Functional Characterization of ycao in Escherichia coli C91 Reveals Its Role in Siderophore Production, Iron-Limited Growth, and Antimicrobial Activity

Background: The emergence of antibiotic-resistant bacteria is one of the top health concerns. Escherichia coli is a Gram-negative bacterium that commonly causes severe infections. However, this research exposed its antibiotic-producing potential. Methods: Rifampicin-resistant mutants of E. coli C91 were generated to activate cryptic BGCs. Mutants (C91-R1, R2 and R3) were tested for antimicrobial production using agar-well diffusion assays. Metabolite profiling was performed by LC-MS/MS. Siderophore production was tested by construction of a Δycao deletion mutant. Growth of this mutant was assessed under iron-limited conditions versus iron-rich conditions using dipyridyl. qRT-PCR was used to analyze gene expression entB, mcmA and mchF. Genome mining was performed using antiSMASH and BAGEL4. Results: Compared to the wild type, Mutant C91-R1(S531L) displayed clear antibacterial activity against Staphylococcus aureus. LC-MS/MS revealed unique metabolites, including a novel peak at m/z 410.5, specific to the mutant C91-R1. A reduction in siderophore production of 61% was demonstrated in the Δycao mutant, and downregulation of entB, mcmA and mchF. Conclusions: Genome mining predicted non-ribosomal peptide, thiopeptide and polyketide BGCs. E. coli C91 offers antibiotic-producing potential that can be activated through ribosome-engineering-type approaches. Moreover, E. coli C91-R1 has unique metabolites and is considered as a promising candidate for novel antibiotic discovery.