Cloning and characterization of a DNA gyrase B gene from Streptomyces sphaeroides that confers resistance to novobiocin.

Abstract

A gyrB gene from Streptomyces sphaeroides, a producer of novobiocin, has been cloned in Streptomyces lividans, where it conferred resistance to novobiocin. The Streptomyces gyrB gene was sufficiently similar to a Bacillus subtilis gyrB probe to be specifically recognized during Southern analysis. Partial purification of DNA gyrase by affinity chromatography revealed the presence of two such activities (differing in their responses to novobiocin) in the clone. The product of the cloned gene, a novobiocin‐resistant DNA gyrase B subunit, was identified in vitro by coupled transcription–translation as a 79‐kd protein.

Andrimid producers encode an acetyl-CoA carboxyltransferase subunit resistant to the action of the antibiotic

“…Although the majority of the self-protective strategies exploited by antibiotic-producing microbes involve intracellular antibiotic modification, cellular target modification, and antibiotic efflux (20)(21)(22), several antibiotic producers have been known to encode antibiotic-resistant target enzymes that contribute to their survival. The bacterial producers of DNA gyrase inhibitors such as aminocoumarins and albicidins are known to encode DNA gyrase-resistant genes that contribute to their self-protection (23,(24)(25)(26), although the molecular mechanism underlying these resistances is still unknown. The D-cycloserine producer Streptomyces lavendulae has been shown to encode an antibiotic-resistant alanine racemase (ALR) that manifests a slower enzymatic conversion to the final cycloserine pyridoxal derivatives that inhibit the catalytic activity of ALR (27,28).…”

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

“…GyrB R and ParY R were first expressed in S. lividans TK24, using the Streptomyces expression vector pUWL201. Affinity chromatography on novobiocinSepharose (Staudenbauer & Orr, 1981;Thiara & Cundliffe, 1988) allowed the purification of the genuine aminocoumarin-sensitive GyrB protein from S. lividans, which could be reconstituted with GyrA from the same organism to yield active gyrase. However, the aminocoumarinresistant GyrB R and ParY R proteins did not bind to the affinity column and apparently eluted with the bulk of the proteins.…”

“…A gyrB R gene is also found in the biosynthetic gene cluster of novobiocin (Steffensky et al, 2000), and the function of this gene and the regulation of its expression has been investigated by Thiara & Cundliffe (1988. The fact that the coumermycin cluster contains not only a gyrB R gene but also a parY R gene (which is absent in the novobiocin cluster) is consistent with the very high affinity of coumermycin A 1 both for gyrase and for topo IV (Peng & Marians, 1993), creating the need to efficiently protect both enzymes in the coumermycin producer.…”

“…This was now investigated in vitro. The gyrase of S. lividans is highly sensitive to novobiocin, with an IC 50 value of approximately 0?5 mg ml 21 (Thiara & Cundliffe, 1988), very similar to the IC 50 of E. coli gyrase, which was determined as approximately 0?6 mg ml 21 (Peng & Marians, 1993). In contrast, we observed an IC 50 of 50 mg ml 21 for the complex of GyrA and GyrB R .…”

“…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).…”