We used a series of gene disruptions and gene replacements to mutagenically characterize 30 kilobases of DNA in the erythromycin resistance gene (ermE) region of the Saccharopolyspora erythraea chromosome. Five previously undiscovered loci involved in the biosynthesis of erythromycin were found, eryBI, eryBII, eryCl, eryCII, and eryH; and three known loci, eryAl, eryG, and ermE, were -further characterized. The new Ery phenotype, EryH, was marked by (i) the accumulation of the intermediate 6-deoxyerythronolide B (DEB), suggesting a defect in the operation of the C-6 hydroxylase system, and (ii) a block in the synthesis or addition reactions for the first sugar group. Analyses of ermE mutants indicated that erinE is the only gene required for resistance to erythromycin, and that it is not required for production of the intermediate erythronolide B (EB) or for conversion of the intermediate 3-a-mycarosyl erythronolide B (MEB) to erythromycin. Mutations in the eryB and eryC loci were similar to previously reported chemically induced eryB and eryC mutations blocking synthesis or attachment of the two erythromycin sugar groups. Insertion mutations in eryAI, the macrolactone synthetase, defined the largest (at least 9-kilobase) transcription unit of the cluster. These mutants help to define the physical organization of the erythromycin gene cluster, and the eryH mutants provide a source for the production of the intermediate DEB.Erythormycin A is a medically important macrolide antibiotic produced by the gram-positive, sporeforming bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus NRRL 2338) (29). As is the case for other antibiotic-producing organisms, the genes for the biosynthesis of erythromycin (ery) are thought to be clustered about the gene for resistance to erythromycin, ermE (22,27,29); however, much remains to be learned about the organization and location of the many individual genes predicted to be involved in the pathway (6, 18).The ermE gene was originally cloned by Thompson et al. (24) and later sequenced by Uchiyama and Weisblum (25); its promoter and the promoter of an adjacent upstream open reading frame were characterized by Bibb et al. (3,4). The adjacent open reading frame has since been sequenced by Dhillon et al. (7), inactivated by gene disruption, and found to be involved in erythromycin biosynthesis as an eryC-type gene. Independently, the open reading frame was shown by Vara et al. (25a) to be an eryC-type gene through complementation of the eryC160 allele, and we obtained the same results as Dhillion et al. (7) by using linker insertion mutagenesis (see below). Other landmarks in the ery gene cluster are (i) eryG, the O-methyltransferase located 6.5 kilobase pairs (kb) downstream of the ermE promoter region (1Sa, 28); (ii) eryAI, the macrolactone synthetase, a large locus beginning 3 kp upstream of eryG (J. Tuan, J. M. Weber, M. Staver, J. 0. Leung, and L. Katz, Gene, in press); and (iii) the eryB25, eryB26, and eryD24 genes, mapped within an 18-kb region upstream o...
A mutant strain derived by chemical mutagenesis of Saccharopolyspora erythraea (formerly known as Streptomyces erythreus) was isolated that accumulated erythromycin C and, to a lesser extent, its precursor, erythromycin D, with little or no production of erythromycin A or erythromycin B (the 3"-O-methylation products of erythromycin C and D, respectively). This mutant lacked detectable erythromycin O-methyltransferase activity with erythromycin C, erythromycin D, or the analogs 2-norerythromycin C and 2-norerythromycin D as substrates. A 4.5-kilobase DNA fragment from S. erythraea originating approximately 5 kilobases from the erythromycin resistance gene ermE was identified that regenerated the parental phenotype and restored erythromycin O-methyltransferase activity when transformed into the erythromycin O-methyltransferase-negative mutant. Erythromycin O-methyltransferase activity was detected when the 4.5-kiobase fragment was fused to the lacZ promoter and introduced into Escherichia coli. The activity was dependent on the orientation of the DNA relative to lacZ. We have designated this genotype eryG in agreement with Weber et al. (J. M. Weber, B. Schoner, and R. Losick, Gene 75:235-241, 1989). It thus appears that a single enzyme catalyzes all of the 3"-O-methylation reactions of the erythromycin biosynthetic pathway in S. erythraea and that eryG codes for the structural gene of this enzyme.
The number and properties of carbamyl phosphate synthetases in Bacillus subtilis have been uncertain because of conflicting genetic results and instability of the enzyme in extracts. The discovery of a previously unrecognized requirement of B. subtilis carbamyl phosphate synthetases for a high concentration of potassium ions for activity and stability permitted unequivocal demonstration that this bacterium elaborates two carbamyl phosphate synthetases. Carbamyl phosphate synthetase A was shown to be repressed by arginine, to have a molecular weight of about 200,000, and to be coded for by a gene that maps near argC4. This isozyme was insensitive to metabolites of the arginine and pyrimidine biosynthetic pathways. Carbamyl phosphate synthetase P was found to be repressed by uracil, to have a molecular weight of 90,000 to 100,000, and to be coded for by a gene that maps near the other pyr genes. This isozyme was activated by phosphoribosylpyrophosphate and guanine nucleotides and was strongly inhibited by uridine nucleotides. Other kinetic properties of the two isozymes were compared. Bacillus thus resembles eucaryotic microbes in producing two carbamyl phosphate synthetases, rather than the enteric bacteria, which produce a single carbamyl phosphate synthetase.
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