A-500359s, produced by Streptomyces griseus SANK60196, are inhibitors of bacterial phospho-N-acetylmuramyl-pentapeptide translocase. They are composed of three distinct moieties: a 5¢-carbamoyl uridine, an unsaturated hexuronic acid and an aminocaprolactam. Two contiguous cosmids covering a 65-kb region of DNA and encoding 38 open reading frames (ORFs) putatively involved in the biosynthesis of A-500359s were identified. Reverse transcriptase PCR showed that most of the 38 ORFs are highly expressed during A-500359s production, but mutants that do not produce A-500359s did not express these same ORFs. Furthermore, orf21, encoding a putative aminoglycoside 3¢-phosphotransferase, was heterologously expressed in Escherichia coli and Streptomyces albus, yielding strains having selective resistance against A-500359B, suggesting that ORF21 phosphorylates the unsaturated hexuronic acid as a mechanism of self-resistance to A-500359s. In total, the data suggest that the cloned region is involved in the resistance, regulation and biosynthesis of A-500359s.

The nucleoside antibiotic A-500359s are produced by Streptomyces griseus SANK 60196. During a screening program of A-500359s high-producing strains, several interesting mutants were isolated and classified into three groups according to two characteristics, spore-forming ability and the retention of a giant linear plasmid SGF180, as follows: , . A-500359s production was markedly decreased in all the mutants and completely lacking in all the baldtype mutants. To understand the regulatory mechanisms of A-500359s production in these mutants, co-cultivation analyses were conducted in several mutant combinations, and the effect of an addition of an EtOAc extract, which was prepared from a culture broth of an A-500359s producer, was tested. A-500359s production was clearly restored when mutant [spore+ SGF180-] was co-cultivated with mutant [bald SGF180+] and the A-500359s production of the mutant [bald SGF180+] was activated by the addition of an EtOAc extract. The results suggested that SGF180 plays an important role in A-500359s production and that A-500359s biosynthesis might be controlled by a low molecular weight compound, such as an A-factor-like compound, synthesized by the product of afsA gene that was lost in all the tested bald-type mutants.A-500359s were discovered in a culture broth of Streptomyces griseus SANK 60196 as members of the translocase I inhibitors (Muramatsu et al., 2003a). Translocase I is an enzyme that catalyzes the first step in the lipid cycle of peptidoglycan biosynthesis. Peptidoglycan is essential and specific for bacteria; therefore, an antibiotic with translocase I inhibitory activity was expected to be appropriate for therapeutic use, especially against Staphylococcus aureus, Pseudomonas aeruginosa, and Mycobacterium tuberculosis. So far, eight derivatives of A-500359s have been isolated and reported by Muramatsu et al. (2003aMuramatsu et al. ( , 2003b. A-500359s mainly comprise three parts, including uridine, sugar, and a caprolactam ring. The origin of the A-500359s structures was determined and reported by Ohnuki et al. (2003). In addition, culture conditions and media compositions suitable for their production were studied with a view toward commercialization (Muramatsu et al., 2006). During a screening program of A-500359s high-producing strains, a high producer, named strain HP, was isolated by random mutation. Strain HP was treated at a high temperature for further screening to isolate higher producing strains. Strain HP was cultured in Trypto Soy Broth (TSB) medium for seven days at 35, 37, or 39 C. After several rounds of high temperature treatment, six strains (strains 35-1, 35-2, 37-2, 37-3, 37-4, and 37-6) were isolated as spore-forming strains and four mutants (mutants 35-4, 37-9, 39-5 and 39-6) were isolated as bald-type mutants. These isolated strains were cultured in a test tube containing 5 ml of PM-1 medium (Muramatsu et al., 2006) for three days at 23 C. One ml of the resulting culture broth was transferred into a 100 ml Erlenmeyer flask containing 20 ml of OM-1 m...