Use of polyvalent phage for reduction of streptomycetes on soil dilution plates

D.I. KURTBÖKE, C.F. CHEN AND S.T. WILLIAMS. 1992. An isolation method was developed in which prior to inoculation soil suspensions were exposed to suspensions of polyvalent phage isolated to Streptomyces spp. The phage susceptibility of streptomycetes provided a selective means of reducing streptomycetes on isolation plates subsequent to inoculation, and this reduction was persistent after long incubation periods. The efficiency and applicability of the method developed were checked with different samples from a range of sources. The increased chances of development of other genera after the reduction of streptomycetes on soil dilution plates were assessed.     

Improved Rapid Plate Method for the Isolation of Bacteriophages from Lysogenic Bacteria

A modification of a recently reported rapid plate method for the isolation of bacteriophages from lysogenic bacteria is described. The velveteen replica plate technique was used for inoculation of mitomycin C-induced colonies onto agar plates, and tetrazolium chloride was used to enhance detection of phage activity on replicated indicator plates.     

Improved procedure for bacteriophage typing of Listeria strains and evaluation of new phages

A modified technique in listerial phage typing, termed reversed phage typing procedure, was developed. Ready-to-use typing plates were prepared by preapplication of phage suspensions on tryptose agar plates. The new procedure offers a number of substantial advantages as compared with the conventional method, being much more reliable, efficient, and convenient to use. More than 1,000 strains of Listeria have thus far been typed with the reversed phage typing procedure, employing an extended set of 21 genus-specific bacteriophages. The overall typability of strains was 89.5%.     

Improved procedure for bacteriophage typing of Listeria strains and evaluation of new phages.

A modified technique in listerial phage typing, termed reversed phage typing procedure, was developed. Ready-to-use typing plates were prepared by preapplication of phage suspensions on tryptose agar plates. The new procedure offers a number of substantial advantages as compared with the conventional method, being much more reliable, efficient, and convenient to use. More than 1,000 strains of Listeria have thus far been typed with the reversed phage typing procedure, employing an extended set of 21 genus-specific bacteriophages. The overall typability of strains was 89.5%.     

Selective isolation of bioactive soil actinomycetes belonging to the Streptomyces violaceusniger phenotypic cluster

AIMS: To devise and evaluate a strategy for isolating members of the Streptomyces violaceusniger phenotypic cluster, which are known to be a promising source of bioactive metabolites. METHODS AND RESULTS: The treatment of four soil samples with 1.5% phenol (30 degrees C, 30 min) prior to inoculation on humic acid-vitamin (HV) agar eliminated most of the streptomycetes and other bacterial populations. The surviving streptomycetes on the HV isolation plates were subcultured, and species-group identification was made according to the probabilistic identification system of Williams et al. (1989). Of the 133 streptomycetes subcultured, 102 (77%), were assigned to the S. violaceusniger cluster. A test with an overlay technique revealed that all of these S. violaceusniger-cluster isolates had broad antimicrobial spectra, as they inhibited the growth of all test Gram-positive bacteria, yeasts and filamentous fungi. Antitumour activity against colon carcinoma cells was found among 68 or 67%, of these S. violaceusniger-cluster isolates, following growth in submerged culture. CONCLUSIONS: Chemical pretreatment of soil samples with phenol reduces the growth of ubiquitous Streptomyces species, thereby facilitating the recovery of less-abundant S. violaceusniger-cluster strains that are characterized by high antimicrobial and antitumour activities. SIGNIFICANCE AND IMPACT OF THE STUDY: The development and application of new methodologies with which to selectively isolate rare, bioactive streptomycete groups is important for discovering novel secondary metabolites with bioactive properties.     

应用分散和差速离心法分离嗜酸和耐酸链霉菌的试验及评价*

新的微生物资源的开发离不开菌种分离技术的改进和分离效率的提高。我们采用一种新的分离方法——分散和差速离心法(Dispersion and Differential Centrifugation,DDC),以传统的振荡法做对照,对12份酸性土壤样品进行了链霉菌的分离。结果表明DDC方法的分离效率是传统方法的2—20倍,且选择性好。用DDC方法共分离出链霉菌249株,归属于12个颜色类群,其中的45株代表菌株的形态和细胞壁类型均符合链霉菌的特征,最适生长pH均为4．5—5．5。结果表明应用DDC方法分离非常见的嗜酸和耐酸链霉菌是可行的。     

16S rDNA analysis reveals low microbial diversity in community level physiological profile assays

The metabolic diversity of microbial communities is fundamental for the multiple soil functions mediated by microorganisms. Community level physiological profiles (CLPPs) based on sole C source oxidation have been used as a fast and reproducible tool to study soil microbial functional diversity because the utilisation of available carbon is the key factor governing microbial growth in soil. Our aim was to assess the phylogenetic affiliation of the microorganisms responsible for C consumption after inoculating Biolog plates. For this purpose, two semi-arid Mediterranean forest soils with significantly different patterns of C consumption and microbial community structure were used. Following the inoculation of the Biolog plates, suspensions from seven wells were sampled after 1, 2 and 7 d of incubation. DNA was extracted and the microbial communities analysed by polymerase chain reaction followed by denaturing gradient gel electrophoresis (PCR-DGGE) and sequencing of excised bands. Despite major differences in the microbial communities of the soils studied, their DGGE banding patterns after incubation were similar for all the analysed C source suspensions. Microorganisms belonging to beta-Proteobacteria (Ralstonia sp. and Burkholderia sp.) and alpha-Proteobacteria (Rhizobium sp.) were dominant. These opportunists had a competitive advantage under the conditions at which the CLPPs were analysed. This study reveals that significantly different CLPP patterns can be generated on the basis of only 3-4 genera, as reflected by PCR-DGGE analysis. Also for this reason, CLPPs based on incubations of soil suspensions should just be used as a screening method and always be accompanied by other techniques for community analysis.     

Application of a method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter

The present paper describes a simple enrichment technique which enables rapid and selective isolation of diverse zoosporic actinomycete genera directly from soil and plant litter. This technique, designated the rehydration and centrifugation (RC) method, consists of immersing the air-dried source material in 10 mM phosphate buffer containing 10% soil extract, letting the preparation stand at 30 °C for 90 min, followed by centrifugation of the fluid at 1,500×g for 20 min. Portions of the supernatant containing actinomycete zoospores are plated on the humic acid-vitamin agar which is supplemented with nalidixic acid and trimethoprim as the selective inhibitors for Gram-negative bacteria and bacilli. The phosphate buffer-soil extract solution significantly promoted liberation of motile zoospores from the source material. The centrifugation stage greatly eliminated streptomycetes and other non-motile actinomycetes from the liquid phase, thereby facilitating selective growth of rare, motile actinomycetes on the isolation plates subsequent to inoculation. Ten different soil and leaf-litter samples, taken from fields, forests, and stream banks, were examined. The RC method consistently achieved preferential isolation of motile actinomycetes in all samples, which accounted for 37–86% of the total microbial population recovered. The most frequently isolated motile actinomycetes were Actinoplanes and Dactylosporangium. Strains of Actinokineospora, Catenuloplanes and Kineosporia were also recovered, depending on the nature of the samples examined. Other motile actinomycetes that were occasionally isolated in small numbers included Actinosynnema, Geodermatophilus and Sporichthya.     

Application of actinomycetes to soil to ameliorate water repellency

AIMS: The aim of this study was to develop a novel isolation technique using a mixture of Bacillus and Streptomyces phages to selectively isolate wax-utilizing non-streptomycete actinomycetes effective in ameliorating water repellency in a problem soil. METHODS AND RESULTS: Phages added to a soil suspension reduced the dominance of Bacillus and Streptomyces isolates and significantly increased the number of non-streptomycete actinomycetes on isolation plates. Promising isolates, grown on a medium containing beeswax as sole carbon source, were selected for application to water repellent soil. Their addition significantly reduced water repellency. CONCLUSIONS: Phage application significantly increased the isolation of non-streptomycete actinomycetes. Wax-utilizing isolates were found to significantly reduce water repellency in a problem soil. SIGNIFICANCE AND IMPACT OF THE STUDY: The phage technique can be used for the routine isolation of non-streptomycete actinomycetes. Beeswax medium can be used to selectively isolate wax-utilizing micro-organisms with the potential to ameliorate water repellency in soil.     

A modified sucrose fractionation procedure for the isolation of frankiae from actinorhizal root nodules and soil samples

Summary The isolation and pure culture of the symbiotic nitrogen-fixing frankiae has always been difficult. In the past the isolation of these actinomycetes directly from soil samples has proven impossible and isolations from root nodules of many genera has been only poorly successful. We report here a modified sucrose fractionation procedure which increased the success of isolations from root nodules and which permitted the isolation ofFrankia directly from soil samples. Crushed nodule suspensions or soil suspensions were incubated briefly in 0.7% phenol (carbolic acid) just before application to a sucrose density gradient. This phenol incubation decreased the number of contaminating eubacteria and fungi but more importantly increased the number ofFrankia developing on the isolation plates. If the phenol incubation was used solely without sucrose fractionation noFrankia were isolated, suggesting the death of the organisms due to phenol toxicity. The use of selective nitrogen-deficient media proved important for the isolation of frankiae from soils.     

The influence of Bacillus subtilis RB14-C on the development of Rhizoctonia solani and indigenous microorganisms in the soil

The effect of soil inoculation with an antagonistic strain Bacillus subtilis RB14-C on the development of Rhizoctonia solani and changes occurring in soil and rhizosphere microbial communities were studied. RB14-C was added to the soil as a water suspension of the cells or as a broth culture. Application of cell suspensions to non-planted soil reduced the number of culturable bacteria. The density of R. solani and the number of filamentous fungi were not significantly affected by RB14-C. A similar effect was observed in the rhizosphere of tomato plants growns in bacterized soil. Broth cultures of RB14-C suppressed R. solani 1 d after inoculation, but after 3 d there was no difference in the pathogen density between soil amended with broth culture and control soil. In microcosm studies, cell suspensions of RB14-C also did not inhibit growth of R. solani on filters buried in soil. However, an inhibitory effect was obtained when a broth culture of the bacterium was used. The effect of RB14-C on fungal biomass was also estimated by measurement of ergosterol concentration in soil. It was found that ergosterol was mostly derived from R. solani and that there were no significant differences in its content between untreated soil and soil treated with RB14-C. The results suggest that suppression of Rhizoctonia damping-off by B. subtilis RB14-C probably is not related to the reduction of the pathogen population in the soil.     

New Method for Extraction of Streptomycete Spores from Soil and Application to the Study of Lysogeny in Sterile Amended and Nonsterile Soil

A new method for the isolation and enumeration of streptomycete spores from soil was developed. This method makes use of a cation-exchange resin to disperse soil particles. It allowed the detection of 10 spores in 100 g of sterile soil, while ca. 10³ could be accurately enumerated in 100 g. This method was applied to studying the fate of a marked actinophage in soil. In sterile amended and nonsterile soil, relatively high numbers of actinophages were only found during the first few days of the experiment when the host streptomycete was in the mycelial form. Later, after sporulation, lysogens could be detected in sterile amended soil and could still be found 60 days after inoculation. Although no lysogens were found in nonsterile soil, the introduced phage could still be detected in the free state after 60 days, albeit at a low titer.     

Proceedings: Preservation and storage of microorganisms in the gas phase of liquid nitrogen

Methods are described which permit the rapid preparation and retrieval of suspensions of microorganisms stored in the gas space of a liquid nitrogen refrigerator.Inocula are currently used for assay plates, turbidimetric assays, bioautograph plates, and various fermentation processes. The most suitable suspending agents, 10% glycerol with 5% of either lactose, maltose, or raffinose, were devised by determining viabilities from alternate freezing and thawing of cell suspensions.Four milliliters each of concentrated cell suspensions were pipetted into cotton-plugged or plastic-capped glass tubes (13 × 100 mm). After rapid noncontrolled freezing in the nitrogen gas phase, the tubes were stored horizontally either in test-tube racks or in bulk containers.Frozen suspensions of vegetative cells of some algae, many genera of bacteria, as well as spores, vegetative cells, and fragmented mycelia of streptomycetes and fungi have been conveniently prepared from either agar or broth cultures. Sufficient inocula have been prepared to be used for periods of a few weeks to several years.     

Method for selective isolation of Mycobacteria using olive oil emulsified with SDS

This paper describes the demulsification of olive-oil/SDS emulsion (DOSE) method for selective isolation of environmental mycobacteria. A soil sample was suspended in olive oil and centrifuged. The supernatant was emulsified on plates together with SDS solution. After incubation, the colonies that had developed on the plates were surrounded by clear zones. The isolates were identified as genus Mycobacterium, and as belonging to a fast-growing group, by 16S rRNA gene sequence analysis.     

Influences of soil acidity on Streptomyces populations inhabiting forest soils.

The Streptomyces populations inhabiting five acidic forest soils were examined. It was found that lowering the pH of a medium selective for streptomycetes (starch-casein agar) to the pH of the particular soil horizon being plated influenced both the total numbers and types of streptomycetes that were isolated from the soils examined in this study. On the acidified medium both the numbers of streptomycetes and the percentage of total bacteria on the plates represented by streptomycetes increased (as compared with the same medium with a pH of 7.2). These differences were greatest on the isolations from the most acid soils. The largest concentrations of streptomycetes were found in the surface horizon (0 to 15 cm) and the litter layer immediately over the surface mineral horizon. Acidity tolerance tests demonstrated that random samplings of isolates contained acidophilic, neutrophilic, and acidoduric strains, with the largest numbers of acidophiles being found on the acidified media from the most acid soils. There were no differences between overall utilization of selected carbohydrates among the isolates taken from either the neutral or acidic media, although a larger proportion of the acid media isolates produced acid from the carbohydrates. Evidence is presented which indicates that different types of streptomycetes were isolated on the acid media, and possible reasons for the presence of these acid-tolerant populations are discussed.     

Influences of soil acidity on Streptomyces populations inhabiting forest soils.

The Streptomyces populations inhabiting five acidic forest soils were examined. It was found that lowering the pH of a medium selective for streptomycetes (starch-casein agar) to the pH of the particular soil horizon being plated influenced both the total numbers and types of streptomycetes that were isolated from the soils examined in this study. On the acidified medium both the numbers of streptomycetes and the percentage of total bacteria on the plates represented by streptomycetes increased (as compared with the same medium with a pH of 7.2). These differences were greatest on the isolations from the most acid soils. The largest concentrations of streptomycetes were found in the surface horizon (0 to 15 cm) and the litter layer immediately over the surface mineral horizon. Acidity tolerance tests demonstrated that random samplings of isolates contained acidophilic, neutrophilic, and acidoduric strains, with the largest numbers of acidophiles being found on the acidified media from the most acid soils. There were no differences between overall utilization of selected carbohydrates among the isolates taken from either the neutral or acidic media, although a larger proportion of the acid media isolates produced acid from the carbohydrates. Evidence is presented which indicates that different types of streptomycetes were isolated on the acid media, and possible reasons for the presence of these acid-tolerant populations are discussed.     

Isolation and characterization of the Streptomyces cattleya temperate phage TG1

A temperate actinophage, TG1, was isolated from soil by growth on Streptomyces cattleya and has been shown to be potentially useful for the cloning of DNA in this organism and other streptomycetes. It forms stable lysogens by integration at a unique site on the chromosome. The phage genome consists of 41 kb of double-stranded DNA with cohesive ends. It has unique sites for ClaI, NdeI, PstI, SmaI, and XbaI. The PstI site has been shown to be in a dispensable region of the phage genome. Deletions (2 kb in length) were obtained which retain this site and should be useful for the cloning of DNA.     

Method for isolating cyanobacteria-lysing streptomycetes from soil

A new technique is described for the isolation and enumeration of cyanobacteria-lysing Streptomyces spp from soil or water. Two cyanobacteria, Anabaena cylindrica (ACTT 27899) and Tolypothrix tenuis (ATCC 27914) were used as the test organisms. Ten-day-old cyanobacterial cultures were vacuum-filtered to form a lawn on a 7 cm Whatman No. 1 filter paper which was then supported on Allen's agar. When the lawn was inoculated with dilutions of a heavy clay prairie soil and incubated at 27 PT 1dEC under constant illumination, white or grey colonies of streptomycetes grew. These colonies were surrounded by zones where a yellowing and lysis of the algal cells occurred. Streptomyces achromogenes was identified as a major lytic species within a population of 5 times 10₃ plaque-producing streptomycetes/g (dry weight) soil.     

An automated technique for most-probable-number (MPN) analysis of densities of phagotrophic protists with lux AB labelled bacteria as growth medium.

An automated modification of the most-probable-number (MPN) technique has been developed for enumeration of phagotrophic protozoa. The method is based on detection of prey depletion in micro titre plates rather than on presence of protozoa. A transconjugant Pseudomonas fluorescens DR54 labelled with a luxAB gene cassette was constructed, and used as growth medium for the protozoa in the micro titre plates. The transconjugant produced high amounts of luciferase which was stable and allowed detection for at least 8 weeks. Dilution series of protozoan cultures and soil suspensions were inoculated into micro titre plates amended with a suspension of the transconjugant. After 45 days measurement of light emission allowed detection of individual wells in the titre plates, where protozoan grazing had removed the inoculated bacteria.     

Characterization of the sporulation control protein SsgA by use of an efficient method to create and screen random mutant libraries in streptomycetes

Filamentous actinomycetes are commercially widely used as producers of natural products. However, the mycelial lifestyle of actinomycetes has been a major bottleneck in their commercialization, and screening is difficult due to their poor growth on microtiter plates. We previously demonstrated that the enhanced expression of the cell division activator protein SsgA results in the fragmented growth of streptomycetes, with enhanced growth rates and improved product formation. We here describe a novel and efficient method to create, maintain, and screen mutant libraries in streptomycetes and the application of this method for the functional analysis of Streptomyces coelicolor ssgA. The variants were amplified directly from deep-frozen biomass suspensions. Around 800 ssgA variants, including single-amino-acid-substitution mutants corresponding to more than half of all SsgA residues, were analyzed for their abilities to restore sporulation to an ssgA mutant. The essential residues were clustered in three main sections, and hardly any were in the carboxy-terminal third of the protein. The majority of the crucial residues were conserved among all SsgA-like proteins (SALPs). However, the essential residues L29, D58, and S89 were conserved only in SsgA orthologues and not in other SALPs, suggesting an SsgA-specific function.