Abstract
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 ∼100-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.
Highlights
We report the identification of citrocin, a 19-amino acid-long antimicrobial lasso peptide from the bacteria Citrobacter pasteurii and Citrobacter braakii
Neither the C. pasteurii nor C. braakii genomes are to date fully assembled, the presence of common plasmid-associated genes on this contig suggest that the cluster may be located on a plasmid or a plasmid that has been integrated into the genome
Like microcin J25 (MccJ25) and unlike the gene clusters of all other lasso peptides characterized to date, citrocin’s gene cluster has the citA precursor gene transcribed in the opposite direction of the rest of the genes in the cluster (Fig. 1C)
Summary
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. We heterologously expressed and structurally and biochemically characterized an antimicrobial lasso peptide, citrocin. Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides [2] that are characterized by their unique lasso structure [6, 7] This lasso structure is formed by an isopeptide bond between the N terminus and an aspartate or glutamate side chain to form a 7–9-membered ring through which the C-terminal end of the peptide is threaded and locked in place. Lasso peptides that exhibit antimicrobial activity have been found to contain a D gene that encodes an ABC transporter. The notable exception to this is microcin J25’s gene cluster, in which the mcjA gene is transcribed in the opposite direction of the mcjBCD
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