Structural features of cell wall teichoic acid and peptidoglycan of Actinomadura cremea INA 292.

The teichoic acid from the cell wall of Actinomadura cremea INA 292 has an unusual structure, being a poly(galactosylglycerol phosphate) chain with glycerol phosphate groups. Monomeric units of 1-O, beta-D-galactopyranosylglycerol monophosphate are joined in the polymer by phosphodiester links involving the glycerol C3 and the galactose C6 atoms. Approximately every second galactosyl substituent has a glycerol phosphate residue at its C3 atom. The teichoic acid structure was established by chemical analysis and 13C-NMR spectroscopy. There also is a peptidoglycan belonging to the A1 gamma type: as well as meso-2,6-diaminopimelic acid it contains small amounts of the LL form and glycine.

Kedarcidin, a new chromoprotein antitumor antibiotic. I. Taxonomy of producing organism, fermentation and biological activity.

Strain L585-6 (ATCC 53650) is an actinomycete isolated from a soil sample collected in Maharastra State, India. It produces a new chromoprotein antitumor antibiotic, designated kedarcidin. Taxonomic studies demonstrated that strain L585-6 is an unidentified and unknown actinomycete. Kedarcidin shows potent antitumor activity against implanted P388 leukemia (3.3 micrograms/ml/kg) and B16 melanoma (2 micrograms/kg) in mice. Kedarcidin also shows potent antimicrobial activity against Gram-positive bacteria but no activity against Gram-negative bacteria.

Tetrazomine, a new antibiotic produced by an actinomycete strain. Taxonomy, fermentation, isolation and characterization.

A new antibacterial antibiotic tetrazomine was found from the fermentation broth of an actinomycete strain which was isolated from beach sand collected at Chichijima, Ogasawara Islands, Tokyo, Japan. The strain Y-09194L, was identified as Saccharothrix mutabilis subsp. chichijimaensis subsp. nov. The antibiotic exhibited broad antimicrobial activity against Gram-positive and Gram-negative bacteria in vitro. It also exhibited strong cytotoxic activity against P388 leukemia cells and showed antitumor activity against P388 leukemia. The apparent molecular formula of tetrazomine was determined as C24H34N4O5. It has a rare structure which consists of six rings including piperidine, piperadine, oxazole, and pyrrolidine.

Polyacrylamide gel electrophoresis analysis of ribosomal protein AT-L30 as a novel approach to actinomycete taxonomy: application to the genera Actinomadura and Microtetraspora.

Actinomycete ribosomal protein AT-L30 exhibits electrophoretic mobility that is specific for each genus. On the basis of this fact, we analyzed ribosomal AT-L30 proteins from 26 type strains of species belonging to the genera Actinomadura and Microtetraspora. The electrophoretic mobilities of AT-L30 preparations from these strains, as determined by two-dimensional polyacrylamide gel electrophoresis, revealed that they could be divided into two groups, one group with relative electrophoretic mobilities of 14.0 to 41.5 and another group with relative electrophoretic mobilities of -6.5 to 0. The first group corresponded to the genus Actinomadura, and the second group corresponded to the genus Microtetraspora. Partial amino acid sequencing of AT-L30 preparations from several strains proved that we were indeed dealing with the specified protein homologous to ribosomal protein L30 of Escherichia coli. Our results strongly supported the conclusions of previous work and thus proved the efficacy of ribosomal protein analysis as a novel approach for taxonomy of actinomycetes.

Isolation and structure of the lipid envelopes from the nitrogen-fixing vesicles of Frankia sp. strain CpI1.

Frankia vesicles are differentiated during nitrogen starvation; they contain nitrogenase whether produced by free-living frankiae or by frankiae in actinorhizal root nodules. Vesicles are surrounded by envelopes of several monolayers of uncharacterized lipid. It has been suggested that the envelope limits diffusion of O2 into the vesicle cytoplasm, thereby preventing inactivation of nitrogenase. Whole vesicles were prepared on sucrose gradients and sonicated, and vesicle envelopes were isolated on top of a cushion of 40% sucrose. Transmission electron microscopy of potassium permanganate-fixed envelopes confirmed the purity of these preparations. Only the outer and inner envelope layers were visible in permanganate-fixed intact vesicles; the laminae were not visible in aldehyde-osmium-fixed, lead citrate-uranyl acetate-stained whole vesicles. However, the laminated nature of the envelope was clearly evident in sonicated vesicles and in envelope fragments fixed with KMnO4. The observations indicate that partial disruption of the vesicle envelope enables its visualization with permanganate fixation, and these observations open the way for further studies on the relationship of the vesicle surface to environmental conditions.

Menaquinone patterns of Amycolatopsis species.

The menaquinones of representative strains of the actinomycete genus Amycolatopsis were examined by reverse phase thin-layer chromatography and mass spectrometry. Representatives of all seven validly described species contained various combinations of di-, tetra- and hexahydrogenated menaquinones with nine isoprene units as predominant isoprenologues. It seems likely that the variation in the predominant menaquinones merely reflects the stages from the growth cycle from which biomass was taken. The detection of major proportions of hydrogenated menaquinones with nine isoprene units serves to distinguish Amycolatopsis strains from most other actinomycetes, notably those belonging to related genera such as Amycolata and Pseudonocardia.

Artritis de rodilla por Streptomyces Somaliensis: un caso / Arthritis of knee for streptomyces somaliensis: a case

Se presenta un caso de artritis de rodilla por Streptomyces somaliensis, raro agente causal de micetomas, que predomina en el continente africano. Es el segundo caso encontrado en la Argentina, con aislamiento e identificación microbiológica del agente causal y el único donde se demostró compromiso intra-articular

Artritis de rodilla por Streptomyces Somaliensis: un caso / Arthritis of knee for streptomyces somaliensis: a case

Se presenta un caso de artritis de rodilla por Streptomyces somaliensis, raro agente causal de micetomas, que predomina en el continente africano. Es el segundo caso encontrado en la Argentina, con aislamiento e identificación microbiológica del agente causal y el único donde se demostró compromiso intra-articular

Isolation of cytotoxic substance, kalafungin from an alkalophilic actinomycete, Nocardiopsis dassonvillei subsp. prasina.

An alkalophilic actinomycete, strain OPC-553 regarded as Nocardiopsis dassonvillei subsp. prasina, produced the cytotoxic substance, TS-1, which showed a marked inhibitory activity against L5178Y mouse leukemic cell in vitro. The cytotoxicity of TS-1 on this cell was very strong and its ID50 was 0.018 micrograms/ml. Through direct comparison of its spectral data with those of an authentic sample, TS-1 was identified as the antifungal antibiotic, kalafungin, already isolated from the culture broth of Streptomyces tanashiensis. However, the isolation of kalafungin from an alkalophilic actinomycete and its cytotoxicity are reported for the first time in this paper.

Cellular fatty acid composition of Oerskovia species, CDC Coryneform groups A-3, A-4, A-5, Corynebacterium aquaticum, Listeria denitrificans and Brevibacterium acetylicum.

Twenty four strains representing eight species of gram positive yellow-pigmented rods (Oerskovia turbata, Oerskovia xanthineolytica, CDC Coryneform groups A-3, A-4, A-5, Listeria denitrificans, Corynebacterium aquaticum and Brevibacterium acetylicum) were divided into two major groups based on the relative amounts of 12 methyltetradecanoate (15:0a) obtained by capillary gas liquid chromatography. O. turbata, O. xanthineolytica, CDC groups A-3 and A-4, L. denitrificans and C. aquaticum were placed in the first group due to the presence of a higher percentage (29-47%) of 15:0a, than CDC group A-5 and B. acetylicum. The latter contained 2-6% of this fatty acid, and were placed in the second group. All species in the two groups except C. aquaticum and CDC group A-4, were further separated from each other based on the qualitative and quantitative differences in their fatty acid compositions. In addition, the eight strains of CDC group A-5 revealed four different patterns and were further divided into four subgroups. This study supports the importance of the composition of cellular fatty acids in differentiating some closely related organisms.

[Polyamines in representative taxa of coryneform and other bacteria]. / Poliaminy predstavitelei nekotorykh taksonov korinepodobnykh i drugikh bakterii.

The composition of polyamines is studied for the first time in representatives of the genus Micrococcus and taxon "conglomeratus", strains Staphylococcus aureus CCM 209, Deinococcus erythromyxa CCM 706 as well as of Erwinia carotovora ATCC 15713 polyamines, which are not extracted by perchloric acid. Considerable amounts of spermine and rarely of spermidine are revealed in cells of Gram positive microorganisms, that differs them from Gram negative bacteria possessing high concentrations of putrescine, spermidine and their derivatives. A procedure is developed for detection of polyamines in cells of Gram positive microorganisms. It is recommended to use the hydrolysis of their cells by 6N HCl for 4 at 120 degrees C or for 8-10h at 100 degrees C with the subsequent electrophoretic separation. Putrescine, as well as comparable with it amount of agmatine and spermidine traces are found in Erwinia carotovora ATCC 15713 cell hydrolyzates, whereas putrescine and agmatine traces are found in perchloric extracts of intact cells. Spermine is not observed in the cells. The binding of polyamines with biopolymers of cells of Gram positive bacteria and their difference by the given character from the Gram negative procaryotes are under discussion.

Actinomadura fibrosa sp. nov. isolated from soil.

A new species of the genus Actinomadura which belongs to the Actinomadura madurae group of Goodfellow et al. was isolated from soil collected in Togo, West Africa. Traditional taxonomic methods plus contemporary fatty acid analysis techniques were used to establish the position of this species. Both physiological characteristics and fatty acid composition differentiate this strain from previously described species. This culture produces a new polyether antibiotic. It is characterized by the production of white to pink aerial hyphae on a limited number of media. The aerial hyphae appear asporogenous, forming thick fibers and projections instead of true spores. The reverse side is a distinctive reddish orange. This organism is resistant to 5% NaCl and grows at temperatures between 20 and 45 degrees C. Whole cells contain meso-diaminopimelic acid, galactose, glucose, mannose, madurose, phosphatidylinositol, and diphosphatidylglycerol. The menaquinones detected were MK-9(H6) and minor amounts of MK-9(H8). The name proposed for this new species is Actinomadura fibrosa; the type strain is strain NRRL 18348.

Ansamitocin P-3, a maytansinoid, from Claopodium crispifolium and Anomodon attenuatus or associated actinomycetes.

Guided by cytotoxicity, ansamitocin P-3, a maytansinoid, was isolated in very low yield from two members of the moss family Thuidiaceae, Claopodium crispifolium (Hook.) Ren. & Card. and Anomodon attenuatus (Hedw.) Hueb. Ansamitocin P-3 showed potent cytotoxicity against the human solid tumor cell lines A-549, HT-29. A possible basis for the occurrence of this compound in mosses is discussed.

[Poly(galactosylglycerophosphate) with mannosyl side residues from the cell wall of Actinoplanes phillipinensis VKM Ac-647]. / Poli(galaktozilglitserofosfat) s mannozil'nymi bokovymi edinitsami iz kletochnoi stenki Actinoplanes philippinensis VKM Ac-647.

The Actinoplanes philippinensis cell wall has several anionic carbohydrate-containing polymers. The major polymer is of poly(glycosylglycerol phosphate) type, its monomeric unit being O-alpha-D-mannopyranosyl-(1----4)-beta-D- galactopyranosyl-(1----1)-glycerol monophosphate. The phosphodiester linkages connect the C3 of glycerol units and the C6 of galactosyl ones, and the mannosyl residues form side branches of the teichoic acid's main chain. Chains without mannosyl residues were found in addition to the major teichoic acid. The structure of the polymers was established by chemical analysis, and 13C and 1H NMR spectroscopy. It is for the first time that a teichoic acid with mannosyl residues was found in bacterial cell walls. The phosphorylated mannan contains, in addition to mannose, 2-O-methylmannose. The main chain has alpha-1,2, alpha-1,3 and alpha-1,6 types of substitution, which was established by 13C NMR spectroscopy.

Characterization of exposure to molds and actinomycetes in agricultural dusts by scanning electron microscopy, fluorescence microscopy and the culture method.

Air samples from 79 farms with 10(5) to 10(11) microorganisms/m3 were analyzed by scanning electron microscopy (SEM), fluorescence microscopy (FM), and the culture method. The total exposure to microorganisms (particularly actinomycetes) was underestimated when assessed as colony-forming units (cfu). The average cfu count was one-sixth of the total count according to SEM or FM, and the individual variability was great. This occurrence was partly explained by the aggregation of spores. Single spores accounted for 2-65% of all spores in 35 samples. There was an average of three spores/particle, and 93 (range 67-100)% of the spores were single or in aggregates of respirable size. Aggregation was more pronounced for actinomycetes and at high spore counts. Actinomycetes and bacteria could not be distinguished by FM. Bacteria (other than actinomycetes) were not detected by SEM, yet the total count of microorganisms was similar for FM and SEM. Most particles were spores from actinomycetes and fungi of the genera Aspergillus or Penicillium.

The inhibition of cytoplasmic acetoacetyl-CoA thiolase by a triyne carbonate (L-660, 631).

The compound L-660, 631 (2-oxo-5-(1-hydroxy-2,4,6-heptatriynyl)-1,3-dioxolane-4 heptanoic acid), a natural product isolated from an Actinomycete culture, was found to inhibit rat liver cytosolic acetoacetyl-CoA thiolase, the first step in the cholesterol biosynthesis pathway, with an IC50 of 1.0 x 10(-8) M. The inhibitor had no effect on other sulfhydryl containing enzymes of lipid synthesis such as HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. When tested in cultured human liver Hep G2 cells the compound inhibited the incorporation of 14C-acetate and 14C-octanoate into sterols 56% and 48% respectively at 3 x 10(-6) M with no effect on fatty acid synthesis. No noticeable effect was seen on fatty acid biosynthesis. This strongly suggests that the locus of inhibition of acetate incorporation into sterols found with this compound is the acetoacetyl-CoA thiolase step in the cholesterol biosynthesis pathway.

[Amino acid composition of peptidoglycans isolated from cells of Coryneform bacteria by various methods]. / Aminokislotnyi sostav peptidoglikanov, vydelennykh razlichnymi metodami iz kletok korinepodobnykh bakterii.

A comparative study has revealed the identity of the amino acid composition of the peptide part of peptidoglycans obtained from the intact cells (the first method) and of the amino acid composition of peptidoglycans isolated from cell walls (the second method). This evidences for the possibility of using the first method when determining types of peptidoglycans for diagnosis of the coryneform bacteria genera.

Taxonomic studies on Kitasatosporia cystarginea sp. nov., which produces a new antifungal antibiotic cystargin.

Taxonomic studies on a new species, Kitasatosporia cystarginea are presented. Among the several species already described in this genus, this strain is characteristic in forming distinct spirals of spore chains. A significant properties of the species is the production of a new antifungal antibiotic, cystargin.

Rapid screening method for inhibitors of protein kinase C.

Specific inhibitors of protein kinase C (PKC) were screened for with a unique detection system, named bleb forming assay. When K562, a human chronic myeloid leukemia cell, was treated with phorbol 12,13-dibutylate (PDBu) or teleocidin which are activators of PKC, many blebs appeared on the cell surface of K562 within 10 minutes. This appearance of blebs is inhibited by staurosporine and H7 which are known to be PKC inhibitors. Teleocidin and PDBu did not induce bleb formation of HL60, a human acute promyelocytic leukemia cell, and the mouse Friend leukemia cell, even though their morphology was changed 24 hours after treatment with teleocidin or PDBu. Many inducers of terminal differentiation of K562 have the same effect on HL60 and Friend cells. However, the bleb inducing activity of PKC activators seems to be specific for K562. The bleb forming assay satisfied the criteria (simplicity and specificity) required for preliminary screening of activators or inhibitors of PKC. Teleocidins A and B, and tautomycin (a new antibiotic isolated in our laboratory) were identified as activators of PKC, and also staurosporine and isoflavones (daidzein and genistein) as inhibitors.

Plasmids in coryneform bacteria of human origin.

A study of plasmids in coryneform bacteria isolated from human sources is reported. Seventy of 269 strains possessed a total of 89 plasmids. These were shown to be of varying sizes and in some cases of varying structures by endonuclease restriction digest. In six of 20 strains antibiotic resistance was cured with loss of the plasmid. The diversity of plasmids is emphasized.