The maturation pathway of microcin B17, a peptide inhibitor of DNA gyrase

Yorgey, Peter; Davagnino, Juan; Kolter, Roberto

doi:10.1111/j.1365-2958.1993.tb01747.x

Cited by 57 publications

(54 citation statements)

References 17 publications

(13 reference statements)

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“…The structural gene, mcbA, is plasmid encoded (11). Downstream of mcbA are mcbBCDEFG, whose products are responsible for posttranslational modification of McbA, microcin B17 export, and immunity and resistance to microcin B17 (20,39,40). MprA, a negative regulator of microcin B17 gene expression (7), PmbA, purported to be involved in leader processing (25), and OmpR, a positive regulator of microcin B17, ompC, and ompF gene expression (14,24), are all chromosomally encoded.…”

Section: Discussionmentioning

confidence: 99%

A newly discovered gene, tfuA, involved in the production of the ribosomally synthesized peptide antibiotic trifolitoxin

1996

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Trifolitoxin (TFX) is a gene-encoded, posttranslationally modified peptide antibiotic. Previously, we have shown that tfxABCDEFG from Rhizobium leguminosarum bv. trifolii T24 is sufficient to confer TFX production and resistance to nonproducing strains within a distinct taxonomic group of the ␣-proteobacteria (E. W. Triplett, B. T. Breil, and G. A. Splitter, Appl. Environ. Microbiol. 60:4163-4166, 1994). Here we describe strain Tn5-2, a Tn5 mutant of T24 defective in the production of TFX, whose insertion maps outside of the tfx cluster. It is not altered in growth compared with T24, nor does it inactivate TFX in its proximity. The wild-type analog of the mutated region of Tn5-2 was cloned. Sequencing, transcriptional fusion mutagenesis, and subcloning were used to identify tfuA, a gene involved in TFX production. On the basis of computer analysis, the putative TfuA protein has a mass of 72.9 kDa and includes a peroxidase motif but no transmembrane domains. TFX production studies show that extra copies of the tfxABCDEFG fragment increase TFX production in a T24 background while additional copies of tfuA do not. Lysate ribonuclease protection assays suggest that tfuA does not regulate transcription of tfxA. Upstream of tfuA are two open reading frames (ORFs). The putative product of ORF1 shows high similarity to the LysR family of transcriptional regulators. The putative product of ORF2 shows high similarity to the cytosine deaminase (CodA) of Escherichia coli.Trifolitoxin (TFX) is a ribosomally synthesized, posttranslationally modified peptide antibiotic produced by Rhizobium leguminosarum bv. trifolii T24 (4, 29). A number of features make TFX an interesting antibiotic for study. Its spectrum of activity is quite narrow but includes bacteria that are plant symbionts and plant and animal pathogens (29,34,35). TFX has already been shown to limit nodules formed by TFXsensitive strains (32-34) and so may provide a solution to the Rhizobium competition problem (9, 36). This problem arises when inoculant strains are not competitive for legume root nodulation against strains indigenous to soil. Its ribosomal synthesis makes it an easy substrate for drug modification (12,16,17,27). Though the structure of TFX has not been completely elucidated, it is known to contain a thiazoline ring and another cyclic chromophore (23). The latter appears to be a novel, pyrimidine-like structure (unpublished results). It may be possible to use the enzymes that modify TFX to modify different substrates, thereby creating new molecules. EpiD from the epidermin system has been used to catalyze the oxidative decarboxylation of heptapeptides (21), thereby showing the very real possibility of using antibiotic posttranslational modification enzymes to catalyze reactions on molecules other than their natural preantibiotic target. Given the TFX system's agricultural, medical, and biochemical potential, elucidation of the genetics of TFX production is important.A number of genes have been shown to be required for TFX production and resistance. T...

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Section: Discussionmentioning

confidence: 99%

A newly discovered gene, tfuA, involved in the production of the ribosomally synthesized peptide antibiotic trifolitoxin

1996

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“…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). The last step in MccB17 maturation is the removal of the leader peptide of pro-MccB17, yielding mature MccB17 (55).…”

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confidence: 99%

“…The PmbA protein might be either the peptidase responsible for maturation or a chaperone promoting the interaction of pro-MccB17 with the specific McbEF pump, where cleavage would take place. Export seems to be distinct from leader peptide removal, as (i) exogenously added MccB17 is pumped out of target bacteria containing the McbEF pump and (ii) an McbA-LacZ fusion is processed in the absence of McbEF (54).…”

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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 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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confidence: 99%

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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The maturation pathway of microcin B17, a peptide inhibitor of DNA gyrase

Cited by 57 publications

References 17 publications

A newly discovered gene, tfuA, involved in the production of the ribosomally synthesized peptide antibiotic trifolitoxin

A newly discovered gene, tfuA, involved in the production of the ribosomally synthesized peptide antibiotic trifolitoxin

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

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

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