The mechanism of microcin C resistance provided by the MccF peptidase

Abstract

The heptapeptide-nucleotide microcin C (McC) is a potent inhibitor of enteric bacteria growth. Inside a sensitive cell, McC is processed by aminopeptidases, which release a nonhydrolyzable aspartyl-adenylate, a strong inhibitor of aspartyl-tRNA synthetase. The mccABCDE operon is sufficient for McC production and resistance of the producing cell to McC. An additional gene, mccF, which is adjacent to but not part of the mccABCDE operon, also provides resistance to exogenous McC. MccF is similar to Escherichia coli LdcA, an L,D-carboxypeptidase whose substrate is monomeric murotetrapeptide L-Ala-D-Glu-meso-A(2)pm-D-Ala or its UDP-activated murein precursor. The mechanism by which MccF provides McC resistance remained unknown. Here, we show that MccF detoxifies both intact and processed McC by cleaving an amide bond between the C-terminal aspartate and the nucleotide moiety. MccF also cleaves the same bond in nonhydrolyzable aminoacyl sulfamoyl adenosines containing aspartyl, glutamyl, and, to a lesser extent, seryl aminoacyl moieties but is ineffective against other aminoacyl adenylates.

FIGURE 1.

Microcin C, the mechanism of its action, the mcc gene cluster, and the primary structure of MccF. A, the structures of intact McC; processed McC, aspartyl-adenylate, an intermediate of reaction catalyzed by AspRS; and DSA, an inhibitor of AspRS, are shown. Arrows indicate bonds cleaved by MccF. B, the organization of the mcc gene cluster is shown. Arrows indicate mcc genes (drawn not to scale). The directions of the arrows indicate the direction of transcription. The mccA–E genes form a single operon. Genes whose products are involved in McC biosynthesis are indicated in solid gray. Genes whose products are involved in McC immunity are highlighted by vertical hatching. C, the sequence of MccF is aligned with E. coli LD-carboxypeptidase LdcA and the AgnC5 protein encoded by the agrocyn 83 biosynthetic cluster. The extent of amino acid residue conservation is indicated by background shading (black, identity; gray, conservative substitution; white, nonconservative substitution). MccF residues that, based on sequence comparisons, form the catalytic triad (Ser¹¹⁸, Glu²⁴³, and His³⁰⁹) are marked with asterisks. Alignment was done using ClustalW2 software. This figure was prepared using GENEDOC.

FIGURE 2.

Overproduced MccF makes cells resistant to McC, XDSA, XESA, and DSA. A, the sizes of growth inhibition zones around 2-μl drops containing McC and XDSA (10 μm solutions), XESA and XLSA (35 μm solutions), and albomycin (10 μm solution) deposited onto cell lawns formed by E. coli BL21(DE3) cells carrying indicated expression plasmids are shown. The error bars show standard deviations of measurements obtained in at least three independent experiments. B, the sizes of growth inhibition zones around 2-μl drops of 10 mm solutions of DSA and LSA deposited on cell lawns formed by E. coli BL21(DE3) cells carrying the indicated expression plasmids are shown.

FIGURE 3.

Recombinant MccF is sufficient to overcome tRNA aminoacylation inhibition caused by the addition of McC and DSA. A, AspRS-catalyzed aminoacylation of tRNA^Asp in S30 extracts prepared from E. coli cells with or without the addition of recombinant MccF. Where indicated, the extracts were supplied with intact McC or DSA. Reactins were indicated for the times indicated, and tRNA^Asp aminoacylation reactions were carried out. A representative result of three independent experiments is shown. B, LeuRS-catalyzed aminoacylation of tRNA^Leu in S30 extracts prepared from E. coli cells with or without the addition of recombinant MccF. Where indicated, the extracts were supplied with LSA, and tRNA^Leu aminoacylation reactions were carried out at the time points indicated. A representative result of three independent experiments is shown.

FIGURE 4.

Incubation with pure MccF abolishes antibacterial activity of McC. A, the sizes of growth inhibition zones around 2-μl drops of McC (10 μm solution) and DSA (10 mm solution) deposited on cell lawns formed by cells expressing wild-type MccF, MccF mutant carrying a S118A substitution, as well as cells harboring the pET vector control are shown. The error bars show standard deviations of measurements obtained in at least three independent experiments. B, McC was incubated with purified MccF in the presence or in the absence of PMSF, or with MccF^S118A mutant as described under “Experimental Procedures.” The reactions were terminated by lyophilization, reaction products were dissolved in water, and aliquots were deposited onto cell lawns formed by McC-sensitive E. coli cells. The sizes of growth inhibition zones observed after overnight incubation are shown. The error bars show standard deviations of measurements obtained in at least three independent experiments.

FIGURE 5.

Mass spectrometric analysis of products of incubation of recombinant MccF with McC and its maturation intermediates and aminoacyl sulfamoyl adenylates. Mature McC (A), its maturation intermediate (B), DSA (C), LSA (D), ESA (E), and seryl-sulfamoyl-adenosine (F) were incubated with or without recombinant MccF as described under “Experimental Procedures.” After incubation, aliquots of each reaction were subjected to MALDI-TOF MS analysis (reflector mode). The numbers indicate the average masses of the mass peaks detected.

FIGURE 6.

Increased antibacterial activity of DSA and ESA treated with MccF in vitro is due to generation of SA. A, DSA or ESA was incubated with purified MccF in the presence or in the absence of PMSF or with MccF^S118A mutant, and the reactions were treated as described under “Experimental Procedures” and Fig. 4B legend. The sizes of growth inhibition zones formed around drops of reaction aliquots deposited on cell lawns formed by E. coli BL21 cells are shown. The error bars show standard deviations of measurements obtained in at least three independent experiments. B, the sizes of growth inhibition zones around drops containing solutions of DSA, SA, or DSA incubated with MccF on cell lawns formed by cells carrying indicated expression plasmids are shown. The error bars show standard deviations of measurements obtained in at least three independent experiments. C–E, each panel shows HPLC elution profiles of DSA, SA, and DSA treated with MccF. The numbers by the HPLC peaks indicate the elution times.