Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Aug;183(15):4543-50.
doi: 10.1128/JB.183.15.4543-4550.2001.

Escherichia coli RNA polymerase is the target of the cyclopeptide antibiotic microcin J25

M A Delgado  1 , M R Rintoul, R N Farías, R A Salomón
Affiliations

Escherichia coli RNA polymerase is the target of the cyclopeptide antibiotic microcin J25

M A Delgado et al. J Bacteriol. 2001 Aug.

Abstract

Escherichia coli microcin J25 (MccJ25) is a plasmid-encoded, cyclic peptide antibiotic consisting of 21 unmodified amino acid residues. It is primarily active on gram-negative bacteria related to the producer strain, inducing cell filamentation in an SOS-independent way. A mutation causing resistance to MccJ25 was isolated. Genetic analysis indicated that it resided in the rpoC gene, encoding the beta' subunit of RNA polymerase, at 90 min on the E. coli genetic map. The mutation was genetically crossed on to a plasmid containing the wild-type rpoC gene. The presence of the recombinant plasmid conferred complete resistance to otherwise sensitive strains. Nucleotide sequencing of the plasmid-borne, mutant rpoC gene revealed a ACC (Thr)-to-ATC (Ile) change at codon 931, within homology block G, an evolutionarily conserved region in the large subunits of all RNA polymerases. MccJ25 decreased RNA synthesis both in vivo and in vitro. These results point to the RNA polymerase as the target of microcin action. We favor the possibility that the filamentous phenotype induced by MccJ25 results from impaired transcription of genes coding for cell division proteins. As far as we know, MccJ25 is the first peptide antibiotic shown to affect RNA polymerase.

PubMed Disclaimer

Figures

FIG. 1
FIG. 1
Schematic representation of the plasmids used in this work. The portion of the rpoBC region contained in the various plasmids is indicated. Only relevant restriction sites are included: BglII (Bg); BamHI (B), HindIII (H); and PstI (P).
FIG. 2
FIG. 2
Genetic context and sequence alteration of the rpoC931 mutation. The heavy bar represents the 1,407-amino-acid β′ subunit of E. coli RNAP. Lettered boxes (A to H) symbolize evolutionarily conserved segments of β′ (33). A relevant sequence stretch (Gly912 to Gly939) from segment G of β′ (E.c.) is expanded underneath. The position of the amino acid alteration (a Thr-to-Ile change at codon 931) caused by the rpoC931 mutation is highlighted by boldface. The top sequence from E. coli is aligned with the corresponding portions of eubacterial, archaeal, and eukaryotic homologues: Pseudomonas putida (P.p.); Bacillus subtilis (B.s.); Helicobacter pylori (H.p.); Mycobacterium leprae (M.l.); Methanobacterium thermoautotrophicum (M.t.); Thermotoga maritima (T.m.); Sulfolobus acidocaldarium (S.a.); Saccharomyces cerevisiae RNAP I, II, and III (Y1, Y2, and Y3, respectively); RNAP II and III of Trypanosoma (T2 and T3, respectively); chloroplasts from spinach (S), tobacco (T), and liverwort (L); RNAP II of Drosophila melanogaster (D2); and RNAP II of mouse (M2). The dots symbolize amino acid sequence identity. The highly conserved 7-amino-acid sequence stretch (RTFHIGG) shown by Borukhov et al. (4) to contact the nascent RNA product is underlined.
FIG. 3
FIG. 3
Effect of MccJ25 on RNA synthesis of parent AB259 and mutant SBG231 strains. RNA synthesis was measured by monitoring incorporation of [3H]uridine into TCA-precipitable material as described in Materials and Methods. Accumulated RNA in the absence (□) and in the presence (■) of microcin is shown.
FIG. 4
FIG. 4
Effect of MccJ25 on protein synthesis of parent AB259 and mutant SBG231 strains. Incorporation of [3H]leucine in the absence (□) and in the presence (■) of microcin is shown and was determined as described in Materials and Methods.
FIG. 5
FIG. 5
Inhibition of RNA polymerase by MccJ25 in vitro. The transcription assay was done as indicated in Materials and Methods. The amount of transcription activity relative to the uninhibited enzyme is indicated as a function of increasing MccJ25 concentration.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Allison L A, Moyle M, Shales M, Ingles C J. Extensive homology among the largest subunits of eukaryotic and prokaryotic RNA polymerases. Cell. 1985;42:599–610. - PubMed
    1. Baracchini E, Bremer H. Determination of synthesis rate and lifetime of bacterial mRNAs. Anal Biochem. 1987;167:245–260. - PubMed
    1. Blond A, Péduzzi J, Goulard C, Chiuchiolo M J, Barthélémy M, Prigent Y, Salomón R A, Farías R N, Moreno F, Rebuffat S. The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli. Eur J Biochem. 1999;259:747–755. - PubMed
    1. Borukhov S, Lee J, Goldfarb A. Mapping of a contact for RNA 3′-terminus in the largest subunit of RNA polymerase. J Biol Chem. 1991;266:23932–23935. - PubMed
    1. Chiuchiolo M J, Delgado M A, Farías R N, Salomón R A. Growth-phase-dependent expression of the cyclopeptide antibiotic microcin J25. J Bacteriol. 2001;183:1755–1764. - PMC - PubMed

Publication types

MeSH terms