Resistance Genes of Aminocoumarin Producers: Two Type II Topoisomerase Genes Confer Resistance against Coumermycin A ₁ and Clorobiocin

Abstract

The aminocoumarin resistance genes of the biosynthetic gene clusters of novobiocin, coumermycin A 1 , and clorobiocin were investigated. All three clusters contained a gyrB R resistance gene, coding for a gyrase B subunit. Unexpectedly, the clorobiocin and the coumermycin A 1 clusters were found to contain an additional, similar gene, named parY R . Its predicted gene product showed sequence similarity with the B subunit of type II topoisomerases. Expression of gyrB R and likewise of parY R in Streptomyces lividans TK24 resulted in resistance against novobiocin and coumermycin A 1 , suggesting that both gene products are able to function as aminocoumarin-resistant B subunits of gyrase. Southern hybridization experiments showed that the genome of all three antibiotic producers and of Streptomyces coelicolor contained two additional genes which hybridized with either gyrB R or parY R and which may code for aminocoumarin-sensitive GyrB and ParY proteins. Two putative transporter genes, novA and couR5, were found in the novobiocin and the coumermycin A 1 cluster, respectively. Expression of these genes in S. lividans TK24 resulted in moderate levels of resistance against novobiocin and coumermycin A 1 , suggesting that these genes may be involved in antibiotic transport.The aminocoumarin antibiotics novobiocin, clorobiocin, and coumermycin A 1 (Fig. 1A) are known as potent inhibitors of gyrase (18). Their equilibrium dissociation constants are in the range of 10 nM (10), i.e., their affinity for gyrase is considerably higher than that of modern fluoroquinolones. Novobiocin is licensed as an antibiotic for clinical use (Albamycin; Pharmacia-Upjohn) and is used for the treatment of infections with multiresistant gram-positive bacteria, e.g., Staphylococcus aureus.Novobiocin is produced by Streptomyces spheroides (synonym S. caeruleus [15]) NCIMB 11891, clorobiocin is produced by S. roseochromogenes var. oscitans DS12.976, and coumermycin A 1 is produced by S. rishiriensis DSM 40489 (2). Obviously, these organisms must protect their gyrases from the inhibitory effect of aminocoumarin during antibiotic formation. reported that the principal resistance mechanism of the novobiocin producer S. sphaeroides is the de novo synthesis of a coumarin-resistant gyrase B subunit, which replaces the sensitive GyrB subunit in the active (GyrA) 2 (GyrB) 2 heterotetramer. Thus, this novobiocin producer contains two gyrB genes, a constitutively expressed gyrB S , encoding the coumarin-sensitive protein, and the gyrB R gene, encoding the resistant protein and expressed in the presence of novobiocin. The promoter of gyrB R appears to be regulated by changes in the superhelical density of DNA (30). Mitchell et al. (20) supplied evidence that additional genes may contribute to novobiocin resistance. They used the novobiocin producer S. niveus, which has recently been identified as a subjective synonym for S. spheroides (15).We cloned and sequenced the novobiocin biosynthetic gene cluster (27), depicted in Fig. 1B. On its right border, the...

Comparative efficacy of novobiocin and amoxicillin in experimental sepsis caused by β-lactam-susceptible and highly resistant pneumococci

“…Novobiocin is an inhibitor of bacterial DNA gyrase. Like other coumarin products, it binds more tightly to gyrase (as reflected by equilibrium dissociation constants in the range of 10 nM [7]) than do typical quinolones, and novobiocin also causes inhibition of ATP binding [8]. Novobiocin was used for the treatment of a variety of infections caused by susceptible organisms, including pneumococcal pneumonia [9].…”

Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParYR and GyrBR from the coumermycin A1 producer Streptomyces rishiriensis DSM 40489

“…For selection of transformants of S. lividans T7, kanamycin (50 mg ml 21 ) was used. Transformants of S. lividans TK24 with pUWL201 expression constructs (Schmutz et al, 2003) were selected with thiostrepton (50 mg ml 21 ).…”

“…These organisms protect their own gyrase from the inhibitory effect of the aminocoumarins by de novo synthesis of an aminocoumarin-resistant gyrase B subunit during antibiotic formation (Schmutz et al, 2003;Thiara & Cundliffe, 1988. The gene encoding this subunit (named gyrB R ) was found in all three gene clusters (Schmutz et al, 2003). Interestingly, the biosynthetic gene clusters for clorobiocin and coumermycin A 1 contained an additional resistance gene (named parY R ) which showed high sequence similarity to parY of S. coelicolor (Fig.…”

“…These aminocoumarin antibiotics are potent inhibitors of the subunit B of gyrase (Maxwell, 1997), and are produced by different Streptomyces strains. These organisms protect their own gyrase from the inhibitory effect of the aminocoumarins by de novo synthesis of an aminocoumarin-resistant gyrase B subunit during antibiotic formation (Schmutz et al, 2003;Thiara & Cundliffe, 1988. The gene encoding this subunit (named gyrB R ) was found in all three gene clusters (Schmutz et al, 2003).…”

“…Recently, we have identified type II topoisomerase genes as resistance genes within the biosynthetic gene clusters of novobiocin, clorobiocin and coumermycin A 1 (Schmutz et al, 2003). These aminocoumarin antibiotics are potent inhibitors of the subunit B of gyrase (Maxwell, 1997), and are produced by different Streptomyces strains.…”