Bacterial DNA gyrases are type H topoisomerases made up of two A subunits and two B subunits. Coumarins are carbohydrate-containing antibiotics that inhibit topolsomerases II by competing with ATP for binding to the-enzymes. figh resistance to coumarins is produced in bacterial species by mutations in gyrB, the gene encoding subunit B. We have found an unusual mechanism of resistance to coumarins in Escherichia coli. This mechanism is exhibited by cells containing the wild-type gyrB, or its 5' half, in high copy number. Since homologous mutant gyrB (coumermycin resistant) truncated genes did not confer drug resistance at all under the same conditions, we propose that this mechanism of resistance is due to drug sequestration by the overproduced wild-type GyrB polypeptides. A corollary of this is that the amino half of GyrB is required and sufficient to fashion the ATP-binding domain of DNA gyrase, a conclusion that was further supported by mapping three independent coumarinresistant mutations at Arg-136 of GyrB. Just upstream of this residue there is a glycine-rich sequence highy conserved in all topoisomerases II, which seems to be a good candidate for the actual ATP-binding site.such as nalidixic acid and oxolinic acid, act by trapping a gyrase-DNA reaction intermediate (13)(14)(15). The enterobacterial peptide microcin B17 acts in the same way (16). In contrast, coumarins, such as coumermycin Al, novobiocin, and chlorobiocin, act by competing with ATP for binding to the enzyme (17). The fact that all the E. coli mutations conferring high resistance to coumarins have been mapped to gyrB strongly supports the theory that subunit B is the primary target of those drugs. As a corollary, this subunit has been thought to be responsible for the ATP-binding and ATP hydrolysis activities of the enzyme (4, 5). Indeed gyrase subunit B has been shown to exhibit a low level of ATPase activity (18,19). By inhibiting the ATPase activity of DNA gyrase, the coumarins block the introduction of supercoils into relaxed DNA and relax supercoiled chromosomal DNA in vivo (20, 21). As a consequence, semiconservative DNA replication and cell growth are arrested.In this paper we report an unusual mechanism of resistance to coumarins and provide significant data concerning the structure-function relationship of the E. coli GyrB protein.DNA topoisomerases are enzymes that catalyze a variety of interconversions between topological isomers of DNA (1-3). All known DNA topoisomerases relax closed circular DNA, but only bacterial topoisomerases II, usually known as DNA gyrases, have been shown to supercoil DNA. This supercoiling activity is achieved at the expense of ATP hydrolysis to ADP and phosphate (for reviews, see refs. 1, 4, and 5). In eubacteria, the ATP-independent relaxing activity of DNA topoisomerase I and the ATP-dependent DNA supercoiling activity of DNA topoisomerase II compete to produce the proper level of superhelical tension in the cell, which is important to biological functions such as DNA replication and transcrip...

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains harbouring plasmid pMccB17. We have isolated two mutations that strongly reduce the production of MccB17. These mutations, which map at 96 min on the E. coli chromosome, define a new gene that we have called pmbA. A chromosomal DNA fragment of about 13 kb, including the wild-type pmbA allele, was cloned into a mini-Mu plasmid vector. pmbA was located within the cloned DNA fragment by insertional mutagenesis and deletion analysis. The nucleotide sequence of a 1.7 kb DNA region containing the gene was determined. pmbA encodes a hydrophilic protein of 450-amino-acid residues with a predicted molecular size of 48375D, which was visualized in polyacrylamide gels. Protein profiles of cellular envelope and soluble fractions from cells with plasmids overproducing PmbA indicated that it is cytoplasmic. Physiological experiments suggested that pmbA mutants synthesize a molecule (pro-MccB17) able to inhibit DNA replication but unable to be released from cells. We propose that PmbA facilitates the secretion of the antibiotic by completing its maturation.

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A TnS insertion decreasing the production of microcin B17 was mapped to 50.2 min on tbe Escherichia coli chromosome map. Sequence analysis showed that the insertion disrupted hisT, the gene encoding pseudouridine synthase I, a tRNA-modifying enzyme. hisT::TnS mutant cells were also shown to be defective for the production of other antibiotic peptides, such as microcin C7, microcin H47, and colicin V.In our studies of specific host-plasmid interactions, we have identified several chromosomal genes alfecting the production of the small enterobacterial plasmid-encoded antibiotics called microcins (4). Microcin B17 (MccB17) is a peptide of 3,200 Da which inhibits DNA replication by blocking DNA gyrase (24). Its production depends on six plasmid genes carried by pMccB17 and other related plasmids (9, 10, 21). Two Escherichia coli chromosomal genes regulate the production of this peptide by activating (ompR) (13) or by reducing (mprA) (7) the transcription of the plasmid genes. A third gene, pmbA, seems to be required for the maturation and secretion of the microcin (20). In this study, we analyzed an E. coli null hisT mutant which is impaired in the production of MccB17 and other small peptides.A chromosomal TnS insertion that caused decreased MccB17 production from strain MC4100(pMM4) was isolated as previously described (20). pMM4 is a pMccB17 derivative which contains a TnJO inserted into a region nonessential for MccB17 production and immunity or plasmid replication. After verifying that this TnS insertion was actually responsible for the phenotype of reduced production (the diameter of the antibiosis halos was twofold smaller), we introduced the transposon by P1 transduction into several Hfr strains. Mating experiments using these TnS Hfr derivatives allowed us to map the TnS insertion to the 50-to 60-min region of the E. coli genetic map (data not shown).To map precisely the position of the insert, we cloned the TnS insertion and the adjacent chromosomal region by the method of Groisman et al. (12). The cloning vector was plasmid pEG109, and the fragments were cloned from strain Hfr KL14 carrying the TnS insertion. Plasmids from six Cmr Kmr muductants were isolated and shown by restriction analysis to contain chromosomal fragments from the hisTpurF region (min 50 to 50.2) (14). The structures of these plasmids (pCID450 to pCID455) are shown in Fig. 1C production and suggest that the inactivation of hisT is the primary cause of the observed microcin phenotype.The product of hisT is a tRNA modification enzyme, pseudouridine synthase I, which catalyzes the formation of the pseudouridine residues x,38, x,39, and x440 in the anticodon stem and loop of at least 30 tRNA isoaccepting species (6,23). It follows that over half of the total cellular tRNA species are undermodified in hisT mutants. This is assumed to be the cause of the reduction in the general rate of translation elongation observed in hisT mutants (19).When we examined the effect of the mutation on the expression (transcription-translation) of an mcbA-lac...