Polyol metabolism by Rhizobium trifolii.

In Rhizobium trifolii 7000, the polyols myo-inositol, xylitol, ribitol, D-arabitol, D-mannitol, D-sorbital, and dulcitol are metabolized by inducible nicotinamide adenine dinucleotide-dependent polyol dehydrogenases. Five different polyol dehydrogenases were recognized: inositol dehydrogenase, specific for inositil; ribitol dehydrogenase, specific for ribitol; D-arabitol dehydrogenase, which oxidized D-arabitol, D-mannitol, and D-sorbitol; xylitol dehydrogenase, which oxidized xylitol and D-sorbitol; and dulcitol dehydrogenase, which oxidized dulcitol, ribitol, xylitol, and sorbitol. Apart from inositil and xylitol, all of the polyols induced more than one polyol dehydrogenase and polyol transport system, but the heterologous polyol dehydrogenases and polyol transport systems were not coordinately induced by a particular polyol. With the exception of xylitol, all of the polyols tested served as growth substrates. A mutant of trifolii 7000, which was constitutive for dulcitol dehydrogenase, could also grow on xylitol.     

Spectrophotometric characteristics of the coccoid forms of actinomycetes]

IR spectra of thw whole cells of the coccoid forms (Mycococcus and Micrococcus) isolated from lithophilous lichen were compared with IR spectra of the collection cultures of Micrococcus and Arthrobacter. Generic spectral characteristics of Mycococcus and Micrococcus are presented. Spectral heterogeneity within the genus Arthrobacter complicates the diagnosis. The cultures of the Mycococcus genus were divided into three groups according to their spectral characteristics. Spectral scans of the studied coccoid forms differ from the scans of the mycelial actinomycetes, and their intensities within the range of 900-1200 cm-1 (lipids) decrease in the series Mycococcus, Arthrobacter, Micrococcus.     

Isolation of Actinomycetes synthesizing proteases with thrombolytic activity

Proteases with the thrombolytic activity were studied in 212 strains of actinomycetes isolated from different soils of the Soviet Union. The cultures belonged to the genera Micromonospora, Nocardia and Streptomyces. Proteases were synthesized by 41% of the studied actinomycetes and some of their strains completely dissolved in vitro artificially obtained blood thrombi within 120-240 min. In the Streptomyces genus, more active strains were found in the groups Flavus, Fradia and Globisporus. The groups Olivaceus, Violaceus and Viridis had less active strains.     

Formation of Dulcitol in Aerobic Dissimilation of d-Galactose by Yeasts

During the study of aerobic dissimilation of galactose by yeasts, polyhydric products were isolated in crystalline form from the fermented broths and identified. Yeast species may be divided into two groups on basis of sugar alcohol production: type I yeasts form the same end products from galactose as from glucose; type II yeasts produce dulcitol from galactose with or without other sugar alcohols but they produce no dulcitol from glucose. Isolation of dulcitol from microorganism has not been previously described.     

Simultaneous measurement of urinary polyols using gas chromatography/mass spectrometry

In the present study, we simultaneously measured several polyols, such as adonitol, arabitol, dulcitol, glucose, myo-inositol, mannitol, sorbitol, and xylitol, in urine by gas chromatography/mass spectrometry-positive chemical ionization. We also examined possible relationship between the levels of these metabolites and age in normal individuals. In order to proceed to its quantification by GC/MS, 200 microL of a urine sample were diluted with 3 ml of distilled water, lyophilized, acetylated, and then analyzed them. Using this method, we were able to quantify as little as 0.5-1.0 ng/microL, and we made the calibration curves to be linear from 0.25 to 250 ng/microL (r(2)>0.991). Analytical recoveries were over 89.4%, and the inter-day and intra-day variability for accuracy and reproducibility was less than 20%. In the normal urine sample, the levels of polyols were gender-differentiated and age-related. This simple GC/MS method is sensitive and allows the measurement of wide ranges of polyols using small amounts of urine. We conclude that the quantitation of urinary polyols using GC/MS appears to be a clinically useful method for assessing polyol-pathway activity.     

pH dependence and thermostability of lipases from cultures from the ARS culture collection

Summary Previously we used a simple, sensitive agar plate method to screen lipase activity from 1229 selected cultures including 508 bacteria, 479 yeasts, 230 actinomycetes and 12 fungi covering many genera and species. About 25% of the cultures tested were lipase-positive. These lipase-positive strains were further classified as good, moderate or weak enzyme producers. We have expanded our screening method to focus specifically on the pH dependence and thermostability of these lipase activities. The lipases exhibited various pH sensitivities and were divided into three groups: (i) lipases which are active at pH 5.5 but not at pH 7.5—produced by 36 bacteria, 23 yeasts and four actinomycetes; (ii) lipases which are active at pH 7.5 but not at pH 5.5—produced by 17 bacteria, four yeasts, two actinomycetes and one fungus; and (iii) lipases which are active at both pH 5.5 and pH 7.5—produced by 112 bacteria, 90 yeasts, 15 actinomycetes and five fungi. By screening at 60°C and pH 9.0, we further identified 50 bacteria and 26 yeasts that produce thermostable alkali-tolerant lipases. Product analyses confirmed our screening results. Lipases with specific pH dependency and thermostability have potential to be developed into industrial enzymes.     

Correlations in the utilization of carbon sources in the range of griseus species of the genus Streptomyces

The author proposes a new scheme for arranging the data about the assimilation of carbon sources in tables; this is illustrated with the Griseus species belonging to the Streptomyces genus and with the information about them presented by Bergey [2]. In the scheme, carbon sources in the tables are arranged in the order of their diminishing availability for streptomycetes, and the lists of species within the morphological groups are constructed according to the property of a carbon source not being assimilated. Such an arrangement of data in the tables is very clear and convenient to use; it allows one to make optimal schemes for determining the assimilation of carbon sources by actinomycetes, and makes it easy to divide any large number of cultures into groups according to a similar assimilation of carbon sources. The scheme has been used to analyse the data available in science and to establish correlations in the assimilation of carbon sources by species of the Griseus series belonging to the Streptomyces genus.     

13C-NMR spectroscopy as a tool to study organic osmolytes in the mangrove red algal genera Bostrychia and Stictosiphonia (Ceramiales)

Field and culture samples of the red algal genera Bostrychia and Stictosiphonia from all around the world were analyzed for the polyols D-sorbitol and dulcitol, that function as osmolytes, as well as for the heteroside digeneaside by using ¹³C-NMR spectroscopy and HPLC. While all plants exhibited D-sorbitol, the occurrence of dulcitol and digeneaside was highly variable. Therefore, different types of low molecular weight carbohydrate distribution patterns were found in Bostrychia and Stictosiphonia. The presence of D-sorbitol seems to be a reliable chemosystematic character for both genera, because no other red alga is known to contain this compound. The lack of dulcitol may be correlated with the geographical origin of the Bostrychia and Stictosiphonia samples: while all tropical isolates exhibited both sugar alcohols, in cold-temperate plants only D-sorbitol was determined. In warm-temperate species, however, both polyol distribution types may occur. These data are discussed in terms of possible temperature sensitivity of the dulcitol pathway. However, the biological function of digeneaside (the main photo-assimilated compound in members of the order Ceramiales) is still obscure.     

Biochemical characteristics of erythrocyte sorbitol dehydrogenase from selected mammals

The erythrocyte sorbitol dehydrogenase (EC 1.1.1.14) activity, regarding its action on sorbitol oxidation to fructose, was studied in 19 species of mammals, showing a striking variability, with high activity in rodents. Enzyme activity was studied against other polyols, namely xylitol, inositol, manitol and dulcitol. Most animals showed activity against all the polyols studied, but hamster and red deer only presented activity on sorbitol and xylitol. Michaelis-Menten constant determinations for sorbitol were performed, and it was observed that animals which presented high activity had a high Km. pH curves were obtained from 8 animals, with an optimum pH ranging from pH 8.0 to pH 10.0; four of the animals presented an optimum pH at 8.5.     

Use of direct-infusion electrospray mass spectrometry to guide empirical development of improved conditions for expression of secondary metabolites from actinomycetes

A major barrier in the discovery of new secondary metabolites from microorganisms is the difficulty of distinguishing the minor fraction of productive cultures from the majority of unproductive cultures and growth conditions. In this study, a rapid, direct-infusion electrospray mass spectrometry (ES-MS) technique was used to identify chemical differences that occurred in the expression of secondary metabolites by 44 actinomycetes cultivated under six different fermentation conditions. Samples from actinomycete fermentations were prepared by solid-phase extraction, analyzed by ES-MS, and ranked according to a chemical productivity index based on the total number and relative intensity of ions present in each sample. The actinomycete cultures were tested for chemical productivity following treatments that included nutritional manipulations, autoregulator additions, and different agitation speeds and incubation temperatures. Evaluation of the ES-MS data from submerged and solid-state fermentations by paired t test analyses showed that solid-state growth significantly altered the chemical profiles of extracts from 75% of the actinomycetes evaluated. Parallel analysis of the same extracts by high-performance liquid chromatography-ES-MS-evaporative light scattering showed that the chemical differences detected by the ES-MS method were associated with growth condition-dependent changes in the yield of secondary metabolites. Our results indicate that the high-throughput ES-MS method is useful for identification of fermentation conditions that enhance expression of secondary metabolites from actinomycetes.     

Comparative evaluation of various bacterial growth inhibitors as selective agents for isolation of soil Actinomyces]

Tobramycin and dioxidine sensitivity of 57 strains belonging to 14 actinomycetes genera was studied. The cultures of Streptomyces were much more sensitive to tobramycin than the cultures of rare genera. The majority of the Streptomyces cultures showed a high resistance to dioxidine (MIC greater than or equal to 50 micrograms/ml). At the same time the majority of the cultures of rare genera were inhibited by low concentrations of dioxidine (no more than 50 micrograms/ml). For isolation of actinomycetes from soil samples, tobramycin, dioxidine, ceftriaxone and novobiocin were used. Tobramycin added in a concentration of 10 micrograms/ml to the Gauze agar organic medium No. 2 promoted a 2-fold increase in detection of actinomycetes of the rare genera as compared to the control. It was especially favourable for detection of cultures belonging to Micromonospora, Amycolatopsis, Streptosporangium and Nocardiopsis. Dioxidine in concentrations of 10 and 50 micrograms/ml inhibited the growth of the cultures belonging to rare genera. Ceftriaxone in the same concentrations inhibited the growth of the cultures of both Streptomyces and the rare genera. Novobiocin favoured detection of the cultures belonging to Amicolatopsis and Micromonospora. Therefore, among the tested compounds tobramycin and novobiocin appeared to be the most useful selective agents for isolation of actinomycetes of rare genera.     

嗜酸丝状放线菌的选择性分离与多样性

摘要:【目的】针对酸性土壤中的嗜酸丝状放线菌,建立有效的选择性分离方法,并了解其多样性。【方法】用不同的样品预处理方式和分离培养基,并添加不同的抑制剂进行分离;根据放线菌的菌落数和出菌率确定最佳分离方法组合。采用最佳分离方法对从江西采集的17份酸性土壤样品进行分离;根据培养特征对分离菌株进行分群,进一步通过对各类群的显微形态观察和pH梯度生长实验确定代表菌株;对代表菌株进行16S rRNA基因序列分析研究其多样性。【结果】嗜酸丝状放线菌的最佳分离方法为：土壤样品经分散差速离心预处理后,涂布添加了放线菌酮、制霉菌素和萘啶酮酸（各50 mg/L）的GTV培养基。用此方法共分离到放线菌369株,归为10个不同的颜色类群,其中6.6%为严格嗜酸放线菌,72.4%为中度嗜酸放线菌,21.0%为耐酸放线菌。52株嗜酸放线菌代表菌株分布于放线菌目中的12个属：链霉菌属(Streptomyces)、小单孢菌属(Micromonospora) 、诺卡氏菌属(Nocardia)、野野村菌属(Nonomuraea) 、韩国生工属(Kribbella) 、小双孢菌属(Microbispora)、马杜拉菌属(Actinomadura)、拟无枝菌酸菌属(Amycolatopsis)、指孢囊菌属(Dactylosporangium)、伦茨氏菌属(Lentzea)、游动四孢菌属(Planotetraspora) 和链嗜酸菌属(Streptacidiphilus),其中链霉菌分离菌株在系统发育树上形成12个不同的进化类群。【结论】所建立的选择性分离方法可用于土壤嗜酸丝状放线菌的高效分离;江西酸性土壤含有丰富多样的嗜酸丝状放线菌种属。     

Various principles of detecting the producers of beta-lactamase inhibitors among soil Actinomycetes

Principles of detecting organisms producing beta-lactamase inhibitors among soil actinomycetes were developed. For detecting such cultures it was recommended to use the Gauze agarized medium No. 1 supplemented with beta-lactam antibiotics. Benzylpenicillin proved to be the most efficient. Various liquid fermentation media for detecting the inhibitory activity of soil actinomycetes were compared. Two media were the most favourable i.e. the glucose-yeast medium No. 18/3 and the soybean-glucose medium with Na2SO4 and CoCl2 No. 20/3. The use of test cultures with relatively low resistance to benzylpenicillin was shown expedient in screening cultures producing beta-lactamase inhibitors. Test cultures with high resistance should be used in more detailed characterization of the selected cultures.     

Differential Growth Inhibition of Diaporthe and Phomopsis Isolates by the Metabolic Activity of Five Actinomycetes

Fifty-five cultures derived from Diaporthe perithecia and Phomopsis pycnidia found on diverse host plant species collected at different times and sites in Vojvodina, Yugoslavia, showed distinguishing quantitative reactions to the fungistatic activity of five actinomycetes obtained as fortuitous laboratory contaminants coming from field material. Streptomyces albidoflavus , S. albus , S. diastaticus , Streptomyces sp., and Streptoverticillium sp. could be ranked by their growth-inhibitory potential, with S. albus showing the strongest, and Streptomyces sp. the lowest. The responses of the fungi varied depending on the tested actinomycetes, but two major groups could be distinguished: A, which encompased the isolates that were less affected by the proximity of the actinomycetes; and B, with those which exhibited high sensitivity in all the experiments. Group A was typically represented by Diaporthe arctii , Phomopsis longicolla, and the Phomopsis type-1 cultures from Xanthium italicum; group B was typically represented by Diaporthe/Phomopsis helianthi, Phomopsis type-2 cultures from X. italicum , and isolates from Lactuca serriola . The results obtained underscore the dissimilarities between D. arctii and D. helianthi , and corroborate the value of the physiological aspects of congeneric isolates in considering taxonomic problems in the coelomicete genus Phomopsis.     

Selective medium with nalidixic acid for isolating antibiotic-producing Actinomyces

The majority of actinomycetes belonging to various genera proved to be resistant to nalidixic acid concentrations having an inhibitory effect on bacteria with trailing growth i.e. B. subtilis and B. mycoides. The bacteria prevented isolation of actinomycetes as pure cultures. The use of a selective medium with nalidixic acid for isolation of soil actinomycetes resulted in 20 per cent increase in the number of the actinomycetes isolated as pure cultures. Preliminary treatment of the soil samples with calcium carbonate under moist conditions followed by the inoculation to the medium with nalidixic acid made it possible to increase isolation of actinomycetes at most 100-fold. With this complex method 495 actinomycete cultures were isolated, their antibiotic properties were studied and their taxonomic position at the genus level was determined. The complex method including the preliminary treatment of soil samples with calcium carbonate followed by inoculation to the selective medium with nalidixic acid is efficient and may be recommended for screening organisms producing new antibiotics.     

Calcium as a regulator of intracellular processes in actinomycetes: a review

Data on the effects of calcium ions (Ca2+) on processes of morphological and physiological differentiation in cultures of actinomycetes have been reviewed, with emphasis on representatives of the genus Streptomyces. Evidence accumulated thus far, of the regulatory role of serine-threonine protein kinases in the differentiation and of the possible involvement of Ca2+-dependent protein kinases in secondary metabolism (including antibiotic biosynthesis) are analyzed. The possibility that regulatory elements of apoptosis (including Ca2+-dependent) function in actinomycetes is discussed. A hypothesis is advanced, according to which determinants of antibiotic resistance play a key role in the network of signal transduction systems of actinomycetes.     

The detection of diverse aminoglycoside phosphotransferases within natural populations of actinomycetes

The conserved nature of the genes that code for actinomycete secondary metabolite biosynthetic pathways suggests a common evolutionary ancestor and incidences of lateral gene transfer. Resistance genes associated with these biosynthetic pathways also display a high degree of similarity. Actinomycete aminoglycoside phosphotransferase antibiotic resistance enzymes (APH) are coded for by such genes and are therefore good targets for evaluating the bioactive potential of actinomycetes. A set of universal PCR primers for APH encoding genes was used to probe genomic DNA from three collections of actinomycetes to determine the utility of molecular screening. An additional monitoring of populations for the predominance of specific classes of enzymes to predict the potential of environmental sites for providing isolates with interesting metabolic profiles. Approximately one-fifth of all isolates screened gave a positive result by PCR. The PCR products obtained were sequenced and compared to existing APH family members. Sequence analysis resolved the family into nine groups of which six had recognizable phenotypes: 6′-phosphotransferase (APH(6)), 3′-phosphotransferase (APH(3)), hydroxyurea phosphotransferase (HUR), peptide phosphotransferase, hygromycin B phosphotransferase (APH(7″)) and oxidoreductase. The actinomycetes screened fell into seven groups, including three novel groups with unknown phenotypes. The strains clustered according to the environmental site from where they were obtained, providing evidence for the movement of these genes within populations. The value of this as a method for obtaining novel compounds and the significance to the ecology of antibiotic biosynthesis are discussed. Journal of Industrial Microbiology & Biotechnology (2002) 29, 60–69 doi:10.1038/sj.jim.7000260 Received 25 June 2001/ Accepted in revised form 26 March 2002     

Use of the method of enriching of soil samples with calcium carbonate for isolation of Actinomyces

Possible application of a modified procedure for treatment of soil samples with calcium carbonate under humid conditions to isolation of actinomycetes was studied. The procedure proved to be rather efficient in regard to increasing the number of the isolates including representatives of rare genera as compared with the routine methods. The number of microorganisms isolated from the soil samples treated with calcium carbonate under humid conditions was 2 orders higher than that from untreated soil samples. 1033 actinomycete cultures were isolated from the soil samples treated in accordance with the above procedure and only 597 cultures were isolated from the untreated soil samples. The antibiotic activity of the isolates was studied and their taxonomic position at the genus level was determined. The preliminary treatment of soil samples equally stimulated development of actinomycetes belonging to different genera. It is advisable that the described procedure for isolation of actinomycetes from soil samples in screening of cultures producing new antibiotics be used in combination with other selective methods of isolation.