<b>The leader peptide is essential for the post‐translational modification of the DNA‐gyrase inhibitor microcin B17</b>

Madison, Lara L.; Vivas, Eugenio I.; Li, Yueming; Walsh, Christopher T.; Kolter, Roberto

doi:10.1046/j.1365-2958.1997.2041565.x

Cited by 58 publications

(60 citation statements)

References 22 publications

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“…It should be emphasized that the roles of these start͞stop motifs are putative, and additional characterization is required. However, the microcin B17 prepeptide has been shown to be essential for proper posttranslational modification (36). Conserved residues in leader sequences are known to be important in the modification of some lantibiotics (30,37,38), and a consensus sequence (GAEPR) found in these prepeptides bears a striking resemblance to the PatE start consensus motif, G(L͞ V)E(A͞P)S. Class I lantibiotics usually seem to possess a proline residue at the Ϫ2 position, although in the case of nisin this proline could be substituted with glycine and valine without impacting production (30,37).…”

Section: Correlation Of the Presence Of The Pat Gene Cluster With Patmentioning

confidence: 99%

Patellamide A and C biosynthesis by a microcin-like pathway inProchloron didemni, the cyanobacterial symbiont ofLissoclinum patella

Schmidt

Nelson

Rasko

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

552

717

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Prochloron spp. are obligate cyanobacterial symbionts of many didemnid family ascidians. It has been proposed that the cyclic peptides of the patellamide class found in didemnid extracts are synthesized by Prochloron spp., but studies in which host and symbiont cells are separated and chemically analyzed to identify the biosynthetic source have yielded inconclusive results. As part of the Prochloron didemni sequencing project, we identified patellamide biosynthetic genes and confirmed their function by heterologous expression of the whole pathway in Escherichia coli. The primary sequence of patellamides A and C is encoded on a single ORF that resembles a precursor peptide. We propose that this prepatellamide is heterocyclized to form thiazole and oxazoline rings, and the peptide is cleaved to yield the two cyclic patellamides, A and C. This work represents the full sequencing and functional expression of a marine natural-product pathway from an obligate symbiont. In addition, a related cluster was identified in Trichodesmium erythraeum IMS101, an important bloom-forming cyanobacterium.heterocycle ͉ tunicate ͉ ascidian ͉ genome sequencing ͉ lantibiotic

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Section: Correlation Of the Presence Of The Pat Gene Cluster With Patmentioning

confidence: 99%

Patellamide A and C biosynthesis by a microcin-like pathway inProchloron didemni, the cyanobacterial symbiont ofLissoclinum patella

Schmidt

Nelson

Rasko

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

552

717

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“…Purified compounds were lyophilized to yield white solids and analyzed by matrix-assisted laser desorption ionization͞time of flight MS. The yields and masses from 6 MccB17 stocks were prepared in DMSO, and aliquots (1 l) were dried in a Speedvac (Savant) and redissolved in 50% acetonitrile and water (500 l). The absorbance at 254 nm was measured, and the concentrations of the stocks were calculated by using the published normalized absorbance unit (NAU) values.…”

Section: Biosynthesis Of Mccb17mentioning

confidence: 99%

“…The production of mature MccB17 ( Fig. 1) involves posttranslational modification of a 69-residue polypeptide-precursor to generate two thiazoles, two oxazoles, and two 4,2-fused bisheterocycles (3,4), followed by cleavage of the 26-residue leader sequence by an unidentified protease (5,6). Exposure of sensitive bacteria to MccB17 results in inhibition of DNA synthesis, induction of the SOS response in cells with active replication, mutagenic effects, and DNA degradation (7).…”

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In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites

Zamble

Miller

Heddle

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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M icrocins are a class of small antibiotics (Ͻ10 kDa) produced and excreted by some strains of Enterobacteriaceae at the stationary phase of growth (1). Microcin B17 (MccB17) is a ribosomally synthesized compound that is produced in Escherichia coli harboring the plasmid-borne mcb operon, which contains the genes necessary for antibiotic synthesis, dedicated export, and immunity (2). The production of mature MccB17 ( Fig. 1) involves posttranslational modification of a 69-residue polypeptide-precursor to generate two thiazoles, two oxazoles, and two 4,2-fused bisheterocycles (3, 4), followed by cleavage of the 26-residue leader sequence by an unidentified protease (5, 6). Exposure of sensitive bacteria to MccB17 results in inhibition of DNA synthesis, induction of the SOS response in cells with active replication, mutagenic effects, and DNA degradation (7). These observations suggest that MccB17 is either a DNAdamaging agent or targets a key DNA-processing pathway such as replication.Genetic experiments designed to identify the target of MccB17 resulted in the isolation and characterization of a mutation in the B subunit of DNA gyrase (8). Strains carrying this mutation, a Trp-Arg substitution at position 751, exhibited increased resistance to MccB17 and a decrease in MccB17-induced DNA cleavage. Gyrase is a prokaryotic type II topoisomerase that introduces negative supercoils into DNA (9, 10). During the reaction, the enzyme passes through a transient intermediate in which two tyrosine hydroxyl groups form phosphodiester bonds with the exposed 5Ј phosphates of doublestrand cleaved DNA. This intermediate allows the enzyme to pull the ends of the DNA break apart to form a gate through which another DNA segment is passed. The quinolone family of drugs traps this covalent intermediate, known as the cleavable complex, and the accumulation of this complex is monitored in vitro by treatment with SDS and proteinase K, which releases linear DNA (11-13). The ternary complex blocks passage of the replication fork (14, 15), which may initiate the bactericidal activity of the quinolones, although the exact irreversible lethal event has not been firmly established (16). Recent in vitro analysis of the interaction between gyrase and MccB17 revealed that this antibiotic also causes accumulation of the cleavable complex in a manner similar to that of the quinolone drugs (17).Gyrase is the target of several classes of clinically successful drugs, including the quinolones and the coumarins (11, 13); however, the emergence of resistant strains is an incentive to elucidate the mechanism of action of existing drugs and develop new candidates. The identification of gyrase as a target of MccB17 provides an opportunity to analyze the structurefunction relationship of this unusual antibiotic. The experiments described here investigate the effects of modifying MccB17 at the sites of the tandem heterocycles, labeled A and B (Fig. 1). The interaction of MccB17 analogs with DNA gyrase was evaluated by measuring the rate of formation of the ...

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“…Premicrocin B17 is modified by the activity of the mcbB, -C, and -D gene products (MccB17 synthetase), yielding pro-MccB17 (32,55). The 26-amino-acid leader peptide is required for these modifications and serves as a scaffold for binding of the synthetase complex (33,43). Modifications result in a stably folded pro-MccB17 and confers antibiotic activity to the molecule (54).…”

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confidence: 99%

The Highly Conserved TldD and TldE Proteins of Escherichia coli Are Involved in Microcin B17 Processing and in CcdA Degradation

et al. 2002

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Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains carrying the pMccB17 plasmid. MccB17 is synthesized as a precursor containing an amino-terminal leader peptide that is cleaved during maturation. Maturation requires the product of the chromosomal tldE (pmbA) gene. Mature microcin is exported across the cytoplasmic membrane by a dedicated ABC transporter. In sensitive cells, MccB17 targets the essential topoisomerase II DNA gyrase. Independently, tldE as well as tldD mutants were isolated as being resistant to CcdB, another natural poison of gyrase encoded by the ccd poison-antidote system of plasmid F. This led to the idea that TldD and TldE could regulate gyrase function. We present in vivo evidence supporting the hypothesis that TldD and TldE have proteolytic activity. We show that in bacterial mutants devoid of either TldD or TldE activity, the MccB17 precursor accumulates and is not exported. Similarly, in the ccd system, we found that TldD and TldE are involved in CcdA and CcdA41 antidote degradation rather than being involved in the CcdB resistance mechanism. Interestingly, sequence database comparisons revealed that these two proteins have homologues in eubacteria and archaebacteria, suggesting a broader physiological role.

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The leader peptide is essential for the post‐translational modification of the DNA‐gyrase inhibitor microcin B17

Cited by 58 publications

References 22 publications

Patellamide A and C biosynthesis by a microcin-like pathway inProchloron didemni, the cyanobacterial symbiont ofLissoclinum patella

Patellamide A and C biosynthesis by a microcin-like pathway inProchloron didemni, the cyanobacterial symbiont ofLissoclinum patella

In vitro characterization of DNA gyrase inhibition by microcin B17 analogs with altered bisheterocyclic sites

The Highly Conserved TldD and TldE Proteins of Escherichia coli Are Involved in Microcin B17 Processing and in CcdA Degradation

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