Extending the Salinilactone Family.

Five new members of the salinilactone family, salinilactones D-H, are reported. These bicyclic lactones are produced by Salinispora bacteria and display extended or shortened alkyl side chains relative to the recently reported salinilactones A-C. They were identified by GC/MS, gas chromatographic retention index, and comparison with synthetic samples. We further investigated the occurrence of salinilactones across six newly proposed Salinispora species to gain insight into how compound production varies among taxa. The growth-inhibiting effect of this compound family on multiple biological systems including non-Salinispora actinomycetes was analyzed. Additionally, we found strong evidence for significant cytotoxicity of the title compounds.

Replacement of Soybean Meal with Animal Origin Protein Meals Improved Ramoplanin A2 Production by Actinoplanes sp. ATCC 33076.

Ramoplanin A2 is the last resort antibiotic for treatment of many high morbidity- and mortality-rated hospital infections, and it is expected to be marketed in the forthcoming years. Therefore, high-yield production of ramoplanin A2 gains importance. In this study, meat-bone meal, poultry meal, and fish meal were used instead of soybean meal for ramoplanin A2 production by Actinoplanes sp. ATCC 33076. All animal origin nitrogen sources stimulated specific productivity. Ramoplanin A2 levels were determined as 406.805 mg L(-1) in fish meal medium and 374.218 mg L(-1) in poultry meal medium. These levels were 4.25- and 4.09-fold of basal medium, respectively. However, the total yield of poultry meal was higher than that of fish meal, which is also low-priced. In addition, the variations in pH levels, protein levels, reducing sugar levels, extracellular protease, amylase and lipase activities, and intracellular free amino acid levels were monitored during the incubation period. The correlations between ramoplanin production and these variables with respect to the incubation period were determined. The intracellular levels of L-Phe, D-Orn, and L-Leu were found critical for ramoplanin A2 production. The strategy of using animal origin nitrogen sources can be applied for large-scale ramoplanin A2 production.

Effects of salinity on antibiotic production in sponge-derived Salinispora actinobacteria.

AIMS: To investigate the effects of growth conditions related to marine habitat on antibiotic production in sponge-derived Salinispora actinobacteria. METHODS AND RESULTS: Media with varying salt concentration were used to investigate the effects of salinity in relation to Salinispora growth and rifamycin production. The chemotypic profiles of the model strain Salinispora arenicola M413 was then assessed using metabolomic fingerprints from high-pressure liquid chromatography with diode array detection (HPLC-DAD) and multivariate data analysis, before extending this approach to two other strains of S. arenicola. Fingerprint data were generated from extracts of S. arenicola broth cultures grown in media of varying salt (NaCl) concentrations. These fingerprints were then compared using multivariate analysis methods such as principal components analysis (PCA) and orthogonal projection to latent structures discriminant analysis (OPLS-DA). From the analysis, a low-sodium growth condition (1% NaCl) was found to delay the onset of growth of the model S. arenicola M413 strain when compared to growth in media with either 3% artificial sea salt or 3% NaCl. However, low-sodium growth conditions also increased cell mass yield and contributed to at least a significant twofold increase in rifamycin yield when compared to growth in 3% artificial sea salt and 3% NaCl. CONCLUSIONS: The integration of HPLC-DAD and multivariate analysis proved to be an effective method of assessing chemotypic variations in Salinispora grown in different salt conditions, with clear differences between strain-related chemotypes apparent due to varying salt concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: The observed variation in S. arenicola chemotypic profiles further suggests diversity in secondary metabolites in this actinomycete in response to changes in the salinity of its environment.

Actinoplanes utahensis ZJB-08196 fed-batch fermentation at elevated osmolality for enhancing acarbose production.

Acarbose, a potent α-glucosidase inhibitor, is as an oral anti-diabetic drug for treatment of the type two, noninsulin-dependent diabetes. Actinoplanes utahensis ZJB-08196, an osmosis-resistant actinomycete, had a broad osmolality optimum between 309 mOsm kg(-1) and 719 mOsm kg(-1). Utilizing this unique feature, an fed-batch culture process under preferential osmolality was constructed through intermittently feeding broths with feed medium consisting of 14.0 g l(-1) maltose, 6.0 g l(-1) glucose and 9.0 g l(-1) soybean meal, at 48 h, 72 h, 96 h and 120 h. This intermittent fed-batch culture produced a peak acarbose titer of 4878 mg l(-1), increased by 15.9% over the batch culture.

Influence of leucine and valine on ramoplanin production by Actinoplanes sp. ATCC 33076.

Ramoplanin is a glycolipodepsipeptide antibiotic obtained by fermentation of the Actinoplanes sp. ATCC 33076, isolated as a complex of three closely related components A1, A2 and A3, which differ in their fatty acid moiety. We have investigated the influence of L-leucine and L-valine, the biosynthetic precursors of the fatty acids in A2 and A3 factors, on the complex composition and antibiotic productivity. Addition of 5 g/litre of L-leucine at the time of inoculation increases antibiotic production and improves the production of A2 factor, which represents the active principle component under clinical development. Addition of L-valine in the same conditions modifies the composition of the complex towards the A3 factor but does not improve total antibiotic productivity. A possible explanation for the different actions of the two amino acids is presented.

Ca2+ -dependence and inhibition of transformation by trifluoperazine and chlorpromazine in Thermoactinomyces vulgaris.

Ca(2+) enhanced the transformation frequency of Thermoactinomyces vulgaris (stock no. 1278) of an auxotrophic strain by the chromosomal DNA isolated from a prototrophic strain (stock no. 1227). The number of transformants showed a marked increase with increasing concentration of CaCl(2) upto 0.05 mM; and above this concentration, the transformation frequency decreased significantly. Antipsychotic drugs that are potent calmodulin inhibitors, like trifluoperazine and chlorpromazine, when applied in the concentration range of 0.01-0.04 mM along with optimal CaCl(2) concentration to the cultures of the recipient cells, resulted in a significant inhibition in the frequency of Ca(2+)-stimulated transformation. The results of present investigation suggest the involvement of a Ca(2+)-dependent protein activator in the development of Ca(2+)-mediated competence, which could have played an important role in the enhancement of genetic transformation in this aerobic spore forming thermophilic actinomycete.

Verrucosispora gifhornensis gen. nov., sp. nov., a new member of the actinobacterial family Micromonosporaceae.

A Gram-positive, aerobic, spore-forming actinomycete strain, HR1-2T, was isolated from a peat bog near Gifhorn, Lower Saxony, Germany. Comparative analysis of the 16S rDNA sequence indicated that HR1-2T was phylogenetically related to members of the family Micromonosporaceae, branching adjacent to Spirilliplanes yamanashiensis, Couchioplanes caeruleus, Catenuloplanes japonicus and members of the genus Micromonospora. The affiliation to the family was supported by the presence of family-specific 16S rDNA signature nucleotides, DNA G + C content of 70 mol%, peptidoglycan of type A1 gamma' (directly crossed-linked, presence of glycine, alanine, glutamic acid and mesodiaminopimelic acid in the peptide side-chain), menaquinone MK-9(H4) as the major respiratory lipoquinone, polar lipid composition PII (phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylserine and phosphatidylinositolmannosides) and a glycolyl type of muramic acid. It differed from genera of the family by the lack of arabinose in whole-cell sugars and a unique nucleotide signature stretch between positions 1132 and 1143 (Escherichia coli numbering), 5' CAAUUCGGUUG 3'. Morphologically strain HR1-2T resembles Micromonospora species but can be distinguished from them by the lack of arabinose in whole-cell sugars, the presence of 10-methyl C17:0 fatty acids and a distinct 16S rDNA sequence. Based on the unique combination of morphological, chemotaxonomic and phylogenetic properties a new genus, Verrucosispora gen. nov., is proposed. The type species of this genus is Verrucosispora gifhornensis sp. nov., and the type strain of V. gifhornensis is strain HR1-2T (= DSM 44337T).

The effect of cations on the thermophilic character of alkaline phosphatase from Thermoactinomyces vulgaris.

The effect of cations on the thermophilic character of alkaline phosphatase from Thermoactinomyces vulgaris, is described. The optimal pH and temperature were 9.5 and 55 degrees C to 65 degrees C, respectively. The partial removal of cations with ethylene diamine tetraacetic acid converted the enzyme to mesophilic and susceptible to chemical denaturation. Their complete removal caused complete inhibition. The addition of 0.3mM cobalt and 10mM magnesium added before heating were found to be optimal for restoring its thermophilic character and its stability to a chemical denaturant.

[Directed screening of aminoglycoside antibiotic producers on selective media with gentamycin]. / Napravlennyi poisk produtsentov aminoglikozidnykh antibiotikov na selektivnykh sredakh s gentamitsinom.

The method of directed screening of aminoglycoside antibiotics is based on the natural phenomenon that fresh isolates of Micromonospora are highly resistant to the antibiotics produced by them. The method consists of plating of the soil samples on selective media with gentamicin (10--25 micrograms/ml). The frequency of Micromonospora against the total number of the isolated actinomycetes increased from 3.3--11.4 to 30.6--78 per cent. The number of the cultures isolated from the control medium and active against gramnegative test-bacteria amounted to 13.4 per cent, while the number of Micromonospora isolated from the selective medium with gentamicin and active against gramnegative test-bacteria amounted to 69.4 per cent. 37 crude antibiotics were isolated with the method of ion exchange concentration usually used for aminoglycoside antibiotics. The new antibiotics were identified as 2-desoxy amine-containing aminoglycosides by cross resistance of aminoglycoside resistant strains of Staphylococci to them, the methods of paper and thin-layer chromatography and mass spectrometry. The majority of the aminoglycosides belonged to the gentamicin group. 2 compounds with molecular weights not described in the literature for aminoglycosides were isolated.

[Use of selective media with lincomycin for the directed screening of antibiotic producers]. / Ispol'zovanie selektivnykh sred s linkomitsinom dlia napravlennogo poiska produtsentov antibiotikov.

Relation between lincomycin resistance of Micromonospora cultures freshly isolated from soil samples and their capacity for production of antibiotics related to lincomycin by the structure or mode of action was shown. 32 cultures of Micromonospora were isolated from soil platings containing 50--100 microgram/ml of lincomycin. Crude antibiotic substances were recovered with the method of organic solvent extraction from 10 cultures possessing pronounced antibiotic activity. Selective inactivity (MIC more 1000 microgram/ml) of the crude substances with respect to the lincomycin resistant variant of Staph. aureus 209 p was observed, 2 of them having no inhibitory effect on the erythromycin resistant variant of the staphylococcus. The crude antibiotics inhibited the growth of the initial strain of the staphylococcus and its other antibiotic resistant variants in concentrations of 0.5--10 microgram/ml. It was demonstrated with the use of gas chromatography and mass spectrometry that one substance was lincomycin and 4 substances were known macrolides. Efficiency of the simple method of directed screening of antibiotics belonging to definite groups is indicated. Resistance of actinomycetes freshly isolated from natural substrates to various antibiotics is used as the criterion for antibiotic screening. The method provides detection of various antibiotics which are analogs in the structure or mode of action of the selecting antibiotic used for the screening.

Phosphate utilization and constitutive synthesis of phosphatases in Thermoactinomyces vulgaris Tsilinsky.

Thermoactinomyces vulgaris utilized both organic and inorganic phosphates with equal efficiency for its growth. The specific activities of the thermophilic acid and alkaline phosphatases were found to be maximum at 1 mM concentration of each phosphate source. All the phosphatase isoenzymes (three alkaline and one acidic) were observed irrespective of the substrate source and concentration, suggesting constitutive synthesis of the enzymes. During growth and differentiation, both acid and alkaline phosphatases exhibited uniformly stable patterns of isoenzymes with slight variations in their specific activities.

[Effect of the composition of the medium and magnesium and calcium ions on the germination of spores of Thermoactinomyces vulgaris]. / Prorastanie spor Thermoactinomyces vulgaris: znachenie sostava sredy, rol' Mg2+ i Ca2+

The germination of the spores of Thermoactinomyces vulgaris formed on a complex medium is stimulated by suspending them in solutions containing Mg2+ and Ca2+ ions. The stimulation is not the result of the initiation of the spores in the presence of the ions since the experiments were carried out at a temperature of 20 degrees C at which the initiation did not virtually take place. The ions of Na+ and K+ have almost no effect on the germination of the spores. The fraction of the resting spores of Thermoactinomyces vulgaris depends on the composition of the growth medium, especially on its amino acid composition. The addition of Mg2+ and Ca2+ ions to a minimal synthetic growth medium stimulates the growth of the cultures and decreases the dormancy of the spores. The spores formed on the synthetic medium are less thermostable than the spores formed on the complex medium. Thermostability of the spores increases upon the addition of Mg2+ to the synthetic medium. Spore suspensions obtained on the synthetic medium with Mg2+ or Ca2+ are initiated more completely than spore suspensions obtained on the complex medium.