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Sequence analysis of the four plasmid genes required to produce the circular peptide antibiotic microcin J25.

ABSTRACT: A 4.8-kb plasmid region carrying the four genes mcjABCD necessary for production of and immunity to the cyclic peptide antibiotic microcin J25 (MccJ25) has been sequenced. mcjA encodes the primary structure of MccJ25 as a precursor endowed with an N-terminal extension of 37 amino acids. The products of mcjB and mcjC are thought to be involved in microcin maturation, which implies cleavage of McjA and head-tail linkage of the 21-residue pro-MccJ25. The predicted McjD polypeptide, which is highly similar to several ABC exporters, was found to be required for MccJ25 secretion, thus explaining its ability to confer immunity to MccJ25.

SUBMITTER: Solbiati JO

PROVIDER: S-EPMC93700 | biostudies-literature | 1999 Apr

REPOSITORIES: biostudies-literature

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Sequence analysis of the four plasmid genes required to produce the circular peptide antibiotic microcin J25.

Solbiati J O JO Ciaccio M M Farías R N RN González-Pastor J E JE Moreno F F Salomón R A RA

Journal of bacteriology 19990401 8

A 4.8-kb plasmid region carrying the four genes mcjABCD necessary for production of and immunity to the cyclic peptide antibiotic microcin J25 (MccJ25) has been sequenced. mcjA encodes the primary structure of MccJ25 as a precursor endowed with an N-terminal extension of 37 amino acids. The products of mcjB and mcjC are thought to be involved in microcin maturation, which implies cleavage of McjA and head-tail linkage of the 21-residue pro-MccJ25. The predicted McjD polypeptide, which is highly ...[more]

PMID: 10198038

Similar Datasets

Computational design of the lasso peptide antibiotic microcin J25.

Project description:Microcin J25 (MccJ25) is a 21 amino acid (aa) ribosomally synthesized antimicrobial peptide with an unusual structure in which the eight N-terminal residues form a covalently cyclized macrolactam ring through which the remaining 13 aa tail is fed. An open question is the extent of sequence space that can occupy such an extraordinary, highly constrained peptide fold. To begin answering this question, here we have undertaken a computational redesign of the MccJ25 peptide using a two-stage sequence selection procedure based on both energy minimization and fold specificity. Eight of the most highly ranked sequences from the design algorithm, each of which contained two or three amino acid substitutions, were expressed in Escherichia coli and tested for production and antimicrobial activity. Six of the eight variants were successfully produced by E.coli at production levels comparable with that of the wild-type peptide. Of these six variants, three retain detectable antimicrobial activity, although this activity is reduced relative to wild-type MccJ25. The results here build upon previous findings that even rigid, constrained structures like the lasso architecture are amenable to redesign. Furthermore, this work provides evidence that a large amount of amino acid variation is tolerated by the lasso peptide fold.

| S-EPMC3038460 | biostudies-literature

Growth-phase-dependent expression of the cyclopeptide antibiotic microcin J25.

Project description:Microcin J25 is a 2,107-Da, plasmid-encoded, cyclopeptide antibiotic produced by Escherichia coli. We have isolated lacZ fusions to mcjA (encoding the 58-amino-acid microcin precursor) and mcjB and mcjC (which are required for microcin maturation), and the regulation of these fusions was used to identify factors that control the expression of these genes. The mcjA gene was found to be dramatically induced as cells entered the stationary phase. Expression of mcjA could be induced by resuspending uninduced exponential-phase cells in spent supernatant obtained from an early-stationary-phase culture. Induction of mcjA expression was not dependent on high cell density, pH changes, anaerobiosis, or the buildup of some inducer. A starvation for carbon and inorganic phosphate induced mcjA expression, while under nitrogen limitation there was no induction at all. These results taken together suggest that stationary-phase induction of mcjA is triggered by nutrient depletion. The mcjB and mcjC genes were also regulated by the growth phase of the culture, but in contrast to mcjA, they showed substantial expression already during exponential growth. Induction of the microcin genes was demonstrated to be independent of RpoS, the cyclic AMP-Crp complex, OmpR, and H-NS. Instead, we found that the growth-phase-dependent expression of mcjA, mcjB, and mcjC may be explained by the concerted action of the positively acting transition state regulators ppGpp, Lrp, and integration host factor. Measurements of microcin J25 production by strains defective in these global regulators showed a good correlation with the reduced expression of the fusions in such mutant backgrounds.

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Structure and organization of plasmid genes required to produce the translation inhibitor microcin C7.

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| S-EPMC177592 | biostudies-other

Fate and Biological Activity of the Antimicrobial Lasso Peptide Microcin J25 Under Gastrointestinal Tract Conditions.

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Antibacterial peptide microcin J25 inhibits transcription by binding within and obstructing the RNA polymerase secondary channel.

Project description:The antibacterial peptide microcin J25 (MccJ25) inhibits transcription by bacterial RNA polymerase (RNAP). Biochemical results indicate that inhibition of transcription occurs at the level of NTP uptake or NTP binding by RNAP. Genetic results indicate that inhibition of transcription requires an extensive determinant, comprising more than 50 amino acid residues, within the RNAP secondary channel (also known as the "NTP-uptake channel" or "pore"). Biophysical results indicate that inhibition of transcription involves binding of MccJ25 within the RNAP secondary channel. Molecular modeling indicates that binding of MccJ25 within the RNAP secondary channel obstructs the RNAP secondary channel. We conclude that MccJ25 inhibits transcription by binding within and obstructing the RNAP secondary channel--acting essentially as a "cork in a bottle." Obstruction of the RNAP secondary channel represents an attractive target for drug discovery.

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Project description:The role of the outer-membrane iron transporter FhuA as a potential receptor for the antimicrobial peptide MccJ25 (microcin J25) was studied through a series of in vivo and in vitro experiments. The requirement for both FhuA and the inner-membrane TonB-ExbB-ExbD complex was demonstrated by antibacterial assays using complementation of an fhuA(-) strain and by using isogenic strains mutated in genes encoding the protein complex respectively. In addition, MccJ25 was shown to block phage T5 infection of Escherichia coli, in vivo, by inhibiting phage adhesion, which suggested that MccJ25 prevents the interaction between the phage and its receptor FhuA. This in vivo activity was confirmed in vitro, as MccJ25 inhibited phage T5 DNA ejection triggered by purified FhuA. Direct interaction of MccJ25 with FhuA was demonstrated for the first time by size-exclusion chromatography and isothermal titration calorimetry. MccJ25 bound to FhuA with a 2:1 stoichiometry and a K(d) of 1.2 microM. Taken together, our results demonstrate that FhuA is the receptor for MccJ25 and that the ligand-receptor interaction may occur in the absence of other components of the bacterial membrane. Finally, both differential scanning calorimetry and antimicrobial assays showed that MccJ25 binding involves external loops of FhuA. Unlike native MccJ25, a thermolysin-cleaved MccJ25 variant was unable to bind to FhuA and failed to prevent phage T5 infection of E. coli. Therefore the Val11-Pro16 beta-hairpin region of MccJ25, which is disrupted upon cleavage by thermolysin, is required for microcin recognition.

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An experimental and computational investigation of spontaneous lasso formation in microcin J25.

Project description:The antimicrobial peptide microcin J25 (MccJ25) is posttranslationally matured from a linear preprotein into its native lasso conformation by two enzymes. One of these enzymes cleaves the preprotein and the second enzyme installs the requisite isopeptide bond to establish the lasso structure. Analysis of a mimic of MccJ25 that can be cyclized without the influence of the maturation enzymes suggests that MccJ25 does not spontaneously adopt a near-lasso structure. In addition, we conducted atomistically detailed replica-exchange molecular dynamics simulations of pro-microcin J25 (pro-MccJ25), the 21-residue uncyclized analog of MccJ25, to determine the conformational ensemble explored in the absence of the leader sequence or maturation enzymes. We applied a nonlinear dimensionality reduction technique known as the diffusion map to the simulation trajectories to extract two global order parameters describing the fundamental dynamical motions of the system, and identify three distinct pathways. One path corresponds to the spontaneous adoption of a left-handed lasso, in which the N-terminus wraps around the C-terminus in the opposite sense to the right-handed topology of native MccJ25. Our computational and experimental results suggest a role for the MccJ25 leader sequence and/or its maturation enzymes in facilitating the adoption of the right-handed topology.

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Structural mechanism of transcription inhibition by lasso peptides microcin J25 and capistruin.

Project description:We report crystal structures of the antibacterial lasso peptides microcin J25 (MccJ25) and capistruin (Cap) bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). Both peptides bind within the RNAP secondary channel, through which NTP substrates enter the RNAP active site, and sterically block trigger-loop folding, which is essential for efficient catalysis by the RNAP. MccJ25 binds deep within the secondary channel in a manner expected to interfere with NTP substrate binding, explaining the partial competitive mechanism of inhibition with respect to NTPs found previously [Mukhopadhyay J, Sineva E, Knight J, Levy RM, Ebright RH (2004) Mol Cell 14:739-751]. The Cap binding determinant on RNAP overlaps, but is not identical to, that of MccJ25. Cap binds further from the RNAP active site and does not sterically interfere with NTP binding, and we show that Cap inhibition is partially noncompetitive with respect to NTPs. This work lays the groundwork for structure determination of other lasso peptides that target the bacterial RNAP and provides a structural foundation to guide lasso peptide antimicrobial engineering approaches.

| S-EPMC6347699 | biostudies-literature

Free Energy Calculations of Microcin J25 Variants Binding to the FhuA Receptor.

Project description:Computer simulations were performed to study the antimicrobial peptide microcin J25 (MJ25), a 21-mer peptide with an unusual lasso structure and high activity against Gram-negative bacteria. MJ25 has intracellular targets. The initial step of MJ25 acquisition in bacterial cells is binding to the outer-membrane receptor FhuA. Molecular dynamics simulations were implemented to study the binding mechanism of MJ25 to FhuA and to search for important binding residues. The absolute binding free energy calculated from combined free energy perturbation and thermodynamic integration methods agrees well with experimental data. In addition, computational mutation analysis revealed that His5 is the key residue responsible for MJ25 and FhuA association. We found that the number of hydrogen bonds is essential for binding of MJ25 to FhuA. This atomistic, quantitative insight sheds light on the mechanism of action of MJ25 and may pave a path for designing active MJ25 analogues.

| S-EPMC9481273 | biostudies-literature

Complete nucleotide sequence and comparative genomic analysis of microcin B17 plasmid pMccB17.

Project description:We present a comprehensive sequence and bioinformatic analysis of the prototypical microcin plasmid, pMccb17, which includes a definitive sequence for the microcin operon, mcb. Microcin B17 (MccB17) is a ribosomally synthesized and posttranslationally modified peptide produced by Escherichia coli. It inhibits bacterial DNA gyrase similarly to quinolone antibiotics. The mcb operon, which consists of seven genes encoding biosynthetic and immunity/export functions, was originally located on the low copy number IncFII plasmid pMccB17 in the Escherichia coli strain LP17. It was later transferred to E. coli K-12 through conjugation. In this study, the plasmid was extracted from the E. coli K-12 strain RYC1000 [pMccB17] and sequenced twice using an Illumina short-read method. The first sequencing was conducted with the host bacterial chromosome, and the plasmid DNA was then purified and sequenced separately. After assembly into a single contig, polymerase chain reaction primers were designed to close the single remaining gap via Sanger sequencing. The resulting complete circular DNA sequence is 69,190 bp long and includes 81 predicted genes. These genes were initially identified by Prokka and subsequently manually reannotated using BLAST. The plasmid was assigned to the F2:A-:B- replicon type with a MOBF12 group conjugation system. A comparison with other IncFII plasmids revealed a large proportion of shared genes, particularly in the conjugative plasmid backbone. However, unlike many contemporary IncFII plasmids, pMccB17 lacks transposable elements and antibiotic resistance genes. In addition to the mcb operon, this plasmid carries 25 genes of unknown function.

| S-EPMC10912980 | biostudies-literature

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