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Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Aims: To devise and evaluate a method for isolating the rare, zoosporic actinomycetes, Actinokineospora spp. in soil and plant litter.

Methods and Results: The newly developed method consists of two enrichment stages followed by plating on a selective medium. The source material is initially incubated with calcium carbonate to multiply the population of Actinokineospora spp., and is then air-dried. The second stage consists of rehydration-centrifugation, in which the amended substrate is immersed in phosphate buffer-soil extract to liberate actinomycete zoospores, and nonmotile microbial associates are then eliminated by centrifugation. Portions of the supernatant enriched with zoospores are plated on humic-acid vitamin agar supplemented with fradiomycin, kanamycin, nalidixic acid and trimethoprim. We examined 39 soil and plant-litter samples taken from fields, forests and stream banks. The proposed method consistently enriched and selectively isolated Actinokineospora spp. in 17 samples. Evidence for antimicrobial activity was found in most of the isolates.

Conclusions: A combination of enrichment and a medium containing selective antibiotics can be used successfully for efficient isolation of certain rare actinomycete taxa.

Significance and Impact of the Study: The development of new methodologies with which to isolate rare actinomycetes is of great importance to extend our understanding of their ecology, taxonomy and bioactivity.


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Besides their ecological significance to biodegradation (Williams et al. 1984), interest in actinomycetes concerns their capacity to produce industrially useful metabolites such as antibiotics and enzymes (Okami and Hotta 1988; Peczynska-Czoch and Mordarski 1988). Much effort has long been focused upon the genus Streptomyces, which is the most abundant and a recoverable actinomycete group in soil. Extensive screening of this taxon has led to the discovery of many novel strains that produce useful secondary metabolites (Tanaka and Omura 1990). Although Streptomyces spp. continue to provide new bioactive products, reliable methodologies are required to isolate rare and unusual actinomycetes, to reduce the re-isolation of strains producing known bioactive compounds and to improve the quality of natural products screened (Goodfellow and Williams 1986).

Members of the genus Actinokineospora (Hasegawa 1988) are aerobic, mesophilic actinomycetes that characteristically produce a long branching substrate mycelium and a relatively short, flexuous aerial mycelium with chains of rod-shaped spores. These spores become motile through lophotrichous flagella under aqueous conditions. This genus currently accommodates five validated species, of which Actinokineospora riparia (strain C-39162) produces a macromolecular compound with antimycoplasmal activity (Hasegawa 1991). Actinokineospora spp. are associated in nature with soil and plant litter (Tamura et al. 1995), but they typically represent only a minor component of the microbial population. Therefore, isolation of this rare actinomycete group using conventional dilution plating procedures is precluded by the presence of numerically dominant and faster growing microbial associates on the plates.

More recently, an effective enrichment technique, designated rehydration and centrifugation (RC), has been developed for the isolation of motile actinomycetes (Hayakawa et al. 2000). The RC method consists of accumulating actinomycete zoospores in a favourable flooding solution above the substrate, centrifuging the fluid, then plating the supernatant on humic acid-vitamin (HV) agar supplemented with nalidixic acid and trimethoprim (Hayakawa and Nonomura 1987). Actinokineospora spp. can be recovered on isolation plates depending on the natural sample source. However, they are still largely outnumbered by Actinoplanes spp. and related associates, causing problems in recognizing and isolating Actinokineospora colonies.

The present study aimed to develop an efficient strategy with which to isolate Actinokineospora spp. by combining the RC method with HV agar containing highly selective agents for this rare actinomycete group and to exploit their bioactivity with respect to antibiotic synthesis. A preliminary survey revealed that all species of the genus Actinokineospora have the ability to grow in the presence of the aminoglycoside antibiotics, fradiomycin and kanamycin. This resistance was utilized to construct a novel isolation method. The applicability of using calcium carbonate (Tsao et al. 1960) for pre-enrichment was also tested to determine whether or not the likelihood of Actinokineospora spp. appearing on isolation plates is increased.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Strains and culture conditions

Forty-two strains of motile bacteria (Table 1), including five of the actinomycete genus Actinokineospora, were stored on oatmeal-YGG agar (Hayakawa et al. 1982) or nutrient agar slopes, and their capability to resist antibacterial agents was investigated. Their origins and culture conditions were as described (Hayakawa et al. 1991a).

Table 1.   Ability of a range of motile actinomycetes and other bacteria to grow in the presence of antibacterial agents Thumbnail image of

Sensitivity test to antibacterial agents

Basal HV-N agar (Hayakawa and Nonomura 1989) was autoclaved, then supplemented with fradiomycin (40 mg l–1; Wako Pure Chemical Ind., Osaka, Japan), kanamycin (40 mg l–1; Wako), or with a mixture of nalidixic acid (10 mg l–1: Sigma Chemical Co., St Louis, USA) and trimethoprim (20 mg l–1; Wako) (Hayakawa et al. 1996a). When appropriate, these four antibacterial agents were all incorporated into autoclaved HV-N agar. Dense suspensions of actinomycetes and other bacteria, prepared as described (Hayakawa and Nonomura 1989), were streaked onto plate surfaces and growth was scored relative to that on basal medium alone, at appropriate intervals up to 14 d.

Recovery of Actinokineospora strains from zoospore suspensions

Basal HV agar was supplemented after being autoclaved with the same mixture of the four antibacterial agents described above. Zoospore suspensions of the test motile actinomycetes were prepared in 10 mmol l–1 phosphate buffer (pH 7·0) containing 10% soil extract under the described conditions (Hayakawa et al. 2000). The suspension was diluted with sterile tap water and 0·2 ml aliquots were plated in triplicate onto HV agar and onto the same agar supplemented with the antibacterial agents. The plates were incubated for 14 d, then appearing colonies were counted. Experiments were performed in triplicate to obtain mean colony counts.

Isolation procedure for Actinokineospora spp. from natural substrates

Isolation media and natural samples.

The isolation media were HV agar with or without a mixture of fradiomycin (40 mg l–1), kanamycin (40 mg l–1), nalidixic acid (10 mg l–1) and trimethoprim (20 mg l–1). The media also contained cycloheximide (50 mg l–1; Wako) to suppress fungal growth (Williams and Davies 1965).

Thirty-five soil samples and four leaf-litter samples (decaying leaves) were collected from several locations in Yamanashi and Nagano prefectures (Japan). Soil samples, as well as leaf-litter samples previously ground with a blender, were sieved and air-dried, then actinomycetes were isolated. The chemical nature (pH, moisture content, and loss on ignition as a function of humus content) of these samples was determined as described (Hayakawa and Nonomura 1987).

Sample preparation, inoculation and isolation.

Calcium carbonate as proposed by Tsao et al. (1960) was applied with slight procedure modifications. Samples of air-dried soil (5 g) or leaf-litter (3 g) were mixed in a mortar with 0·5 g or 0·3 g, respectively, of powdered calcium carbonate and spread over the surface of a glass-fibre filter (70 mm in diameter, 0·44 mm thickness; ADVANTEC Inc, Tokyo, Japan) that was moistened with 3 ml of sterilized tap water and placed in a Petri dish. Where necessary, 1–2 ml of sterilized tap water was added to the sample to give a moisture content of about 20% (w/w). The Petri dish functioned as a moist chamber, and was maintained at 26°C for 14 d. The sample was then air-dried at room temperature to a constant weight.

Samples (0·5 g) processed or not (control) with calcium carbonate, were placed in a glass vessel and flooded with 50 ml of 10 mmol l–1 phosphate buffer (pH 7·0) containing 10% soil extract at 30°C for 2 h to liberate actinomycete zoospores. A portion (8 ml) of the flooding mixture was transferred into a screw-cap test tube and centrifuged at 1500 × g for 20 min in a swinging bucket rotor. After settling for 30 min, a portion of the supernatant enriched with zoospores was serially diluted with sterile tap water, then 0·2 ml aliquots were plated in triplicate onto plates of HV agar with or without antibacterial agents (Hayakawa et al. 2000).

All plates were incubated at 30°C for 2–3 weeks before counting actinomycete colonies, and all experiments were performed in triplicate. Actinomycetes were examined by eye and by using a light microscope equipped with a 40 × long working distance objective (model ULWDCDPlan; Olympus, Tokyo, Japan) and tentatively identified up to genus rank based on morphological criteria (Labeda 1987; Cross 1989; Lechevalier 1989). Actinokineospora strains were identified as those isolates forming thin, flat colonies with sparse white aerial hyphae. Microscopic observation subsequently confirmed the formation of long branching substrate hyphae and relatively short, tufted aerial hyphae with chains of rod-shaped arthrospores (Hasegawa 1988). Spore motility was confirmed in hanging drops by light microscopy.

Taxonomic analyses

Strains.

Twenty-nine representative isolates with the morphology typical of the genus Actinokineospora according to light microscopy, were subcultured and their taxonomic properties examined in greater detail. Type strains of Actinokineospora diospyrosa, A. globicatena, A. inagensis, A. riparia and A. terrae were simultaneously compared. Distilled water suspensions of spores and hyphae, prepared from stock culture slants, were used as inocula (Shirling and Gottlieb 1966). All cultures were incubated at 28°C for 14 d.

Morphological, cultural and physiological characterization.

Cultures were grown on HA agar (Nonomura et al. 1979), or on oatmeal-YGG agar supplemented with soil extract (10%, v/v; Henrich 1947), and morphology was observed by light and scanning electron microscopy (Hayakawa et al. 1996b). Spore motility and flagellation were observed as described (Hayakawa et al. 2000). Substrate mycelial colour and soluble pigment production were observed on yeast extract-malt extract agar (Shirling and Gottlieb 1966). Colours were identified with reference to the Guide to Color Standard (Japan Color Research Institute 1954).

Carbohydrate utilization was tested by the method of Shirling and Gottlieb (1966) using C2 (Nonomura and Ohara 1971) agar. The degradation of starch (0·5 g l–1) and calcium malate (0·5 g l–1) in Bennett’s agar (Jones 1949) was detected as described by Williams et al. (1983). Gelatin hydrolysis and nitrate reduction were examined as described by Nonomura et al. (1979) and Gordon (1968), respectively.

Chemotaxonomic analyses.

Cell-wall sugars, polar lipids, isoprenoid quinones, and diaminopimelic acid isomer were analysed as described (Hayakawa et al. 2000).

16S rDNA sequence analysis.

Genomic DNA was extracted as described by Saito and Miura (1963) and amplification of 16S ribosomal DNA (rDNA) genes was PCR (Saiki et al. 1988) mediated using TaKaRa Taq polymerase (Takara Shuzo, Kyoto, Japan) and the primer pairs, 9F (5′-GAGTTTGATCCTGGCTCAG) and 1541R (5′-AAGGAGGTGATCCAGCC). Amplified 16S rDNA (1·5 kb) was purified and directly sequenced using an ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, Calif., USA) as specified by the manufacturer. The sequencing primers were: 9F (5′-GAGTTTGATCCTGGCTCAG), 515F (5′-GTGCCAGCAGCCGCCGCGGT), 536R (5′-GTATTACCGCGGCTGCTG), 785F (5′-GGATTAGATACCCTGGTAGTC), 802R (5′-TACCAGGGTATCTAAT CC), 1099F (5′-GCAACGAGCGCAACCC), 1115R (5′-AGGGTTGCGCTCGTTG), and 1541R (5′-AAGGAGGTGATCCAGCC). An Applied Biosystems PRISM 310 Genetic Analyser performed electrophoresis of the sequencing reaction mixtures.

The 16S rDNA sequences determined in this study were manually aligned with the published sequences of reference strains available from the EMBL/GenBank/DDBJ databases. The CLUSTAL W software package (Thompson et al. 1994) generated evolutionary distances (the Knuc value of Kimura 1980) and similarity values. A phylogenetic tree was constructed by neighbour-joining (Saitou and Nei 1987) from Knuc values. The topology of the phylogenetic tree was evaluated by bootstrap re-sampling as described by Felsenstein (1985) with 1000 replicates.

Nucleotide sequence accession numbers.

The 16S rDNA sequences of strains YU 873-1 and YU 923-201 are available from the EMBL/GenBank/DDBJ databases under accession numbers AB048863 and AB048864, respectively.

Determination of antimicrobial activity

The ability to inhibit the growth of Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed using an overlay method (Williams et al. 1983). The tested bacteria were Bacillus subtilis IFO 13719, Micrococcus luteus IFO 12708, Staphylococcus aureus IFO 3061, Agrobacterium rhizogenes IFO 14554, Escherichia coli IFO 3044 and Pseudomonas fluorescens IFO 14106. The yeasts examined were Candida krusei (laboratory strain) and Saccharomyces cerevisiae IFO 10217. The filamentous fungi included Aspergillus niger ATCC 9642 and Aspergillus oryzae (laboratory strain). Bennett’s agar supplemented with humic acid (0·5 g l–1) (Hayakawa et al. 1995) was the antibiotic production medium. Spot-inoculated, 10-d-old colonies on the plates were inverted over 1·5 ml chloroform for 40 min. Killed colonies were overlaid with 5 ml of sloppy Bennett’s agar inoculated with the test organisms. Zones of inhibition around the colonies were recorded after 24 h at 30°C.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Susceptibility to fradiomycin and kanamycin

Using dense cell suspensions as inocula, we investigated the growth of a range of motile actinomycetes (29 strains: five genera and 29 species) and nonfilamentous bacteria (13 strains: four genera and 12 species), that represent types found in soil, in the presence of antibacterial agents (Table 1). Fradiomycin (40 mg l–1) and kanamycin (40 mg l–1) had no effect on the growth of all the test Actinokineospora strains. In contrast, the growth of other test strains was inhibited or restricted by fradiomycin and/or kanamycin, except for Actinosynnema mirum, Agrobacterium rhizogenes, Pseudomonas fluorescens and Rhizobium leguminosarum strains which were less sensitive to both antibiotics. Growth of the Gram-negative bacteria, Agrobacterium rhizogenes, Pseudomonas fluorescens and Rhizobium leguminosarum, was suppressed when a mixture of fradiomycin and kanamycin was combined with that of nalidixic acid (10 mg l–1) and trimethoprim (20 mg l–1), which are selective inhibitors of Gram-negative bacteria and bacilli (Hayakawa et al. 1996a).

Improvement to the isolation medium

Diluted zoospore suspensions of the actinomycete strains listed in Table 1 were plated onto HV agar with or without a mixture of fradiomycin (40 mg l–1), kanamycin (40 mg l–1), nalidixic acid (10 mg l–1) and trimethoprim (20 mg l–1) to improve medium selectivity. We then confirmed the recovery of each strain on the two media. Viable counts, as well as colony sizes and aerial hyphae formation of all the Actinokineospora reference strains tested were not affected by the antibacterial agents (data not shown). Virtually no colonies of other actinomycete strains appeared on the HV agar containing antibacterial agents, except for Actinosynnema mirum and Actinoplanes missouriensis. Colony counts of these strains on the amended HV agar were 82% and 7% of those on the HV agar control, respectively.

Enrichment and selective isolation of Actinokineospora spp.

Sample preparation and selective media were used to preferentially isolate Actinokineospora spp. from a sample of forest soil no. 838 (Table 2). Actinokineospora spp. were recovered on HV agar by rehydration-centrifugation (RC) (Hayakawa et al. 2000). However, microbes, including undesirable zoosporic actinomycetes and motile unicellular bacteria, highly contaminated the isolation plates and Actinokineospora spp. (2·9 × 104 cfu g–1 of dried soil) constituted only 1% of the total microbial population recovered. We therefore attempted to increase the quantitative recovery of Actinokineospora spp. The number of Actinokineospora colonies on HV agar significantly increased when the calcium carbonate procedure (Tsao et al. 1960; El-Nakeeb and Lechevalier 1963) was applied before the RC method. Although this protocol concomitantly and moderately increased the number of undesirable motile bacteria including actinoplanetes such as Actinoplanes spp., further growth was severely curtailed by incorporating a mixture of fradiomycin, kanamycin, nalidixic acid and trimethoprim into the HV agar. These antibacterial agents had no adverse effect on the emergence and development of Actinokineospora colonies. Thus, the integrated procedure consisting of calcium carbonate soil treatment, the RC technique, and plating on the HV agar supplemented with antibacterial agents, yielded a high colony count (2·9 × 105 cfu g–1 of dried soil) of Actinokineospora spp. that accounted for 44% of the total colonies recovered.

Table 2.   Effects of calcium carbonate and antibacterial agents on isolation of Actinokineospora spp. from soil* Thumbnail image of

The efficiency of the integrated method for isolating Actinokineospora spp. was confirmed when applied to 39 different soil and leaf-litter samples collected from fields, forests and stream banks (Table 3). From 17 samples (15 of soils and two of leaf-litter materials), the integrated method selectively isolated Actinokineospora spp., constituting 4–86% of the total microbial population recovered. Among the samples examined, the most favourable isolation sources for Actinokineospora spp. were forest soils that were rich in humus and not too acidic.

Table 3.   Selective isolation of Actinokineospora spp. from various soil and plant-litter samples using newly developed method* Thumbnail image of

A typical isolation plate prepared by the integrated method is shown in Fig. 1. The Actinokineospora colonies that developed were thin and flat with sparse white aerial hyphae. Microscopic observation revealed long, straight substrate hyphae with branching and relatively short, tufted aerial hyphae.

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Figure 1.  Isolation plate prepared from soil no. 924 (level-land forest, pH 5·5; 14·8% loss on ignition) using newly developed method (a) after 2 weeks incubation at 28°C (arrows indicate representative Actinokineospora colonies), and light microscopy of Actinokineospora colony on the plate (b) showing long branching substrate hyphae, from which aerial hyphae arise in tufts. Bar, 10 μm

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Taxonomic evaluation of the isolates

Confirmation of generic identification.

To validate the presumptive generic identification according to light microscopy, 29 putative Actinokineospora strains isolated from 17 samples and randomly selected, were further tested by scanning electron microscopy (SEM) and chemical analyses. In addition, two representative isolates were characterized by 16S rDNA sequence analysis to establish their phylogenetic positions.

SEM showed that all 29 test isolates produced branched, flexuous aerial hyphae that divided to form the chains of rod-shaped arthrospores. The spores of all the test strains were smooth-surfaced and became motile through lophotrichous flagella under aqueous conditions. Detailed spore-chain morphology of typical strains is illustrated in Fig. 2. Spore chains of strain YU 924-201 at maturity tended to aggregate into clusters resembling sporodochia. Chemical analyses on the other hand, revealed that the test 29 strains all contained meso-diaminopimelic acid as the cell-wall diamino acid. The principal whole-cell sugars were arabinose, galactose and rhamnose. The predominant menaquinone component of the test strains was MK-9(H4), and they also contained phosphatidylethanolamine (PE) as well as hydroxy-PE (phospholipid type PII sensuLechevalier et al. 1981). These morphological and chemical properties are consistent with the classification of the 29 test isolates into Actinokineospora.

image

Figure 2.  Isolates of Actinokineospora, sporulating aerial hyphae. Scanning electron microscopy. (a–c) Strain YU 923-201. (d–f ) Strain YU 961–201. Bar: a, d, 5 μm; b, c, e, 2 μm; f, 1 μm

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The 16S rDNA sequences (> 1400 bases; positions 28–1524, according to the Escherichia coli numbering system of Brosius et al. 1978) of the isolates YU 873-1 and YU 923-201 were almost completely determined and compared with the sequences of selected members of the family Actinosynnemataceae (Labeda and Kroppenstedt 2000). The phylogenetic dendrogram derived from evolutionary distances determined by neighbour-joining is shown in Fig. 3. A total of 1339 nucleotides were analysed after eliminating all sites that were not determined in any sequence. Isolates YU 873-1 and YU 923-201 were closely related to Actinokineospora riparia NRRL B-16432 (= IFO 14541; type strain of the genus Actinokineospora) and these three strains formed a coherent cluster supported by bootstrap analysis at a confidence level of 88%. The values of 16S rDNA sequence similarity between the test isolates and Actinokineospora riparia NRRL B-16432 were 96·7–98·6%.

image

Figure 3.  Neighbour-joining tree based on 16S rDNA gene sequences, showing relationships among strains YU 873-1 and YU 923-201 and representatives of the family Actinosynnemataceae. Bar, 0·01 nucleotides substitutions per site. Numbers on branches are confidence limits estimated from bootstrap analysis of 1000 replicates

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Characterization based on phenotypic properties.

The 29 test isolates were classified according to the phenotypic criteria recommended by Tamura et al. (1995) as being useful for differentiating Actinokineospora species (Table 4). The results divided the isolates into 10 phenotypic groups including four groups of single members. Of the 29 test isolates, 15 shared all test morphological, cultural and physiological characteristics with Actinokineospora diospyrosa IFO 15665, Actinokineospora globicatena IFO 15664, or Actinokineospora terrae IFO 15668. However, the remaining 14 strains, which represented seven phenotypic groups, were differentiated from any known Actinokineospora strain by some taxonomic properties.

Table 4.   Some morphological and physiological characteristics of Actinokineospora isolates (29 strains) Thumbnail image of

Antimicrobial activity

The Actinokineospora isolates were tested for antimicrobial activity. Of the 29 test strains, 28 (97%) exhibited inhibitory activity. Most of this activity was directed against Gram-positive bacteria, which were inhibited by 25 (86%) of the isolates. Within this bacterial group, Micrococcus luteus was the most susceptible, followed closely by Staph. aureus. Thirteen isolates (45%) inhibited Gram-negative bacteria. Agrobacterium rhizogenes was the least susceptible of the Gram-negative bacteria tested, as it was inhibited by only one isolate. Among the 13 active isolates, nine strains also inhibited Gram-positive bacteria. Antimycotic activity was found in 11 isolates (38%), all of which inhibited Aspergillus oryzae, whereas only four had anti Candida activity. These 11 strains were also active against Gram-positive and/or Gram-negative bacteria.

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

Methodologies that favour the selective isolation of actinomycetes have been extensively reviewed (Cross 1982; Williams and Wellington 1982; McCarthy 1985; Goodfellow and O’Donnel 1989; Labeda and Shearer 1990). One reasonable approach is to multiply actinomycete propagules (enrichment) in the source materials prior to plating. The prior incubation of soil samples with calcium carbonate (Tsao et al. 1960), or with organic materials such as chitin (Porter and Wilhelm 1961; Williams et al. 1972), significantly increases the actinomycete population, thereby yielding higher total and relative plate counts. Enrichment can also be used for the isolation of specific actinomycete taxa that rarely appear on spread plates prepared by conventional dilution procedures. The most popular means of isolating motile actinoplanetes such as Actinoplanes spp. rely upon baiting with pollen and hair (Couch 1954; Nonomura and Takagi 1977; Hayakawa et al. 1991c) and accumulating zoospores in glass capillaries containing chemoattractants (Palleroni 1980; Hayakawa et al. 1991b). Rehydration-centrifugation (RC) is a novel isolation technique that universally favours diverse motile actinomycetes. The procedure involves an enrichment stage that promotes the liberalization of zoospores from a substrate by flooding (Hayakawa et al. 2000).

Successful isolation of Actinokineospora spp. depends on combining the calcium carbonate and RC procedures. Incubating source materials moistened with calcium carbonate prior to RC significantly increased the number of Actinokineospora spp. on HV plates, thus offering a considerable advantage for yielding a high number and greater diversity of subcultures. A subsequent taxonomic study has found that Actinokineospora isolates can be categorized into diverse phenotypes. Although the rationale behind the calcium carbonate effect remains to be studied, Tsao et al. (1960) have stressed that the pH of the isolation sources after mixing with powdered calcium carbonate would be altered in favour of the growth of actinomycete propagules. Calcium ions reportedly stimulate the formation of aerial mycelia by several actinomycete cultures (Natsume et al. 1989).

Since enrichment usually allows for the concomitant increase of impeding microbes, selective media must be used for isolation. In the present study, undesirable zoosporic actinomycetes and motile unicellular bacteria that arose on the HV isolation plates were significantly reduced by supplementing the medium with specific antibacterial agents. Fradiomycin and kanamycin were included because they inhibit the growth of a wide range of motile actinomycetes apart from Actinokineospora strains. Nalidixic acid and trimethoprim were also included, since Actinokineospora strains were entirely insensitive to these inhibitors of Gram-negative bacteria and bacilli (Hayakawa et al. 1996a). The efficiency of using all four of these antibacterial agents in reducing a wide range of contaminating bacteria may be due to differences in their antimicrobial spectra. Their synergistic antibacterial effect may also account for the efficient de-contamination. Tests of pure cultures showed a synergistic association of the four antibacterials with Pseudomonas fluorescens, a commonly encountered motile bacterium (Table 1).

The development of new methodologies with which to isolate actinomycetes has altered understanding about their ecology, taxonomy and bioactivity (Goodfellow 1992; Williams et al. 1993). While the isolation procedure described here cannot be used to estimate the actual population of Actinokineospora spp. in natural substrates, it has already revealed considerable information about their distribution. Actinokineospora spp. are more common in soil and plant-litter than was previously estimated. Evidence for the frequent occurrence of Actinokineospora spp. in forest soils and decaying leaves suggests that they play roles in the degradation of plant remains, but more detailed studies are required to determine their significance in such ecosystems. The proposed isolation method has provided various Actinokineospora cultures, which can be clearly differentiated from known species in terms of phenotypic properties. Further investigation, including numerical phenetic and molecular systematic analyses, could reveal their exact taxonomic status at the species rank. Actinokineospora isolates may also be worthy of investigation in natural product screening programmes, since most of the test strains have significant antibiotic activity against diverse microbes.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References

The authors thank Dr T. Tamura and Dr K. Hatano of Institute for Fermentation, Osaka and Mr H. Kobayashi of Mercian Co. Ltd. for their helpful suggestions regarding 16S rDNA analysis. Appreciation is also given to Mr M. Kimura for technical assistance.

This study was partly supported by the TV Yamanashi science development fund.

References

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. References
  • 1
    Brosius, J., Palmer, J.L., Kennedy, J.P., Noller, H.F. (1978) Complete nucleotide sequences of a 16S ribosomal RNA gene from Escherichia coli. Proceedings of the National Academy of Sciences USA 75 , 48014805.
  • 2
    Couch, J.N. (1954) The genus Actinoplanes and its relatives. Transactions New York Academy of Sciences 16 , 315318.
  • 3
    Cross, T. (1982) Actinomycetes: a continuing source of new metabolites. Developments in Industrial Microbiology 23 , 118.
  • 4
    Cross, T. (1989) Growth and examination of actinomycetes—some guidelines. In Bergey’s Manual of Systematic Bacteriology Vol. 4, eds Williams, S.T., Sharpe, M.E. and Holt, J.P. pp. 2340–2343. Baltimore: Williams & Wilkins.
  • 5
    El-Nakeeb, M.A. & Lechevalier, H.A. (1963) Selective isolation of aerobic actinomycetes. Applied Microbiology 11 , 7577.
  • 6
    Felsenstein, J. (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39 , 783791.
  • 7
    Goodfellow, M. (1992) The family Streptosporangiaceae. In The Prokaryotes eds Balows, A., Trüper, H.G., Dworkin, M., Harder, W. and Schleifer, K.-H. pp. 1115–1138. Berlin: Springer Verlag.
  • 8
    Goodfellow, M. & O'Donnel, A.G. (1989) Search and discovery of industrially significant actinomycetes. In Microbial Products: New Approaches, Society for General Microbiology Symposium No. 44 eds Baumberg, S., Hunter, I.S. and Rhodes, P.M. pp. 343–383. Cambridge: Cambridge University Press.
  • 9
    Goodfellow, M. & Williams, E. (1986) New strategies for the selective isolation of industrially important bacteria. Biotechnology and Genetic Engineering Reviews 4 , 213262.
  • 10
    Gordon, R.E. (1968) The taxonomy of soil bacteria. In The Ecology of Soil Bacteria eds Gray, T.R.G. and Parkinson, D. pp. 293–321. Liverpool: Liverpool University Press.
  • 11
    Hasegawa, T. (1988) Actinokineospora: a new genus of the Actinomycetales. Actinomycetologica 2 , 3145.
  • 12
    Hasegawa, T. (1991) Studies on motile arthrospore-bearing rare actinomycetes. Actinomycetologica 5 , 6471.
  • 13
    Hayakawa, M. & Nonomura, H. (1987) Humic acid-vitamin agar, a new medium for the selective isolation of soil actinomycetes. Journal of Fermentation Technology 65 , 501509.
  • 14
    Hayakawa, M. & Nonomura, H. (1989) A new method for the intensive isolation of actinomycetes from soil. Actinomycetologica 3 , 95104.
  • 15
    Hayakawa, M., Iino, S., Nonomura, H. (1982) Heavy metal resistance and melanoid pigment production in the streptomycete flora of copper-polluted vineyard soils. Hakkokogaku 60 , 19.
  • 16
    Hayakawa, M., Sadakata, T., Kajiura, T., Nonomura, H. (1991a) New methods for the highly selective isolation of Micromonospora and Microbispora from soil. Journal of Fermentation and Bioengineering 72 , 320326.
  • 17
    Hayakawa, M., Tamura, T., Nonomura, H. (1991b) Selective isolation of Actinoplanes and Dactylosporangium from soil by using ã-collidine as the chemoattractant. Journal of Fermentation and Bioengineering 72 , 426342.
  • 18
    Hayakawa, M., Tamura, T., Iino, H., Nonomura, H. (1991c) Pollen-baiting and drying method for highly selective isolation of Actinoplanes spp. from soil. Journal of Fermentation and Bioengineering 72 , 433438.
  • 19
    Hayakawa, M., Ishizawa, T., Yamazaki, T., Nonomura, H. (1995) Distribution of antibiotic-producing Microbispora strains in soils with different pHs. Actinomycetes 6 , 7579.
  • 20
    Hayakawa, M., Takeuchi, T., Yamazaki, T. (1996a) Combined use of trimethoprim with nalidixic acid for the selective isolation of actinomycetes from soil. Actinomycetologica 10 , 8090.
  • 21
    Hayakawa, M., Momose, Y., Yamazaki, T., Nonomura, H. (1996b) A method for the selective isolation of Microtetraspora glauca and related four-spored actinomycetes from soil. Journal of Applied Bacteriology 80 , 375386.
  • 22
    Hayakawa, M., Otoguro, M., Takeuchi, T., Yamazaki, T., Iimura, Y. (2000) Application of a method incorporating differential centrifugation for selective isolation of motile actinomycetes in soil and plant litter. Antonie Van Leeuwenhoek 78 , 171185.
  • 23
    Henrich, A.T. (1947) Methods for studying molds, yeasts, and actinomycetes. In Molds, Yeasts, and Actinomycetes eds Skinner, C.E., Emmons, C.W. and Tsuchiya, H.M. pp. 49–80. New York: Wiley and Sons.
  • 24
    Japan Color Research Institute (1954) Guide to Color Standard. Tokyo: Nippon Silisai Co., Ltd.
  • 25
    Jones, K.L. (1949) Fresh isolates of actinomycetes in which the presence of sporogenous aerial mycelia is a fluctuating characteristic. Journal of Bacteriology 57 , 141145.
  • 26
    Kimura, M. (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16 , 111120.
  • 27
    Labeda, D.P. (1987) Actinomycete taxonomy: generic characterization. Developments in Industrial Microbiology 28 , 115121.
  • 28
    Labeda, D.P. & Kroppenstedt, R.M. (2000) Phylogenetic analysis of Saccharothrix and related taxa: proposal for Actinosynnemataceae fam. nov. International Journal of Systematic and Evolutionary Microbiology 50 , 331336.
  • 29
    Labeda, D.P. & Shearer, M. (1990) Isolation of actinomycetes for biotechnological applications. In Isolation of Biotechnological Organisms from Nature ed. Labeda, D.P. pp. 1–19. New York: McGraw-Hill.
  • 30
    Lechevalier, H.A. (1989) A practical guide to generic identification of actinomycetes. In Bergey’s Manual of Systematic Bacteriology Vol. 4, eds Williams, S.T., Sharpe, M.E. and Holt, J.P. pp. 2344–2347. Baltimore: Williams & Wilkins.
  • 31
    Lechevalier, M.P., Stern, A.E., Lechevalier, H.A. (1981) Phospholipids in the taxonomy of actinomycetes. In: Actinomycetes eds Schaal, K.P. and Pulverer, G. pp. 111–116. New York: Gustav Fisher-Verlag.
  • 32
    McCarthy, A.J. (1985) Developments in the taxonomy and isolation of thermophilic actinomycetes. Frontiers in Applied Microbiology 1 , 114.
  • 33
    Natsume, M., Yasui, K., Marumo, S. (1989) Calcium ion regulates aerial mycelium formation in actinomycetes. The Journal of Antibiotics 42 , 440447.
  • 34
    Nonomura, H. & Ohara, Y. (1971) Distribution of actinomycetes in soil. IX. New species of the genera Microbispora and Microtetraspora, and their isolation method. Journal of Fermentation Technology 49 , 887894.
  • 35
    Nonomura, H. & Takagi, S. (1977) Distribution of actinoplanetes in soils of Japan. Journal of Fermentation Technology 55 , 423428.
  • 36
    Nonomura, H., Iino, S., Hayakawa, M. (1979) Classification of actinomycetes of genus Ampullariella from soils of Japan. Journal of Fermentation Technology 57 , 7985.
  • 37
    Okami, Y. & Hotta, K. (1988) Search and discovery of new antibiotics. In Actinomycetes in Biotechnology eds Goodfellow, M., Williams, S.T. and Mordarski, M. pp. 33–67. London: Academic Press.
  • 38
    Palleroni, N.J. (1980) A chemotactic method for the isolation of Actinoplanaceae. Archives of Microbiology 128 , 5355.
  • 39
    Peczynska-Czoch, W. & Mordarski, M. (1988) Actinomycete enzymes. In Actinomycetes in Biotechnology eds Goodfellow, M., Williams, S.T. and Mordarski, M. pp. 220–283. San Diego, CA: Academic Press.
  • 40
    Porter, J.N. & Wilhelm, J.J. (1961) The effect of Streptomyces populations of adding various supplements to soil samples. Developments in Industrial Microbiology 2 , 253259.
  • 41
    Saiki, R.K., Gelfand, D.H., Stoffe, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Erlich, A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239 , 487491.
  • 42
    Saito, H. & Miura, K. (1963) Preparation of transforming deoxyribonucleic acid by phenol treatment. Biochimica et Biophysica Acta 72 , 619629.
  • 43
    Saitou, N. & Nei, M. (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4 , 406425.
  • 44
    Shirling, E.B. & Gottlieb, D. (1966) Methods for characterization of Streptomyces species. International Journal of Systematic Bacteriology 16 , 313340.
  • 45
    Tamura, T., Hayakawa, M., Nonomura, H., Yokota, A., Hatano, K. (1995) Four new species of the genus Actinokineospora: Actinokineospora inagensis sp. nov., Actinokineospora globicatena sp. nov., Actinokineospora terrae sp. nov. and Actinokineospora diospyrosa sp. nov. International Journal of Systematic Bacteriology 45 , 371378.
  • 46
    Tanaka, Y. & Omura, S. (1990) Metabolism and products of Actinomycetes—an introduction. Actinomycetologica 4 , 1314.
  • 47
    Thompson, J.D., Higgins, D.G., Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research 22 , 46734680.
  • 48
    Tsao, P.H., Leben, C., Keitt, G.W. (1960) An enrichment method for isolating actinomycetes that produce diffusible antifungal antibiotics. Phytopathology 50 , 8889.
  • 49
    Williams, S.T. & Davies, F.L. (1965) Use of antibiotics for selective isolation and enumeration of actinomycetes from soil. Journal of General Microbiology 38 , 251261.
  • 50
    Williams, S.T. & Wellington, E.M.H. (1982) Principles and problems of selective isolation of microbes. In Bioactive Microbial Products 1: Search and Discovery eds Bullock, J.D., Nisbet, L.J. and Winstanley, D.J. pp. 9–26. London: Academic Press.
  • 51
    Williams, S.T., Shameemullah, M., Watson, E.T., Mayfield, C.I. (1972) Studies on the ecology of actinomycetes in soil. VI. The influence of moisture tension on growth and survival. Soil Biology and Biochemistry 4 , 215225.
  • 52
    Williams, S.T., Goodfellow, M., Alderson, G., Wellington, E.M.H., Sneath, P.H.A., Sackin, M.J. (1983) Numerical classification of Streptomyces and related taxa. Journal of General Microbiology 129 , 17431813.
  • 53
    Williams, S.T., Lanning, S., Wellington, E.M.H. (1984) Ecology of actinomycetes. In The Biology of Actinomycetes eds Goodfellow, M., Mordarski, M. and Williams, S.T. pp. 481–528. London: Academic Press.
  • 54
    Williams, S.T., Locci, R., Beswick, A., Kurtböke, D.I., Kuznetsov, V.D., Le Monnier, F.J., Long, P.F., Maycroft, K.A., Palma, R.A., Petrolini, B., Quaroni, S., Todd, J.I., West, M. (1993) Detection and identification of novel actinomycetes. Research in Microbiology 144 , 653656.