Abstract:
The aminocoumarin antibiotics like novobiocin, clorobiocin and coumermycin A1 are produced by various Streptomyces strains. Novobiocin has been licensed in the USA for the treatment of serious infections especially with multiresistant Staphylococci. Aminocoumarins are inhibitors of the bacterial DNA gyrase by interaction with the B subunit of this tetrameric enzyme consisting of two A and two B subunits.
The first task of my thesis was to identify putative resistance genes of the aminocoumarin producers, and heterologous expression of the respective genes. The biosynthetic gene clusters of novobiocin, clorobiocin and coumermycin A1 were investigated. All three clusters contained a resistance gene gyrBR, coding for a gyrase B subunit. The clusters of clorobiocin and coumermycin A1 were found to contain an additional, similar gene named parYR. Its predicted gene product showed sequence similarity with the B subunit of type II topoisomerases.
Expression of gyrBR in Streptomyces lividans TK24 resulted in high resistance against novobiocin and coumermycin A1. The minimal inhibitory concentration (MIC) for transformants with gyrBR was greater than 750 µg/ml novobiocin (wild type 50-100 µg/ml) and 500 µg/ml coumermycin A1 (wild type 50 µ/ml). Resistance conferred by parYR was slightly lower and MICs were 500 µg/ml novobiocin and 300 µg/ml coumermycin A1, respectively.
Southern hybridization experiments showed that the genome of all three antibiotic producers, and of Streptomyces coelicolor, contained two additional genes, which hybridized with either gyrBR or parYR, 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 A1 cluster, respectively. Expression of these genes in S. lividans TK24 resulted in moderate levels of resistance against novobiocin (MIC novA: 250-500 µg/ml; couR5: 250 µg/ml) and coumermycin A1 (MIC novA: 250 µg/ml; couR5: 250 µg/ml), suggesting that these genes may be involved in antibiotic transport.
GyrBR and ParYR, as well as the corresponding A subunits GyrA and ParX of S. coelicolor were overexpressed as hexahistidine fusion proteins in S. lividans T7 and purified by metal affinity chromatography. Reconstitution of the holo enzymes was achieved by mixing the corresponding subunits in vitro. The complex of GyrA and GyrBR was found to catalyze ATP-dependent supercoiling of DNA, i.e. to function as gyrase. No relaxation and decatenation activity was detected. The complex of ParX and ParYR catalyzed ATP-dependent decatenation and relaxation, i.e. the functions of topoisomerase IV (topo IV). This represents the first topo IV identified in the class of actinobacteria. At the same time, it is the first demonstration of the formation of a topo IV as a resistance mechanism of an antibiotic producer, underlining the role of topo IV as target for aminocoumarin antibiotics. The organization of parX and parY in actinobacteria which contain a topo IV is different from the organization of parC and parE on other Gram-positive bacteria.
The task of the second part of my thesis was to investigate the formation of the two amide bonds throughout the biosynthesis of coumermycin A1. The biosynthetic gene cluster of coumermycin contains a putative amide synthetase gene, couL, encoding a protein of 529 amino acids. CouL was overexpressed as hexahistidine fusion protein in E. coli and purified by metal affinity chromatography, resulting in a nearly homogenous protein. CouL catalyzed the formation of both amide bonds of coumermycin A1, i.e. between the central 3-methylpyrrole-2,4-dicarboxylic acid and two aminocoumarin moieties. Gel exclusion chromatography showed that the enzyme is active as a monomer with a molecular weight of 59-60 kDa (calculated 58.9 kDa). The activity was strictly dependent on the presence of ATP and Mn2+ or Mg2+. The apparent Km values were determined as 26 µM for the 3-methylpyrrole-2,4-dicarboxylic acid and 44 µM for the aminocoumarin moiety, respectively. Several analogues of the pyrrole dicarboxylic acid were accepted as substrates. In contrast, pyridine carboxylic acids were not accepted. 3-Dimethylallyl-4-hydroxybenzoic acid, the acyl component in novobiocin biosynthesis, was well accepted, despite its structural difference to the genuine acyl substrate of CouL.