We report the identification of citrocin, a 19-amino acid-long antimicrobial lasso peptide from the bacteria and We refactored the citrocin gene cluster and heterologously expressed it in 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 and strains. We then performed an RNA polymerase (RNAP) inhibition assay using citrocin and microcin J25 against RNAP. Citrocin has a higher minimal inhibition concentration than microcin J25 does against but surprisingly is ∼100-fold more potent as an RNAP inhibitor. This suggests that citrocin uptake by is limited. We found that unlike MccJ25, citrocin's activity against 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.