Proteome analysis of the membrane proteins from Streptomyces coelicolor A3 (2) was carried out. By two-dimensional polyacrylamide gel electrophoresis, over 100 protein spots were detected by Coomassie staining. Among them, nine proteins gave clear amino acid sequences after digestion with trypsin or lysylendopeptidase followed by the separation on HPLC and sequence analyses. Homology analyses of the peptides indicate that protein numbers 1, 2, 3, 4, 5 and 6 are AtpD, AtpA, GroEL2, GroELl, BldKB and BldKB, respectively. Proteins 5 and 6 are supposed to the same proteins with different modification or isoproteins. However, sequence analyses of the peptides from protein numbers 7, 8, and 9 gave no information about homologous proteins. It is suggested, therefore, that some major proteins in a large amount of protein in the membrane have their characteristic functions in the membrane of Streptomyces coelicolor A3(2), which are not known in other eubacteria.

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Fosfomycin (FM, Fig. 1) is a medically important antibiotic produced by various species of Streptomyces¹⁾ as well as Pseudomonas syringae PB-5123²⁾ and Ps. viridiflava PK-5³⁾. FM is characterized by unique structural functions such as a carbon-phosphorus (C-P) bond and an epoxide⁴⁾. Acting as an analog of phosphoenolpyruvate, it irreversibly inhibits phosphoenolpyruvate UDP-N-acetylglucosamine-3-O-enolpyruvyltransferase (enolpyruvyltransferase), the enzyme that catalyzes the first step of peptidoglycan biosynthesis, and shows almost no toxicity to humans⁵⁾. We review here FM biosynthesis and self-resistance, and the biosynthetic genes in S. wedmorensis.

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Special attention has been paid on actinomycetes because they produce various beneficial compounds including antibiotics. Recently, it is revealed that not only morphology but also physiology of actinomycetes are close to those of fungi. Actinomycetes may be a missing link between prokaryotes and eukaryotes. In this paper, some examples exhibiting that actinomycetes are more close to eukaryotes than previously concerned are described based on their secondary metabolites.

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Isolation method for the purpose of obtaining kinds of actinomycetes in plant leaves has been devised. All isolates were classified into genus based on their taxonomical characteristics. They included not only the genus Streptomyces but also the rare genera. In particular, strains belonging to the genus Microbispora represented more than 50% of the total share belonging to the rare isolates¹⁾. Two strains of isolates produced new herbicidal antibiotics, herbicidin H produced by Streptomyces sp. SANK 63997 and γ-glutamylmethionine sulfoximine by Microbispora sp. SANK 62597.

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Our recent search for bioactive compounds from pathogenic Nocardia resulted in the isolation of a new antibiotic, brasilicardin A, from Nocardia brasiliensis IFM 0406, a clinical isolate from a lung nocardiosis patient. Brasilicardin A is a novel tricyclic diterpenoid containing an amino acid moiety, 3-hydroxybenzoate, a rhamnose and an N-acetylglucosamine unit. The antibiotic exhibited immunosuppressive activity in a mouse mixed lymphocyte reaction (MLR) assay system and its IC₅₀ value was 0.057 μg/ml. Its immunosuppressive mechanism was considered to be different from that of the reference drug, cyclosporin A (CyA), because brasilicardin A has no inhibitory activity against IL-2 production in mouse MLR assay. Also new bioactive substances which were recently isolated by our research groups from pathogenic Nocardia are reviewed.

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Benzoisochromanequinone (BIQ) antibiotics are a class of aromatic polyketides produced by Streptomyces spp. A polyketide synthase (PKS, Type II) is involved in the formation of each BIQ chromophore. The first PKS genes were cloned in S. coelicolor A3(2), producer of the typical BIQ antibiotic, actinorhodin; sequence analysis of the PKS genes revealed their clustering with all the other relevant biosynthetic genes (the act cluster). A number of PKS genes have been discovered in the producers not only of other BIQs, but also of other classes of aromatic polyketides, using the act PKS genes as hybridisation probes. Among them similar genetic organisations were identified for the genes encoding the minimal PKS components -KS (ketosynthase), CLF (chain length factor), ACP (acyl carrier protein)- and their closely associated proteins: KR (ketoreductase), ARO (aromatase), and CYC (cyclase). In spite of the increasing knowledge of PKS genes themselves, various biosynthetic problems remain to be solved. As one of the most extensively studied examples at the genetic level, BIQ antibiotics are useful model compounds to be studied to understand the whole biosynthetic pathways of aromatic polyketides. This review describes the genes from the act and gra (granaticin biosynthetic gene cluster in S. violaceoruber Tü22) clusters involved in post-PKS modifying (tailoring) steps to complete BIQ biosynthesis.

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Liposidomycins are atypical lipid-bearing nucleoside antibiotics that inhibit bacterial peptidoglycan synthesis. A producing strain was identified as a Streptomyces sp. from its cultural characteristics and physiological properties. It produced new types of liposidomycins that lacked sulfate and/or 3-methylglutaric acid moieties present in known liposidomycins by changing medium components. Liposidomycins which have no sulfate moiety, exhibited more potent antimicrobial activity. Thus liposidomycins are the unique antibiotics showing highly specific inhibition toward bacterial peptidoglycan synthesis and could be a useful clinical antibiotic agent.

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Leptomycin B (LMB) is a Streptomyces metabolite that causes specific inhibition of the cell cycle of yeast and mammalian cells. The target molecule of LMB in fission yeast has been shown by genetic analysis to be CRM1, a highly conserved protein in eukaryotes. We isolated a human homolog of CRM1 (hCRM1) which was expressed ubiquitously in human tissues and showed that hCRM1 was essential for nuclear export of proteins. CRM1 was shown to be a receptor for the nuclear export signal (NES) of proteins in both yeast and mammalian cells. LMB binds directly to CRM1, which resulted in dissociation of the NES from the nuclear export machinery containing CRM1. Thus, LMB serves as a potent tool for understanding the molecular mechanisms of nucleo-cytoplasmic transport of proteins.

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Bacteria possess arrays of efflux systems that confer resistance to a wide variety of toxic compounds. Several of the multidrug resistance (MDR) systems exist in Streptomyces, but are normally under tight regulation. Therefore, drug resistant mutants of Streptomyces arise only through mutations that derepress the MDR genes. This enigma supports the notion that the tested drugs may only be fortuitous substrates of the MDR systems, of which the normal physiological targets may be something else.

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