TY - JOUR
T1 - Discovery and structure of the antimicrobial lasso peptide citrocin
AU - Cheung-Lee, Wai Ling
AU - Parry, Madison E.
AU - Cartagena, Alexis Jaramillo
AU - Darst, Seth A.
AU - James Link, A.
N1 - Funding Information:
1 Supported in part by a Dodds Fellowship from Princeton University.
Funding Information:
2Supported by a Robert D. Watkins Graduate Research Fellowship from the American Society of Microbiology.
Funding Information:
This work was supported in part by National Institutes of Health Grants GM107036 (to A. J. L.) and GM118130 (to S. A. D.). This work was also sup-ported in part by Princeton University SEAS Innovation Funds, the Helen Shipley Hunt Fund, and the Focused Research Team for Precision Antibi-otics. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2019 Cheung-Lee et al. Published under exclusive license by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2019/4/26
Y1 - 2019/4/26
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.RA118.006494
DO - 10.1074/jbc.RA118.006494
M3 - Article
C2 - 30846564
AN - SCOPUS:85065333118
SN - 0021-9258
VL - 294
SP - 6822
EP - 6830
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 17
ER -