We report the identification of citrocin, a 19-amino acid-long antimicrobial lasso peptide from the bacteria Citrobacter pasteurii and Citrobacter braakii. We refactored the citrocin gene cluster and heterologously expressed it in Escherichia coli. We determined citrocin's NMR structure in water and found that is reminiscent of that of microcin J25 (MccJ25), an RNA polymerase-inhibiting lasso peptide that hijacks the TonB-dependent transporter FhuA to gain entry into cells. Citrocin has moderate antimicrobial activity against E. coli and Citrobacter strains. We then performed an in vitro RNA polymerase (RNAP) inhibition assay using citrocin and microcin J25 against E. coli RNAP. Citrocin has a higher minimal inhibition concentration than microcin J25 does against E. coli but surprisingly is ϳ100-fold more potent as an RNAP inhibitor. This suggests that citrocin uptake by E. coli is limited. We found that unlike MccJ25, citrocin's activity against E. coli relied on neither of the two proton motive force-linked systems, Ton and Tol-Pal, for transport across the outer membrane. The structure of citrocin contains a patch of positive charge consisting of Lys-5 and Arg-17. We performed mutagenesis on these residues and found that the R17Y construct was matured into a lasso peptide but no longer had activity, showing the importance of this side chain for antimicrobial activity. In summary, we heterologously expressed and structurally and biochemically characterized an antimicrobial lasso peptide, citrocin. Despite being similar to MccJ25 in sequence, citrocin has an altered activity profile and does not use the same outer-membrane transporter to enter susceptible cells.

We report the heterologous expression, structure, and antimicrobial activity of a lasso peptide, ubonodin, encoded in the genome of Burkholderia ubonensis. The topology of ubonodin is unprecedented amongst lasso peptides, with 18 of its 28 amino acids found in the mechanically bonded loop segment. Ubonodin inhibits RNA polymerase in vitro and has potent antimicrobial activity against several pathogenic members of the Burkholderia genus, most notably B. cepacia and B. multivorans, causative agents of lung infections in cystic fibrosis patients.