Investigation of novel clusters of bacterial head-to-tail cyclized peptides from the Bacillus pumilus genome

Abstract

Bacterial headtotail cyclized peptides are a group of ribosomally synthesized and post -translationally modified peptides (RiPPs) of relatively large size. During maturation, the peptideundergoes cyclization, forming a peptide bond between its Cterminus and Nterminus. Comparedto other cyclic RiPPs bacterial cyclized peptides are distinguished by their larger cyclic structureand likely by their cyclization mechanism. The sequences of these peptides are highly diverse, andare not similar to each other. However, they share key properties: remarkable resistance to hightemperatures, proteases, and pH fluctuations. These features make them promising candidates forapplications in food preservation and medicine, for example, as antimicrobial agents. All thesepeptides are hydrophobic, and their mechanism of action is thought to involve disrupting the targetorganism’s membrane. It is believed that they form pores in the membrane, altering ionpermeability and ultimately leading to cell death. However, information on their mode of actionremains scarce, and the mechanisms for specific representat ives of this group are not fullyunderstood. Recently, our research team identified two novel clusters of bacterial headtotailcyclized peptides in the genome of Bacillus pumilus. The clusters differ from each other in boththe primary sequence of the precursor peptide and the number of genes within the cluster.Moreover, one of the clusters contains two precursor peptides, which is highly unusual for a clusterof this type. We tested the antibacterial activity of this strain against a range of Gram positive andGram negative bacteria, observing zones of growth inhibition likely linked to the production ofcyclized peptides. To further investigate their antibacterial activity, functions of genes, andcyclization mechanism, we constructed a recombinant shuttle vector based on the pHT01 plasmidfor heterologous expression of these clusters in another Bacillus strain.BeCELS 2025: Belgrade Conference for Early-Career Life Scientists, taking place on Friday, September 5, 2025, at the Institute of Molecular Genetics and Genetic Engineering (IMGGE) in Belgrad