Self‐Protection Mechanisms in Antibiotic Producers
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Abstract:
Various ways in which antibiotic-producing organisms are able to resist the actions of their products are discussed. Examples are given of antibiotic inactivation and also the modification of antibiotic target sites (most notably, ribosomes) to which drugs would otherwise bind and thereby exert their usual inhibitory effects. An interesting variation on the latter theme involves the duplication of target enzymes so that both sensitive and resistant versions are produced, the latter inducibly. Speculative discussion of antibiotic efflux leads to examples of cloned resistance determinants that probably encode components of efflux systems. Although of interest in their own right, resistance mechanisms should not be viewed narrowly when the physiology of antibiotic producers is considered. Thus, chemical modification of drug molecules may not only fulfil a protective role within the cell but may also provide substrates for efflux. Recent evidence that such considerations apply to macrolide antibiotics is presented. The control of resistance in producing organisms is also discussed with particular reference to the induction of novobiocin resistance in Streptomyces sphaeroides. This involves the interplay of novobiocin-sensitive and -resistant forms of DNA gyrase and features a promoter that displays a dramatic response to changes in DNA topology.Keywords:
Novobiocin
Efflux
Viomycin
DNA gyrase has been purified to near homogeneity from Escherichia coli. The enzyme consists of two subunits of molecular weights 90,000 and 100,000 present in roughly equimolar amounts. The subunits can be identified as the products of two genes, determining resistance to coumermycin A1 and novobiocin (cou) and to nalidixic acid and oxolinic acid (nalA), respectively. These antibiotics were previously shown to be specific inhibitors of DNA gyrase. The ATPase activity of DNA gyrase is stimulated by double-stranded DNA and strongly inhibited by novobiocin but is relatively insensitive to oxolinic acid. Covalent attachment of an ATP derivative to the smaller (coumermycin-specific) subunit is also inhibited by novobiocin, suggesting that this drug interferes with the energy-coupling aspect of the DNA supercoiling reaction by blocking the access of ATP to the enzyme.
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Novobiocin is a member of the coumermycin family of antibiotics and is a well-established inhibitor of DNA gyrase. Recent studies have shown that novobiocin binds to a previously unrecognized ATP-binding site at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins at ∼700 μM. In an effort to develop more efficacious inhibitors of the C-terminal binding site, a library of novobiocin analogues was prepared and initial structure−activity relationships revealed. These data suggested that the 4-hydroxy moiety of the coumarin ring and the 3'-carbamate of the noviose appendage were detrimental to Hsp90 inhibitory activity. In an effort to confirm these findings, 4-deshydroxy novobiocin (DHN1) and 3'-descarbamoyl-4-deshydroxynovobiocin (DHN2) were prepared and evaluated against Hsp90. Both compounds were significantly more potent than the natural product, and DHN2 proved to be more active than DHN1. In an effort to determine whether these moieties are important for DNA gyrase inhibition, these compounds were tested for their ability to inhibit DNA gyrase and found to exhibit significant reduction in gyrase activity. Thus, we have established the first set of compounds that clearly differentiate between the C-terminus of Hsp90 and DNA gyrase, converted a well-established gyrase inhibitor into a selective Hsp90 inhibitor, and confirmed essential structure−activity relationships for the coumermycin family of antibiotics.
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Thirteen quinolone antibacterial agents were investigated as to their ability to inhibit Micrococcus luteus DNA gyrase and cell growth, and compared to those of novobiocin and coumermycin. Among the quinolones tested, the most active were found to be CI-934 and ciprofloxacin, which inhibited gyrase full supercoiling activity at concentrations of 100 and 200 μ g/ml, respectively, while inhibiting cell growth at a concentration of 1 μ g/ml. However, both novobiocin and coumermycin inhibited gyrase full supercoiling activity at concentrations of 0.5 and 1.0 μ g/ml, respectively, which were comparable to those concentrations causing inhibition of cell growth.
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Elimination of plasmid pMG110 from Escherichia coli by novobiocin and other inhibitors of DNA gyrase
The ability of novobiocin to eliminate (cure) the wild-type plasmid pMG110 from Escherichia coli has been compared with that of other inhibitors of the gyrase B subunit and of the gyrase A subunit. Novobiocin eliminated pMG110 , producing over 99% plasmid loss at concentrations two- to eightfold below the MIC for bacterial growth. Structurally related compounds ( clorobiocin , coumermycin A1, isobutyryl novenamine , and decarbamyl novobiocin) varied in their ability to eliminate pMG110 . Higher concentrations of drugs were required to eliminate pMG110 from a gyrB( Cour ) strain, implicating DNA gyrase in the curing phenomenon. For these drugs, the ratio of the concentration effecting maximal plasmid elimination to the MIC varied from 0.16 to 1.1, indicating that curing cannot be explained simply by inhibition of a pool of DNA gyrase equally available for replication of the bacterial chromosome and the plasmid DNA molecule. Inhibitors of the gyrase A subunit, nalidixic acid and oxolinic acid, eliminated pMG110 only to variable low levels. The differences in the ability of the gyrase A and B subunit antagonists to eliminate plasmids are discussed.
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The sequence of the gyrase B subunit gene from Staphylococcus aureus strains resistant to the gyrase B subunit inhibitors cyclothialidine, coumermycin, and novobiocin has been determined. The residues altered in the resistant gyrase B subunits map to the ATP-binding region, suggesting that the drugs inhibit ATP binding and hydrolysis. The pattern of cross-resistances indicates that the detailed binding mode of the compounds differs.
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DNA gyrase and topoisomerase IV (topo IV) are the two essential type II topoisomerases of Escherichia coli. Gyrase is responsible for maintaining negative supercoiling of the bacterial chromosome, whereas topo IV's primary role is in disentangling daughter chromosomes following DNA replication. Coumarins, such as novobiocin, are wide-spectrum antimicrobial agents that primarily interfere with DNA gyrase. In this work we designed an alteration in the ParE subunit of topo IV at a site homologous to that which confers coumarin resistance in gyrase. This parE mutation renders the encoded topo IV approximately 40-fold resistant to inhibition by novobiocin in vitro and imparts a similar resistance to inhibition of topo IV-mediated relaxation of supercoiled DNA in vivo. We conclude that topo IV is a secondary target of novobiocin and that it is very likely to be inhibited by the same mechanism as DNA gyrase.
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We investigated how cyclothialidine (Ro 09-1437), a novel DNA gyrase inhibitor belonging to a new chemical class of compounds, acts to inhibit Escherichia coli DNA gyrase. Cyclothialidine up to 100 micrograms/ml showed no effect on DNA gyrase when linear DNA was used as a substrate. Under the same conditions, quinolones, which inhibit the resealing reaction of DNA gyrase, caused a decrease in the amount of linear DNA used. No effect of cyclothialidine was observed on the accumulation of the covalent complex of DNA and the A subunit of DNA gyrase induced by ofloxacin in the absence of ATP. The effect of cyclothialidine on the DNA supercoiling reaction was antagonized by ATP, reducing the inhibitory activity 11-fold as the ATP concentration was increased from 0.5 to 5 mM. Cyclothialidine competitively inhibited the ATPase activity of DNA gyrase (Ki = 6 nM). The binding of [14C]benzoyl-cyclothialidine to E. coli gyrase was inhibited by ATP and novobiocin, but not by ofloxacin. These results suggest that cyclothialidine acts by interfering with the ATPase activity of the B subunit of DNA gyrase. Cyclothialidine was active against a DNA gyrase resistant to novobiocin, suggesting that its precise site of action might be different from that of novobiocin.
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Novobiocin, coumermycin A1, and clorobiocin, structurally related compounds that antagonize the B subunit of the essential bacterial enzyme DNA gyrase, were compared with 18 of their analogs for the inhibition of Escherichia coli DNA gyrase supertwisting activity in vitro and of bacterial multiplication. This family of compounds has a 4-hydroxy-8-methylcoumarin core substituted in the 7 and 3 positions. Important for enzyme inhibition in vitro is a 7 ether linkage to a 3'-substituted noviose sugar. The 3'-ester-linked 5-methylpyrrole, found in the coumermycin series, conferred at least 10-fold more inhibitory activity than did the similarly linked amide, found in the novobiocin series; lack of the pyrrole and amide results in the loss of inhibitory activity. Of many aryl and alkyl substituents linked as an amide at the 3 position, the 4-hydroxyl-3-(3-methyl-2-butenyl)benzoic acid moiety, found in novobiocin and clorobiocin, and the reduplication of the coumarin-noviose-5-methylpyrrole, found in coumermycin A1, were most effective in gyrase inhibition. In vivo, the ability of these compounds to inhibit the growth of E. coli varied greatly. The enhanced inhibition of gyrase in vitro conferred by a 5-methylpyrrole relative to an amide in the 3'-noviose position was reflected in inhibition of bacterial multiplication. Several substitutions at the 3 position of the coumarin core conferring similar antagonism of gyrase in vitro resulted in substantially different inhibitory activities for E. coli, suggesting that these moieties at the 3 position affect drug access to the intracellular target. This target was shown for isobutyryl PNC-NH2 (PNC-NH2 is 3-amino-4-hydroxy-8-methyl-7-[3-O-(5-methyl-2-pyrrolylcarbonyl)noviosyloxy] coumarin) and confirmed for novobiocin, coumermycin A1, and clorobiocin to be in the B subunit of DNA gyrase.
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Novobiocin and coumermycin are known to inhibit the replication of DNA iing of DNA catalyzed by E. coli DNA gyrase, a recently discovered enzyme that introduces negative superhelical turns into covalently circular DNA. The activity of DNA gyrase purified from a coumermycin-resistant mutant strain is resistant to both drugs. The inhibition by novobiocin of colicin E1 plasmid DNA replication in a cell-free system is partially relieved by adding resistant DNA gyrase. Both in the case of coliclls. DNA molecules which are converted to the covalently circular form in thepresence of coumermycin remain relaxed, instead of achieving their normal supercoiled conformation. We conclude that DNA gyrase controls the supercoiling of DNA in E. coli.
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