Hideyuki Muramatsu

Antibacterial discovery in actinomycetes strains with mutations in RNA polymerase or ribosomal protein S12.

We show that selection of drug-resistant bacterial mutants allows the discovery of antibacterial compounds. Mutant strains of a soil-isolated Streptomyces species that does not produce antibacterials synthesize a previously unknown class of antibacterial, which we name piperidamycin. Overall, 6% of non-Streptomyces actinomycetes species and 43% of Streptomyces species that do not produce antibacterials are activated to produce them. The antibacterial-producing mutants all carried mutations in RNA polymerase and/or the ribosomal protein S12.

FR131535, a novel water-soluble echinocandin-like lipopeptide: synthesis and biological properties

The synthesis and biological properties of a novel water-soluble echinocandin-like lipopeptide, FR131535, are described. This compound displayed potent in vitro and in vivo antifungal activities. The hemolytic activity of FR901379 was reduced by replacing the acyl side chain. This compound showed good water-solubility, comparable to the natural product FR901379.

Vulgatibacter incomptus gen. nov., sp. nov. and Labilithrix luteola gen. nov., sp. nov., two myxobacteria isolated from soil in Yakushima Island, and the description of Vulgatibacteraceae fam. nov., Labilitrichaceae fam. nov. and Anaeromyxobacteraceae fam. nov.

Two myxobacterial strains (designated B00001(T) and B00002(T)) were isolated from forest soil samples collected from Yakushima Island, Kagoshima, Japan. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains B00001(T) and B00002(T) respectively formed independent branches within the suborders Cystobacterineae and Sorangiineae and were most closely related to Cystobacter armeniaca DSM 14710(T) (90.4% similarity) and Byssovorax cruenta DSM 14553(T) (91.3%). Neither strain showed typical features of myxobacteria such as bacteriolytic action or fruiting body formation, but both had high DNA G+C contents (66.3-68.3 mol%). Swarming motility was observed in strain B00002(T) only. Cells of both strains were vegetative, chemoheterotrophic, mesophilic, strictly aerobic, Gram-negative, motile rods, and both strains exhibited esterase lipase (C8), leucine arylamidase, naphthol-AS-BI-phosphohydrolase and β-galactosidase activities. Strain B00001(T) contained MK-7 as the predominant respiratory quinone and the major fatty acid was iso-C15:0. In contrast, strain B00002(T) contained MK-8 as the major cellular quinone and the major fatty acids were C16 : 1ω5c and iso-C17 : 0. Based on the phenotypic and genotypic data presented, strains B00001(T) and B00002(T) represent novel genera and species, for which we propose the names Vulgatibacter incomptus gen. nov., sp. nov. and Labilithrix luteola gen. nov., sp. nov., respectively. The type strains of Vulgatibacter incomptus and Labilithrix luteola are B00001(T) ( = NBRC 109945(T) = DSM 27710(T)) and B00002(T) ( = NBRC 109946(T) = DSM 27648(T)), respectively. The new genera are assigned to the new families Vulgatibacteraceae fam. nov. and Labilitrichaceae fam. nov., respectively. In addition, Anaeromyxobacteraceae fam. nov., is proposed to accommodate the genus Anaeromyxobacter, which is related to the genus Vulgatibacter.

Streptomyces tsukubensis sp. nov., a producer of the immunosuppressant tacrolimus.

Streptomyces is well-known as a good producer of pharmaceutically important chemical compounds, not only antibiotics but also physiologically active compounds. Tacrolimus was found as a strong immunosuppressant from a culture broth of an actinomycete strain, ‘Streptomyces tsukubaensis’ 9993T, which was isolated from a soil sample collected from Mount Tsukuba, Ibaraki, Japan, in 1984. The first report of this compound was published in 1987,1 and the drug was launched in 1993 in Japan as an immunosuppressive agent for the prevention of graft rejection after liver transplantation. To date, it has been sold in about 90 countries in several therapeutic areas, including organ transplantation, myasthenia gravis, articular rheumatism, lupus nephritis, ulcerative colitis and atopic dermatitis, among others. After initial discovery of the compound, several taxonomically diverse streptomycete strains were reported as tacrolimus producers, including S. tacrolimicus ATCC 55098T,2 S. clavuligerus CKD 11193 and S. kanamyceticus KCC S-0433T.4 Further, biosynthetic gene clusters of tacrolimus in some strains have been reported.5–7 To date, however, no effective taxonomic description of the species ‘S. tsukubaensis’ has been available. Clarification of the taxonomic position of strain 9993T is important not only for streptomycete taxonomy, but also for evolutionary studies of secondary metabolite biosynthesis gene clusters. We previously revealed that strain 9993T does not belong to any described species according to its 16S rRNA gene sequence phylogeny.4 We therefore investigated its morphological and physiological characteristics and phylogeny to confirm its taxonomic position. Here, we report this taxonomic study of strain 9993T and propose S. tsukubensis sp. nov. Strain 9993T, the original strain of ‘S. tsukubaensis’, was isolated from a soil sample collected from Mount Tsukuba, Ibaraki, Japan in 1984,1 and has been stored as a lyophilized ampule for 27 years. The suspension of lyophilized spores was inoculated on inorganic salts–starch agar (ISP medium 4) and incubated at 28 1C for 1–2 weeks. The culture was stored at 4 1C during this study. S. clavuligerus NBRC 13307T was obtained from Biological Resource Center, National Institute of Technology and Evaluation, Japan. Media preparations, morphological observations and carbon sources utilization tests were performed by the methods of Shirling and Gottlieb.8 For determination of morphological characteristics, the strain was incubated at 28 1C for 14 days, then morphologically examined using light and scanning electron microscopy (Hitachi S-2600N, Hitachi High-Technologies Corp., Tokyo, Japan). Color determinations were referenced against the Methuen Handbook of Color (Methuen London Ltd., London, UK). Growth temperature was determined on ISP medium 4 at 5, 13, 15, 20, 25, 28, 30, 32, 34, 35, 36, 37 and 38 1C, while tolerance to sodium chloride was determined on yeast extract-malt extract agar (ISP medium 2) with 0, 4, 7, 10 and 13% of sodium chloride. Amino acids in cell wall hydrolysates, cellular fatty acids, menaquinones and GþC content of the genomic DNA and DNA–DNA reassociation value were determined at TechnoSuruga Laboratory Co., Ltd. (Shizuoka, Japan). Amino acids, menaquinones and GþC content were analyzed by high-performance liquid chromatography. Cellular fatty acids were analyzed using the standard MIDI system. Whole-cell sugar composition was determined by Thin Layer Chromatography, following the method of Hasegawa et al.9 Polar lipids were analyzed by two-dimensional Thin Layer Chromatography method.10 The 16S rRNA gene sequence of the strain was determined in our previous study (Accession no.; AB217600).4 Homology search was performed using the FASTA program.11 DNA sequence data was downloaded from the NCBI web site (http://www.ncbi.nlm.nih.gov/). BLAST search12 was performed at the DDBJ web site (http:// www.ddbj.nig.ac.jp/). Validity of bacterial names was referenced against the List of Prokaryotic Names with Standing in Nomenclature (http://www.bacterio.cict.fr/). The phylogenetic trees were constructed with 68 closest valid species using the neighborjoining13 or maximum-parsimony methods14 in CLUSTAL X15 or MEGA package.16 Strain 9993T grew well on ISP medium 4 and ISP medium 2. The color of the substrate mycelium was pinkish white to grayish orange. The strain produced a grayish aerial mycelium abundantly on ISP medium 4, and moderately on ISP medium 2. It formed flexuous

Bioconversion of AS1387392: screening and characterization of actinomycetes that convert AS1387392 to AS1429716.

AS1387392 was a novel and powerful histone deacetylase inhibitor with an excellent oral absorption profile, but this compound was lacking in active moieties, which are essential to synthesize more derivatives. In our screening program to identify actinomycetes capable of converting AS1387392 to AS1429716, which has an active moiety to synthesize more derivatives, we identified 12 strains capable of efficient hydroxylation. Results of phylogenetic analysis of 16S rDNA sequences suggested that these strains belonged to the genera Lentzea, Saccharopolyspora, Sphaerisporangium and Amycolatopsis. Morphological and chemical characteristics as well as results of phylogenetic analysis suggested that strain No. 7980 was a new species belonging to the genus Amycolatopsis, according to the FASTA search result of 16S rDNA gene sequence. Using these strains, we can easily produce AS1429716 as a chemical template for further chemical modifications, which may provide more effective and safer immunosuppressant.

Bioconversion of FR901459, a novel derivative of cyclosporin A, by Lentzea sp. 7887.

FR901459, a product of the fungus Stachybotrys chartarum No. 19392, is a derivative of cyclosporin A (CsA) and a powerful immunosuppressant that binds cyclophilin. Recently, it was reported that CsA was effective against hepatitis C virus (HCV). However, FR901459 lacks active moieties, which are essential for synthesizing more potent and safer derivatives of this anti-HCV agent. Here we identified an actinomycete strain (designated 7887) that was capable of efficient bioconversion of FR901459. Structural elucidation of the isolated bioconversion products (1–7) revealed that compounds 1–4 were mono-hydroxylated at the position of 1-MeBmt or 9-MeLeu, whereas compounds 5–7 were bis-hydroxylated at both positions. The results of morphological and chemical characterization, as well as phylogenetic analysis of 16S ribosomal DNA (rDNA), suggested that strain 7887 belonged to the genus Lentzea. Comparison of the FR901459 conversion activity of strain 7887 with several other Lentzea strains revealed that although all examined strains metabolized FR901459, strain 7887 had a characteristic profile with respect to bioconversion products. Taken together, these findings suggest that strain 7887 can be used to derivative FR901459 to produce a chemical template for further chemical modifications that may provide more effective and safer anti-HCV drugs.

Phylogenetic diversity of acidophilic actinomycetes from Malaysia

Actinomycetes produce various physiologically active compounds, including antibiotics. Although the discovery of new active compounds has slowed recently, some novel compounds from non-soil borne actinomycetes; for example, marine actinomycetes, have been reported.1 Thus, discoveries of new taxa will expand the diversity of resources available for screening programs. Basilio et al.2 reported that actinomycetes isolated in alternative selections of pH and salinity present a significant capacity to produce compounds with antimicrobial activity. Like other actinomycetes, marine actinomycetes and other rarely targeted strains, for example, acidophilic actinomycetes, are expected to produce a diverse array of active compounds.

Naphthalecin, a Novel Antibiotic Produced by the Anaerobic Bacterium, Sporotalea colonica sp. nov.

A novel antibiotic naphthalecin was purified and isolated from the cells of an anaerobic bacterium isolated from a soil sample. This antibiotic contained a naphthalene moiety, so named as naphthalecin, and showed antibacterial activity against gram positive species. The producing strain, an obligate anaerobe, was identified as a new species of the genus Sporotalea. Identification of the bacterium, cultivation, purification, structure determination, and antibacterial activity are shown.

KB425796-A, a novel antifungal antibiotic produced by Paenibacillus sp. 530603

The novel antifungal macrocyclic lipopeptidolactone, KB425796-A (1), was isolated from the fermentation broth of bacterial strain 530603, which was identified as a new Paenibacillus species based on morphological and physiological characteristics, and 16S rRNA sequences. KB425796-A (1) was isolated as white powder by solvent extraction, HP-20 and ODS-B column chromatography, and lyophilization, and was determined to have the molecular formula C79H115N19O18. KB425796-A (1) showed antifungal activities against Aspergillus fumigatus and the micafungin-resistant infectious fungi Trichosporon asahii, Rhizopus oryzae, Pseudallescheria boydii and Cryptococcus neoformans.

Bioconversion of AS1387392: bioconversion studies involving Amycolatopsis azurea JCM 3275

We screened actinomycetes capable of converting AS1387392 to AS1429716 and identified those strains capable of hydroxylation. Amycolatopsis azurea JCM 3275 was found to be a particularly efficient strain, capable of converting AS1387392 to AS1429716, with a yield of 44% after 9 h. This strain can metabolize not only the hydroxylation of phenylalanine at the meta and para positions but also the reduction of hydroxyketones, as shown by the isolation of bioconversion products. Examination of more suitable conversion conditions showed that pH 7.8 and 25 °C were the optimum pH and temperature for bioconversion, respectively. We also demonstrated the effect of carbon and nitrogen sources in the culture media on hydroxylation. Using this strain, we were able to efficiently produce AS1429716 as a chemical template. Further derivatization studies may provide more effective, safer immunosuppressants than those that are currently on-market.