Reiterative Synthesis by the Ribosome and Recognition of the N-Terminal Formyl Group by Biosynthetic Machinery Contribute to Evolutionary Conservation of the Length of Antibiotic Microcin C Peptide Precursor

Maximum likelihood phylogenetic tree of MccB and MccB-like proteins. The unrooted tree was generated using RAxML (24) with 400 bootstrap replicates. The numbers at the nodes indicate the bootstrap values. Only bootstrap values greater than 70% are shown. Scale bar shows the number of inferred amino acid substitutions per site. Triangle marks the outgroup consisting of PaaA homologs not involved in the production of McC-like compounds. Each terminal node of the tree corresponds to the sequence representing a cluster of identical proteins and is labeled by the full systematic name of an organism (for the full data set see Table S1 in the supplemental material). The names of the organisms bearing the validated mcc clusters are shown in red. For other organisms, precursor peptides are putative and obtained by manual sequence analysis of mcc-like clusters. The lengths (in amino acids) of the verified or predicted microcin C precursor peptides are indicated both by labels and by the color strip. The color palette is given on the bottom left. White indicates that we were unable to predict the peptide. The blue and the red branches correspond to MccB subfamilies associated with 7-amino-acid and longer MccA precursors, respectively.

Bioactivity of secreted and intracellular McC and McC-like compounds. (A) Organization of gene clusters used in this study and structures of the expected compounds produced by complete and partial mcc gene clusters shown at the top. R, MRTGNA (pA7 and pA7-ap plasmids), MGGGMRTGNA (pA11 and pA11-ap plasmids), or MASTAGGGMRTGNA (pA15 and pA15-ap plasmids). (B) Growth-inhibiting activity of culture media from E. coli cells harboring plasmids encoding wild-type MccA heptapeptide (pA7-ap and pA7) or extended peptides (pA11-ap, pA11, pA15-ap, and pA15). (C) Inhibitory activity of cell extracts obtained from E. coli cells harboring indicated plasmids.

In vivo production of mccA monocistronic transcripts. (A) The structure of minimized mcc operon and expected transcripts. The mcc operon promoter P_mcc and transcription terminator located between the mccA and mccB genes are indicated. Ribosomes bound to ORFs are schematically shown. SD, Shine-Dalgarno sequences. Binding of the ribosome to mccA induces transcription termination (12). (B) Northern blot hybridization analysis of short monocistronic mccA mRNA abundance in cells harboring mcc plasmids with different mccA gene lengths. Radiolabeled hybridization probe was complementary to an invariant fragment of the mccA gene.

In vitro translation efficiency of mccA mRNA. Results of translation reiterative initiation experiment with different mccA mRNAs are shown. In vitro translation reactions in the presence or in the absence of ribosome-sequestering competitor mRNA were performed in parallel. Reaction aliquots were taken at different time points, and the number of MccA peptides synthesized per active ribosome was calculated. Amount of peptide synthesized at each time point without competition (N) was plotted versus amount of peptide synthesized with competition (n) at the same time point. Dotted lines indicate a case when n = N (ribosome never leaves mRNA, n_rec = ∞). Data were fitted according to the model described in reference 13, fitting curves are shown as black lines, and respective n_rec is shown by the colored dashed lines.

In vitro adenylation of MccA peptides of different lengths by MccB. HPLC traces (260 nm) showing product formation after incubation of equal molar amounts of A7 (MRTGNAN), A11 (MGGGMRTGNAN), and A15 (MASTAGGGMRTGNAN) peptides and their N-terminally formylated variant peptides (marked with the f prefix) with MccB in the reaction buffer for 20 min at 28°C.