Selective isolation of actinomycetes of the genus Actinomadura from soil

Some approaches to the selective isolation of actinomycetes of the genus Actinomadura from soil are described. The approach that involves thermal treatment of soil samples and their plating onto Gauze 1 medium with the antibiotics nystatin, nalidixic acid, and rubomycin provides for an increased amount of actinomaduras isolated from the soil actinomycete complex and for a decreased amount of streptomycetes.     

Selective isolation of bacteria with dipeptidyl aminopeptidase type I activity from the sheep rumen

Five-hundred-and-six fresh isolates of rumen bacteria were tested for their ability to hydrolyse the synthetic substrate for dipeptidyl aminopeptidase type I, GlyArg-4-methoxy-2-naphthylamide (GlyArg-MNA), using a gel overlay technique. Twelve positive isolates were small Gram-negative rods which resembled Bacteroides ruminicola in their biochemical and morphological properties. SDS-PAGE of whole cell extracts indicated that two were similar to B. ruminicola strain B14, six resembled B. ruminicola strain M384, and four were similar to B. ruminicola GA33. All hydrolysed GlyArg-MNA, Ala2 and Ala5, and showed no activity against Leu-MNA. Ala3 and Ala2, but no Ala4, was produced from Ala5. The different groups had different, distinctive activity profiles. The two remaining positive isolates were Lactobacillus spp. with an exceptionally high Leu-MNA activity. It was concluded that, although different strains may only be distantly related, B. ruminicola forms the most important group of bacteria in the rumen to possess a dipeptidyl aminopeptidase type I activity.     

Selective isolation of members of the Streptomyces violaceoruber clade from soil

Large numbers of filamentous actinomycetes which formed distinctive red coloured colonies were isolated from three out of four composite soil samples using a medium designed to be selective for members of the Streptomyces violaceoruber clade, a taxon which includes the model organisms "Streptomyces coelicolor" A3(2) and "Streptomyces lividans" 66. The isolation medium, dextran-histidine-sodium chloride-mineral salts agar supplemented with antibacterial and antifungal antibiotics, also supported the growth of representatives of the S. violaceoruber clade. One hundred and ninety one representatives of the isolates that produced red colour colonies on the isolation medium were distributed into four colour groups based on their ability to form distinctive pigments and morphological properties typical of members of the S. violaceoruber clade, an assignment that was confirmed by corresponding 16S rRNA gene sequencing studies. The selective isolation and characterisation procedures used in the present investigation provide a practical means of determining the taxonomic diversity, geographical distribution and roles of representatives of the S. violaceoruber clade in natural habitats.     

不同培养条件对海洋沉积环境细菌的选择性分离

海洋环境中存在着大量未被培养和利用的微生物资源。本研究对一份南海沉积物样品采用不同培养温度、盐度、pH、样品稀释倍数和营养浓度条件进行可培养细菌的多样性研究。经过16S rRNA基因序列分析,获得825株菌分属于5个门,8个纲,17个目,26个科,57个属。分离到最多的属级类群为芽胞杆菌属（Bacillus）。通过不同培养条件的分离实验发现,厚壁菌类群在4 °C~60 °C、0%~15%盐度、pH 5~8及不同的样品稀释倍数和营养浓度实验条件中均为优势培养类群,具有广泛的环境适应性,但2~200的样品稀释倍数可以大大减少分离培养基中厚壁菌的数量。放线菌类群在4 °C低温和0%NaCl添加条件下的可培养多样性较高,同时pH 6和寡营养培养基有助于分离获得稀有放线菌类群。另外,本研究发现新物种资源的获取几率分别在寡营养培养基、5%~10%较高盐度和60°C高温培养有所增加。分离获得的5个主要细菌门类分别为厚壁菌门（Firmicutes,86%）、放线菌门（Actinobacteria,13%）、变形菌门（Proteobacteria,1%）、拟杆菌门（Bacteroidetes）和异常球菌-栖热菌门（Deinococcus-Thermus）。本研究共分离得到29株潜在新种,分别属于芽胞杆菌纲（Bacilli）、放线菌纲（Actinobacteria）、酸微菌纲（Acidimicrobiia）、嗜热油菌纲（Thermoleophilia）、红色杆菌纲（Rubrobacteria）和异常球菌纲（Deinococci）。海洋环境微生物新类群、海洋放线菌稀有类群等微生物新资源的选择性分离培养提供了有效的方法和方案,为后期的深入开展打下良好的基础。     

Selective isolation of mycobacteria from soil: a statistical analysis approach

We compared four decontamination methods for the isolation of mycobacteria from soil specimens. Different media were used: L?wenstein-Jensen, Ogawa and various modified Ogawa media. Statistical analysis demonstrated that the best results (low contamination and high positivity rates) were obtained when the specimens were incubated in trypticase soy broth, treated with solutions containing malachite green and cycloheximide, then decontaminated with sodium hydroxide and inoculated onto Ogawa media. The lowest contamination rates were obtained with Ogawa medium containing 500 micrograms cycloheximide ml-1. The use of these techniques is proposed for the isolation of mycobacteria from heavily contaminated clinical specimens as well as from soil.     

Selective medium for isolation of Xanthomonas maltophilia from soil and rhizosphere environments

A selective medium (XMSM) was developed for isolation of Xanthomonas maltophilia from bulk soil and plant rhizosphere environments. The XMSM basal medium contained maltose, tryptone, bromthymol blue, and agar. Antibiotics added to select for X. maltophilia were cycloheximide, nystatin, cephalexin, bacitracin, penicillin G, novobiocin, neomycin sulfate, and tobramycin. A comparison was made between XMSM and 1/10-strength tryptic soy broth agar for recovery of X. maltophilia from sterile and nonsterile soil infested with known X. maltophilia isolates. A recovery rate of 97% or greater for XMSM was demonstrated. XMSM was used to isolate X. maltophilia from a variety of soil and rhizosphere environments.     

Selective medium for isolation of Xanthomonas maltophilia from soil and rhizosphere environments.

A selective medium (XMSM) was developed for isolation of Xanthomonas maltophilia from bulk soil and plant rhizosphere environments. The XMSM basal medium contained maltose, tryptone, bromthymol blue, and agar. Antibiotics added to select for X. maltophilia were cycloheximide, nystatin, cephalexin, bacitracin, penicillin G, novobiocin, neomycin sulfate, and tobramycin. A comparison was made between XMSM and 1/10-strength tryptic soy broth agar for recovery of X. maltophilia from sterile and nonsterile soil infested with known X. maltophilia isolates. A recovery rate of 97% or greater for XMSM was demonstrated. XMSM was used to isolate X. maltophilia from a variety of soil and rhizosphere environments.     

Selective isolation and distribution of Actinobispora strains in soil

A simplified enrichment method for selective isolation of Actinobispora strains from soil is described. Actinobispora spores were tolerant to dry-heat treatment at 110 degrees C for 15 min. Actinobispora was more resistant to 1 microgram/mL leucomycin, 1 microgram/mL novobiocin, and 0.5 microgram/mL tunicamycin than Streptomyces dominant in soil, which prevents selective isolation of Actinobispora. Percentages of Actinobispora colonies on the isolation plate were increased by addition of antibiotics and dry-heat treatment of the soil samples. By combining the techniques described above, this genus was isolated from 105 out of 574 soil samples (18% of the samples tested). It was recovered from the soil samples with pH values ranging 5.0 to 8.9, and 78% of strains were isolated from neutral soil (pH 6.0-8.0). A number of Actinobispora strains were isolated from various soils around the world. Actinobispora strains are widely distributed in the world at relatively high frequency.     

Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase

The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase.     

Use of a packed-column bioreactor for isolation of diverse protease-producing bacteria from antarctic soil

Seventy-five aerobic heterotrophs have been isolated from a packed-column bioreactor inoculated with soil from Antarctica. The column was maintained at 10 degrees C and continuously fed with a casein-containing medium to enrich protease producers. Twenty-eight isolates were selected for further characterization on the basis of morphology and production of clearing zones on skim milk plates. Phenotypic tests indicated that the strains were mainly psychrotrophs and presented a high morphological and metabolical diversity. The extracellular protease activities tested were optimal at neutral pH and between 30 and 45 degrees C. 16S ribosomal DNA sequence analyses showed that the bioreactor was colonized by a wide variety of taxons, belonging to various bacterial divisions: alpha-, beta-, and gamma-Proteobacteria; the Flexibacter-Cytophaga-Bacteroides group; and high G+C gram-positive bacteria and low G+C gram-positive bacteria. Some strains represent candidates for new species of the genera Chryseobacterium and Massilia. This diversity demonstrates that the bioreactor is an efficient enrichment tool compared to traditional isolation strategies.     

天山冻土产低温脂肪酶菌株的筛选及其多样性分析

【目的】通过天山冻土细菌的分离和产低温脂肪酶菌株的筛选,了解天山冻土微生物的物种多样性和产脂肪酶菌株的系统发育多样性,为高效低温脂肪酶生物技术奠定基础。【方法】采用稀浓度的R2A、TSB平板涂布分离天山冻土中可培养细菌,通过选择性培养基筛选产低温脂肪酶的菌株。采用细菌常规生理生化实验、最适生长温度、耐盐性、产酶性能对分离菌株的生理学进行研究,通过16S rRNA基因序列分析确定产脂肪酶菌种的系统进化地位,通过BOX-PCR指纹技术对16S rRNA基因高度同源性的菌株进一步区分。【结果】分离筛选到78株可培养低温菌,选择培养基显示有17株可产低温脂肪酶,其中8株在两种选择培养基中均可产脂肪酶和酯酶。17株产酶菌分别隶属于5个系统发育类群、6个属,其中假单胞菌属(Pseudomonas)占大多数(58.9%)。【结论】天山冻土中产低温脂肪酶的细菌具有较丰富的系统发育多样性,依据生长温度,均属于耐冷菌。     

Selective plant growth suppression by shoot application of soil bacteria

Selected rhizosphere bacterial isolates, previously determined as plant growth deleterious, were tested for their ability to suppress plant growth after foliar spray applications, for selectivity with regard to plant species, and in pilot field experiments for their potential as weed biocontrol agents. Inundative foliar applications of aqueous bacterial suspension were performed on a range of weed and crop species. Plant symptoms after spraying ranged from rapid necrosis and wilting to an overall growth suppression or stunting. Significant and selective reductions in biomass of up to 90% fresh weight, as well as large reductions in plant survival and plant height were recorded in greenhouse pot experiments. However, monocotyledonous plants were affected weakly or not at all by two isolates extensively tested. Effects of these were dose- and plant age-dependent, and were for some plants enhanced by high relative humidity. For one isolate, A153, effects were also expressed in cell-free culture filtrates pointing to involvement of specific metabolites. In pilot field experiments, strong growth suppression was observed on broad-leaved plants, while barley crop plants were unaffected.     

Emulsifying agents from bacteria isolated during screening for cells with hydrophobic surfaces

Summary The culture supernatants of 126 bacterial strains isolated during screening for hydrophobic cell surfaces, were tested for the production of emulsifying agents. Forty-eight strains were found to produce effective emulsion-stabilizing substances during growth on glucose. The most effective emulsifying agents were isolated and could be divided into two chemical groups. The first group was separated from the isolated extracts by the use of thin-layer chromatography and detected as ninhydrin-negative, 4,4'-tetramethyldiamino-diphenylmethane-positive spots. The amino acid composition indicated surfactin and iturin, produced by one Bacillus species, and viscosin, produced by a Pseudomonas species. The second group was identified as polymeric substances. The chemical characterization of five polymers showed polysaccharides that were able to stabilize emulsions. From these the neutral and charged monosaccharides were determined qualitatively. The constituents of the five isolated polysaccharides were: strain 5, glucose, strain 17, rhamnose, glucose, glucuronic acid; strain 33, rhamnose, galactose, glucose. glucuronic acid; strain 113, fucose, galactose, glucose, galacturonic acid, glucosamine; strain 259, one unknown compound, rhamnose, galactose, glucuronic acid.Offprint requests to: K. Poralla     

Use of mycelia as paths for the isolation of contaminant‐degrading bacteria from soil

Mycelia of fungi and soil oomycetes have recently been found to act as effective paths boosting bacterial mobility and bioaccessibility of contaminants in vadose environments. In this study, we demonstrate that mycelia can be used for targeted separation and isolation of contaminant‐degrading bacteria from soil. In a ‘proof of concept’ study we developed a novel approach to isolate bacteria from contaminated soil using mycelia of the soil oomycete Pythium ultimum as translocation networks for bacteria and the polycyclic aromatic hydrocarbon naphthalene (NAPH) as selective carbon source. NAPH‐degrading bacterial isolates were affiliated with the genera Xanthomonas, Rhodococcus and Pseudomonas. Except for Rhodococcus the NAPH‐degrading isolates exhibited significant motility as observed in standard swarming and swimming motility assays. All steps of the isolation procedures were followed by cultivation‐independent terminal 16S rRNA gene terminal fragment length polymorphism (T‐RFLP) analysis. Interestingly, a high similarity (63%) between both the cultivable NAPH‐degrading migrant and the cultivable parent soil bacterial community profiles was observed. This suggests that mycelial networks generally confer mobility to native, contaminant‐degrading soil bacteria. Targeted, mycelia‐based dispersal hence may have high potential for the isolation of bacteria with biotechnologically useful properties.     

Improved method of lipopolysaccharide isolation from gram-negative bacteria

A modification of the traditional method for lipoplysaccharide isolation from the cells of grammnegative bacteria was elaborated on the basis of extraction by the hot water solution of phenol (the method of Westfahl). To make the method simpler and to raise the yield of the product it was proposed to use the water-phenol extract without its division for plases. The nucleic acids are eliminated by precipitation from dialyzed extract at pH 3,2-3,4 achieved by addition of acetic acid. The comparative isolation of lipopolysaccharides by the classic and modified methods has confirmed the advantages of a new technique.