0.28069-0.

Murawska E, Fiedoruk K, Swiecicka I. 2014. Modular genetic architecture of the toxigenic plasmid pIS56-63 harboring cry1Ab21 in Bacillus thuringiensis subsp. thuringiensis strain IS5056. Pol J Microbiol 63:147–156. https://doi.org/10.33073/pjm-2014-020.

Mahillon J, Chandler M. 1998. Insertion sequences. Microbiol Mol Biol Rev 62:725–774. https://doi.org/10.1128/MMBR.62.3.725-774.1998.

Pinto D, da Fonseca RR. 2020. Evolution of the extracytoplasmic function s factor protein family. NAR Genom Bioinform 2:lqz026. https://doi.org/10.1093/nargab/lqz026.

Schmidt TR, Scott EJ, Dyer DW. 2011. Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group. BMC Genomics 12:430. https://doi.org/10.1186/1471-2164-12-430.

Van Schaik W, Tempelaars MH, Wouters JA, de Vos WM, Abee T. 2004. The alternative sigma factor sB of Bacillus cereus: response to stress and role in heat adaptation. J Bacteriol 186:316–325. https://doi.org/10.1128/JB.186.2.316-325.2004.

Fayad N, Kambris Z, El Chamy L, Mahillon J, Kallassy Awad M. 2021. A novel antidipterian Bacillus thuringiensis strain: unusual Cry toxin genes in a high dynamic plasmid environment. Appl Environ Microbiol 87:e02294-20. https://doi.org/10.1128/AEM.02294-20.

Carroll LM, Wiedmann M, Kovac J. 2020. Proposal of a taxonomic nomenclature for the Bacillus cereus group which reconciles genomic definitions of bacterial species with clinical and industrial phenotypes. mBio 11: e00034-20. https://doi.org/10.1128/mBio.00034-20.

Yoshida K-i, Yamaguchi M, Morinaga T, Kinehara M, Ikeuchi M, Ashida H, Fujita Y. 2008. myo-Inositol catabolism in Bacillus subtilis. J Biol Chem 283:10415–10424. https://doi.org/10.1074/jbc.M708043200.

Gonzalez-Uarquin F, Rodehutscord M, Huber K. 2020. myo-Inositol: its metabolism and potential implication for poultry nutrition – a review. Poult Sci 99:893–905. https://doi.org/10.1016/j.psj.2019.10.014.

Xin B, Zheng J, Xu Z, Song X, Ruan L, Peng D, Sun M. 2015. The Bacillus cereus group is an excellent reservoir of novel lanthipeptides. Appl Environ Microbiol 81:1765–1774. https://doi.org/10.1128/AEM.03758-14.

Wu J, Samara NL, Kuraoka I, Yang W. 2019. Evolution of inosine-specific endonuclease V from bacterial DNase to eukaryotic RNase. Mol Cell 76:44–56. https://doi.org/10.1016/j.molcel.2019.06.046.

Caulier S, Nannan C, Gillis A, Licciardi F, Bragard C, Mahillon J. 2019. Overview of the antimicrobial compounds produced by members of the Bacillus subtilis group. Front Microbiol 10:302. https://doi.org/10.3389/fmicb.2019.00302.

de la Fuente-Salcido N, Guadalupe Alanís-Guzmán M, Bideshi DK, SalcedoHernández R, Bautista-Justo M, Barboza-Corona JE. 2008. Enhanced synthesis and antimicrobial activities of bacteriocins produced by Mexican strains of Bacillus thuringiensis. Arch Microbiol 190:633–640. https://doi.org/10.1007/s00203-008-0414-2.

Abriouel H, Franz CMAP, Omar NB, Gálvez A. 2011. Diversity and applications of Bacillus bacteriocins. FEMS Microbiol Rev 35:201–232. https://doi.org/10.1111/j.1574-6976.2010.00244.x.

Raymond B, Wyres KL, Sheppard SK, Ellis RJ, Bonsall MB. 2010. Environmental factors determining the epidemiology and population genetic structure of the Bacillus cereus group in the field. PLoS Pathog 6: e1000905. https://doi.org/10.1371/journal.ppat.1000905.

Bolger AM, Lohse M, Usadel B. 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30:2114–2120. https://doi.org/10.1093/bioinformatics/btu170.

Page AJ, Cummins CA, Hunt M, Wong VK, Reuter S, Holden MTG, Fookes M, Falush D, Keane JA, Parkhill J. 2015. Roary: rapid large-scale prokaryote pan genome analysis. Bioinformatics 31:3691–3693. https://doi.org/10.1093/bioinformatics/btv421.

Kaas RS, Friis C, Ussery DW, Aarestrup FM. 2012. Estimating variation within the genes and inferring the phylogeny of 186 sequenced diverse Escherichia coli genomes. BMC Genomics 13:577. https://doi.org/10.1186/1471-2164-13-577.

Minkin I, Patel A, Kolmogorov M, Vyahhi N, Pham S. 2013. Sibelia: a scalable and comprehensive synteny block generation tool for closely related microbial genomes. p 215–229. In Darling A, Stoye J (ed), Lecture Notes in Computer Science vol 8126. Springer, Berlin, Germany.

Jesus TF, Ribeiro-Gonçalves B, Silva DN, Bortolaia V, Ramirez M, Carriço JA. 2019. Plasmid ATLAS: plasmid visual analytics and identification in highthroughput sequencing data. Nucleic Acids Res 47:D188–D194. https://doi.org/10.1093/nar/gky1073.

Arndt D, Grant JR, Marcu A, Sajed T, Pon A, Liang Y, Wishart DS. 2016. PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res 44:W16–W21. https://doi.org/10.1093/nar/gkw387.

Xie Z, Tang H. 2017. ISEScan: automated identification of insertion sequence elements in prokaryotic genomes. Bioinformatics 33:3340–3347. https://doi.org/10.1093/bioinformatics/btx433.

Bertels F, Silander OK, Pachkov M, Rainey PB, van Nimwegen E. 2014. Automated reconstruction of whole-genome phylogenies from short-sequence reads. Mol Biol Evol 31:1077–1088. https://doi.org/10.1093/molbev/msu088.

Lees JA, Harris SR, Tonkin-Hill G, Gladstone RA, Lo SW, Weiser JN, Corander J, Bentley SD, Croucher NJ. 2019. Fast and flexible bacterial genomic epidemiology with PopPUNK. Genome Res 29:304–316. https://doi.org/10.1101/gr.241455.118.

Huerta-Cepas J, Szklarczyk D, Heller D, Hernández-Plaza A, Forslund SK, Cook H, Mende DR, Letunic I, Rattei T, Jensen LJ, von Mering C, Bork P. 2019. eggNOG 5.0: a hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2 502 viruses. Nucleic Acids Res 47:D309–D314. https://doi.org/10.1093/nar/gky1085

Kanehisa M, Sato Y, Morishima K. 2016. BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J Mol Biol 428:726–731. https://doi.org/10.1016/j.jmb.2015.11.006.

Murawska E, Fiedoruk K, Bideshi DK, Swiecicka I. 2013. Complete genome sequence of Bacillus thuringiensis subsp. thuringiensis IS50056, an isolate highly toxic to Trichoplusia ni. Genome Announc 21:e0010813. https://doi.org/10.1128/genomeA.00108-13.

9

1

1

16