Heterologous expression and functional characterization of novel microcin B17 congeners from environmental Pseudomonas isolates.

Microcin B17 (MccB17) is a ribosomally synthesized and post-translationally modified peptide (RiPP) with antibacterial activity, natively produced by Escherichia coli. Despite its unique gyrase-inhibiting activity, therapeutic development has been limited, primarily due to its low solubility and narrow spectrum of activity. Here, we employed a genome mining approach to identify and functionally characterize novel MccB17-like biosynthetic gene clusters (BGCs) from uncharacterized environmental Pseudomonas isolates. Four BGCs sharing substantial sequence similarity were cloned and heterologously expressed in E. coli. Regardless of their similarity, the clusters triggered congener-dependent growth retardation and cell elongation, despite lacking detectable antibacterial activity in overlay assays. Transcription and production of modified MccB17-like peptides or their unmodified precursors were confirmed by reverse transcription-quantitative PCR (RT-qPCR) and liquid chromatography-mass spectrometry (LC-MS), respectively. Transcriptome profiling indicated differential induction of stress responses and metabolic shifts in host cells, with limited overlap among the different BGCs. Our findings suggest that structurally similar MccB17 congeners can elicit distinct physiological responses. This study expands the known range of MccB17-like RiPPs and highlights the importance of functionally exploring RiPP diversity beyond known antimicrobial activity, even among congeners. KEY POINTS: • In silico identification of uncharacterized MccB17 congeners from Pseudomonas spp. • Functional analysis of novel variants reveals phenotypic effects on expression host • Transcriptomics reveals congener-specific effects despite similarity to E. coli MccB17.