Exploratie en karakterisering van nieuwe bacteriocinen uit sporenvormende Bacilli en Clostridia

Antimicrobial resistance (AMR) is one of the most urgent global health challenges of our time. As the effectiveness of existing antibiotics declines and resistant infections rise, the need for novel antimicrobial compounds has become critical. Despite this growing threat, only a few new antibiotics have been introduced in the past three decades, leaving a shrinking repertoire of effective treatments. Bacteria have been an early source of antimicrobial compounds and, due to their enormous diversity and widespread presence across various ecosystems that has been only partly unravelled, they remain one of the most promising sources for the future discovery of novel antimicrobials. With recent advances in whole-genome sequencing and bioinformatics, scientists are now better equipped than ever before to explore the untapped biosynthetic potential of newly discovered and lesser-studied microbes for novel antimicrobial discovery. A group of bacterial antimicrobials that have been known for several decades but are currently receiving renewed interest are the bacteriocins. They are characterised by a vast structural diversity, high potency and stability, but unlike antibiotics, they are ribosomally synthesised peptides with usually a narrow spectrum of activity and low toxicity, which makes them attractive candidates for application as clinical or veterinary antimicrobials. This project aims to isolate and characterise novel bacteriocins from three bacteria that were identified as potential bacteriocin producers in previous work, and that belong to the genera Bacillus and Clostridium. These are Gram-positive, spore-forming bacteria that inhabit diverse environments, including soil, water, and the gastrointestinal tracts of humans and animals. While several members of the genus Bacillus have been recognised as prolific producers of antibiotics, the Clostridium genus has been relatively understudied in antimicrobial research, highlighting its potential as a rich reservoir for new bioactive compounds. The first strain selected for detailed analysis was C. botulinum HA5, a non-toxic group I isolate from an environmental sample. This strain displayed antimicrobial activity against several clostridia, including pathogens like group I and II C. botulinum, C. perfringens and Clostridioides difficile. Based on its heat stability (≥90 ֯C for 2 h), pH stability (pH 4-7), sensitivity to proteinase K and moderate UV sensitivity, the compound was expected to be a bacteriocin. As a first step towards identification, the biosynthetic gene clusters (BGCs) potentially encoding the bacteriocin were identified by bioinformatic analysis, and their systematic inactivation was initiated. This led to the emergence of a spontaneous mutant that lost antimicrobial activity. Whole-genome sequencing revealed that this loss was due to the absence of a plasmid (pHA5-1), which carried a BGC encoding a putative ranthipeptide, which we named botocidin HA5. The BGC exhibited no similarity to any known ranthipeptides or other RiPPs, suggesting that botocidin HA5 is a novel compound. The BGC was organised into two operons: one encoding the core peptide, modification enzymes, an ABC transporter and proteases; and another, with opposite orientation, encoding an MFS transporter and a possible additional modification enzyme. Botocidin HA5 immunity was also encoded on the pHA5-1 plasmid, but while production of the bacteriocin was regulated by the Spo0A transcription factor, immunity was not, suggesting that the MFS transporter operon was probably conferring immunity. Botocidin HA5 was isolated from culture supernatant using TCA precipitation, and LC-MS/MS analysis identified three peptide masses consistent with three possible peptides generated from the pHA5-1 BGC precursor peptide by proteolytic cleavage at different positions and the additional formation of two or three thioether linkages. Botocidin HA5 exhibited a bactericidal effect on vegetative C. botulinum cells, and time-lapse microscopy with propidium iodide staining revealed that it causes cell leakage by targeting the cell membrane. While botocidin HA5 did not inhibit spore germination, it effectively blocked spore outgrowth at an early stage, but without killing the germinated spores. These findings confirm that botocidin HA5 is a novel antimicrobial compound with potential to combat specific clostridia that cause disease or food spoilage. The second investigated organism was C. botulinum DSM 1985, a non-toxic group II strain isolated from an environmental sample. This strain exhibited pronounced antimicrobial activity against a limited number of Clostridium genus members, including other Group II C. botulinum strains, C. perfringens, and Clostridioides difficile. The active compound was stable across a wide range of environmental conditions but was protease-sensitive, confirming it was also probably a bacteriocin. Bioinformatic analysis of whole genome sequences revealed that the strain had one unique BGC that was absent in all the sensitive related C. botulinum strains, and that was therefore likely to be responsible for the production of the compound. This cluster encoded a putative two-peptide Class IIb bacteriocin, which we designated botocin 1985. LC-MS of partially purified culture supernatant fractions revealed masses corresponding to the three predicted core peptides in the gene cluster. Pairwise combination of the de novo synthesised peptides allowed the identification of one specific combination that showed strong antibacterial activity. Time-lapse microscopy further validated its bactericidal properties against vegetative cells and germinated spores. Attempts at gene inactivation were unsuccessful due to the strain's inability to accept plasmids via conjugation. Together, these data support the conclusion that botocin 1985 is a novel anticlostridial Class IIb bacteriocin. Its activity against specific foodborne and gut pathogenic clostridia suggests promising potential for food or clinical applications. Finally, the last strain studied in this work, Bacillus thuringiensis DSM 2046, exhibited broad-spectrum antimicrobial activity against a wide range of Gram-positive and Gram-negative bacteria, including multidrug-resistant strains. Remarkably, the active compound was not produced in liquid culture, but only in submerged cultures in semi-solid medium or in surface cultures on solid agar. Biochemical characterisation revealed that the compound was heat-stable, UV-stable, and pH-stable, but sensitive to protease treatment, consistent with a bacteriocin-like inhibitory substance (BLIS). Functional assays, including time-lapse phase-contrast and fluorescence microscopy, showed that the compound was bactericidal and targeted the bacterial cell membrane. Additionally, it inhibited the early-stage germination of both its own spores and those of sensitive indicator strains, without affecting spore viability. Genome mining of B. thuringiensis DSM 2046 revealed multiple BGCs, two of which were eliminated as candidates encoding the compound by knockout analysis. Further analysis revealed the presence of a plasmid encoding two previously described antimicrobials: zwittermicin A and thuricin. Because of its broad-spectrum activity, zwittermicin A is most likely to be the active compound of DSM 2046. Due to time constraints and the likelihood of low novelty, further work on this strain was halted. In conclusion, this work reports the identification and characterisation of two novel bacteriocins, a ranthipeptide or sactipeptide from a group I C. botulinum, and a class IIb two-peptide bacteriocin from a group II C. botulinum. Further study of these compounds may provide an interesting viewpoint on the ecology of these bacteria in their natural environment, which has been poorly studied hitherto. Additionally, the specific anticlostridial activity of the compounds provides perspectives for both clinical and industrial applications. Furthermore, an antimicrobial compound from a B. thuringiensis strain was found to have the unique ability to inhibit bacterial spore germination, raising the question of the possible biological function of this property and its possible applications.