The gut-i mutant has acquired a functional transport system that allows transport of glucose, 2-deoxyglucose, and galactose, as shown by hexose competition experiments. The gut-i transport system concentrates glucose inside the cell at least 10-to 20-fold and is strongly inhibited by respiratory inhibitors, which prevent the establishment of a proton motive force, and by proton-conducting ionophores, suggesting that it is energized by a proton motive force. The new transport system is not completely sugar specific (transporting galactose and glucose through the same system), as opposed to the hexose-specific system reported in wild-type Streptomyces griseus.

A gene, pcbC, encoding the isopenicillin N synthase of Streptomyces griseus NRRL 3851, has been cloned in a 6.4-kb BgllI DNA fragment and located in an internal 1.55-kb PvuII segment by hybridization with the Penicilium chrysogenum pcbC gene. Hybridization studies revealed the presence of homologous sequences in the DNAs of several Streptomyces strains and Nocardia lactamdurans. The S. griseus pcbC gene was not expressed in Streptomyces lividans but was expressed in Streptomyces clavuligerus and complemented a mutation, nce2, that impaired isopenicillin N synthase and cephamycin biosynthesis. The pcbC gene contained an open reading frame of 990 nucleotides that encodes a protein of 329 amino acids with a deduced Mr of 37,371. The isopenicillin N synthase formed after expression of the pcbC gene in the S. clavuligerus nce2 mutant strain was found to have an Mr of 38,000 by gel filtration. A protein of about 38 kDa was observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels of extracts of a transformant of the nce2 mutant strain; this protein was absent from the untransformed mutant strain. The G+C content of the pcbC gene was 63.6%, and the strongly biased codon usage was typical of that of Streptomyces strains. A transcription initiation site was found 44 nucleotides upstream of the ATG translation initiation triplet. A transcript of 1.1 kb was observed in the donor S. griseus strain and also in the S. clavuligerus nce2 mutant strain transformed with the pcbC gene, suggesting that it is transcribed as a monocistronic mRNA. P-Lactam antibiotics are microbial metabolites that in-clude the classical penicillins and cephalosporins and the more recently discovered cephamycins, nocardicins, carbapenems, clavams, and monobactams. The biosynthetic pathways of penicillins and cephalosporins are rather well understood, since most of the enzymes involved have been characterized (24), whereas less attention has been paid to the biosynthesis of cephamycins and the newer P-lactams. Penicillins, cephalosporins, and cephamycins have in common the initial steps forming the tripeptide B(L-a-aminoadipyl)-L-cysteinyl-D-valine (ACV), mediated by the enzyme ACV synthetase, and cyclizing ACV to isopenicillin N, mediated by isopenicillin N synthase (IPNS). The latter enzyme is encoded by the gene pcbC (23). After isopenicillin N the pathway diverges in penicillin G producers (1) and in cephalosporin and cephamycin producers (23, 26).A gene (pcbC) encoding IPNS has been cloned from Penicillium chrysogenum (2, 4), Cephalosporium acremonium (Acremonium chrysogenum) (32), Aspergillus nidulans (28), and the cephamycin producers Streptomyces clavuligerus (20), Streptomyces lipmanii (38), and Streptomycesjumonjinensis (35). Cloning and characterization ofpcbC genes from eucaryotic and procaryotic P-lactam producers are of great interest as a means of understanding the divergent evolution of similar genes. In addition, knowledge of the conserved nucleotide sequences in pcbC genes from different microorganisms will be helpful...

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.