Identification of widely conserved biosynthetic gene cluster involved in pigment production of Bacillus subtilis

Authors: STANNIUS, RUNE - Leiden University; Dunlap, Christopher; MORVAN, ESTELLE - University Of Bordeaux; BERBON, MELANIE - University Of Bordeaux; LECOMTE, SOPHIE - University Of Bordeaux; LOQUET, ANTOINE - University Of Bordeaux; KOVACS, AKOS - Leiden University

Submitted to: mSystems
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/8/2025
Publication Date: 7/3/2025
Citation: Stannius, R.O., Dunlap, C.A., Morvan, E., Berbon, M., Lecomte, S., Loquet, A., Kovacs, A.T. 2025. Identification of widely conserved biosynthetic gene cluster involved in pigment production of Bacillus subtilis. mSystems. https://doi.org/10.1128/msystems.00759-25.
DOI: https://doi.org/10.1128/msystems.00759-25

Interpretive Summary: Bacteria are the source of many bioactive molecules that are used to promote plant health or act as crop protection agents. There is a constant need to identify new molecules to manage resistance to crop protection products and provide new functionalities. A common approach to find new molecules is looking into the genomes of bacteria for unique gene clusters. The objective of the current study was to identify the gene cluster in a plant-associated bacteria that is responsible for the production of a pigmented molecule. The pigment was first reported more than 125 years ago, when it was observed on potatoes, and the chemical structure and the function of this molecule are still being evaluated. Understanding the genes that are responsible for producing different molecules is important, because it allows us to predict the type of molecules these bacteria may produce just from knowing their genes. This research will make genome mining of bacteria more accurate and speed the discovery of novel compounds from bacteria.

Technical Abstract: Bacillus subtilis is widely studied in the microbial secondary metabolite (SM) field due to its rich variety of important natural products and genetic tractability. However, identification of novel SMs and their biosynthetic gene cluster (BGCs) has become increasingly difficult, especially in Bacilli, as the tools for screening and genome mining are dependent on clear function or similarity to already known BGCs. Pigments are SMs identified by their absorption of visible light, resulting in a certain color perceived by our eyes at sufficient concentrations. Thereby, pigments provide the evidence of a BGC without knowing the sequence or function. Expanding the known repertoire of SM BGCs with novel BGCs will further reinforce identification of a broader set of BGCs by mining tools such as antiSMASH. Here, we study a pigment observed in B. subtilis soil isolate MB9_B4 on certain media. We characterize the conditions where this pigment is produced and identify the corresponding BGC using a comparative genomic approach exploiting our strain collection containing other isolates with pigment production ability. The responsible BGC carried several genes, which were annotated as parts of the tryptophan biosynthesis pathway, possibly originating from a duplication and divergence of an originally primary metabolism. Identification of the pigment gene cluster additionally lead to the discovery of additional pigment BGC carrier B. subtilis isolates, some of which were described at the earliest in 1896 under the name Bacillus aterrimus, with a name referring to a dark pigmentation (the Latin “aterrimus” meaning very black). In addition, we employed solid-state nuclear magnetic resonance and Fourier transform infrared spectroscopies to characterize the chemical groups of the pigment. This study describes the chemical and biological features of a new class of SM BGC, which we hope will serve to improve the current BGC discovery pipelines in Bacilli.