Journal of Applied Microbiology 2003, 95, 677685

doi:10.1046/j.1365-2672.2003.02025.x

Application of sucrose-gradient centrifugation for selective isolation of Nocardia spp. from soil
H. Yamamura, M. Hayakawa and Y. Iimura
Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University, Kofu, Japan
2002/484: received 5 December 2002, revised 14 April 2003 and accepted 16 April 2003

ABSTRACT
H . Y A M A M U R A , M . H A Y A K A W A A N D Y . I I M U R A . 2003.

Aims: To devise and evaluate a method for selective isolation of the less abundant actinomycetes, Nocardia spp. in soil. Methods and Results: This newly developed method is based on differentiating Nocardia from other actinomycete taxa by centrifugation. A water suspension of air-dried soil is centrifuged through a gradient consisting of 10, 20, 30, 40 and 50% sucrose at 240 g for 30 min. The 20% sucrose layer, which is enriched with Nocardia spp., is then diluted and plated on humic acidvitamin agar supplemented with antibacterial agents. The proposed method consistently achieved selective isolation of Nocardia spp. in all 14 soil samples tested, which accounted for 589% of the total microbial population recovered. Tentative taxonomic characterization based on a restriction fragment length polymorphism (RFLP) analysis of the 16S ribosomal DNA suggested that many of the soil isolates could belong to N. asteroides, N. salmonicida or N. uniformis. Conclusions: Differential centrifugation can successfully and efciently isolate soil Nocardia populations that are suppressed by conventional dilution plating approaches. Signicance and Impact of the Study: The development and application of new methodologies with which to isolate less-explored actinomycete taxa is important for improving our knowledge about their taxonomy, ecology and industrial applications. Keywords: 16S rDNA, differential centrifugation, Nocardia, RFLP, selective isolation, soil bacteria.

INTRODUCTION Members of the genus Nocardia are aerobic, Gram-positive, mesophilic actinomycetes that characteristically produce branched substrate hyphae often fragmenting into rod to coccoid-shaped elements (Trevisan 1889; Goodfellow and Lechevalier 1989). Aerial hyphae are usually formed and differentiate into arthrospores. The genus belongs to the suborder Corynebacterineae (Stackebrandt et al. 1997), which currently encompasses the mycolic acid group of genera, Corynebacterium, Dietzia, Gordonia, Mycobacterium, Nocardia, Rhodococcus, Skermania, Tsukamurella, Turicella and Williamsia (Chun et al. 1996; Goodfellow et al. 1998). A combination of biochemical, chemical and morphological
Correspondence to: M. Hayakawa, Department of Applied Chemistry and Biotechnology, Faculty of Engineering, Yamanashi University, Takeda-4, Kofu 400-8511, Japan (e-mail: hayakawa@ab11.yamanashi.ac.jp).

tests is necessary to distinguish between these mycolata genera (Goodfellow et al. 1999). Nocardia spp. are widely distributed throughout soil (Cross et al. 1976; Orchard et al. 1977), although some species have been isolated from clinical specimens and animal infections as opportunistic pathogens. In the soil environment, Nocardia spp. appear to play a signicant role as saprophytic organisms in the turnover of naturally occurring organic substances (Goodfellow 1992). They are also of industrial importance as producers of biologically active secondary metabolites, which notably include antibiotics. Valuable antibiotics, such as nocardicin (Aoki et al. 1976) and brasilinolide (Tanaka et al. 1997), have been obtained from Nocardia spp. Therefore, the isolation and subsequent characterization of pure cultures of these organisms from natural habitats is important in order to understand their ecological role, to assess health hazards that

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they may present, and to identify useful strains that produce novel bioactive metabolites. Nocardia spp. are usually found only in small numbers on conventional actinomycete selective agar media, which were primarily designed to isolate Streptomyces spp., and thus several methods have been developed to facilitate efcient recovery. Modications of the parafn-baiting technique of So hnghen (1913) have been used to isolate Nocardia spp. from soil (Portaels 1976; Schaal and Bickenbach 1978). Nocardia spp. in soil do attack parafn baits but are usually associated with numerous other bacteria and fungi, causing problems when the colonized baits are used as inocula for pure culture isolation. Based on the differential antibiotic resistance of Nocardia spp. (Goodfellow and Orchard 1974), Orchard and Goodfellow (1974) devised a useful isolation method, in which soil suspensions are plated and incubated on diagnostic sensitivity test (DST) agar supplemented with an antifungal antibiotic, cycloheximide, and various combinations of antibacterial antibiotics including demethylchlortetracycline, methacycline and chlortetracycline. Use of this modied DST agar method resulted in the isolation of Nocardia spp. in 15 of 47 soil samples examined (Orchard et al. 1977). The present paper describes a new method that successfully eliminates nontarget microbes and results in highly selective isolation of Nocardia spp. from soil. The method is based on the differentiation of Nocardia from other actinomycete taxa, particularly the most common genus Streptomyces, by centrifugation. Sucrose-gradient centrifugation was used in combination with a selective medium [humic acidvitamin (HV) agar] supplemented with chlortetracycline.

the sucrose gradient, and the tube was centrifuged (room temperature, 30 min, 240 g) in a swinging bucket rotor. After centrifugation, each sucrose layer (1 ml) was removed sequentially from the top of the gradient using a different sterile pipette for each layer, and then diluting in a 10-fold series in sterile tap water. Aliquots (02 ml) of this diluted suspension were plated in triplicate on HV agar. Plates were incubated at 30C for 10 days before counting the colonies. As a control, puried spore suspensions were directly diluted with sterile tap water and plated in the same way. Experiments were performed in triplicate in order to obtain mean colony counts. Isolation procedure for Nocardia spp. from soil Isolation media were HV agar (Hayakawa and Nonomura 1987) with or without a mixture of nalidixic acid (10 mg l)1; Sigma Chemical Co., St Louis, MO, USA) and chlortetracycline (10 mg l)1; Wako Pure Chemical Ind., Osaka, Japan) (Orchard and Goodfellow 1974). Media also contained cycloheximide (50 mg l)1; Wako) to suppress fungal growth (Williams and Davies 1965). Sixteen soil samples were collected from forests, and elds of corn, peach, vegetable and paddy rice in Yamanashi and Nagano prefectures (Japan). Each soil sample, about 500 g, was taken from the A or A1-horizon of the soil (Hayakawa et al. 1988). A portion of the soil was passed through a 2 mm mesh sieve, air-dried at room temperature for 7 days, and subsequently used for actinomycete isolation. A gradient of 10, 20, 30, 40 and 50% sucrose was prepared in a screw-cap centrifuge tube (165 105 mm), as described above. A 10)1 dilution of an air-dried soil sample was prepared in sterile tap water and 1 ml was applied to the discontinuous sucrose gradient, which was then centrifuged (room temperature, 30 min, 240 g) in a swinging bucket rotor. After centrifugation, each sucrose section was removed, diluted and aliquots (02 ml) plated on HV agar or the same agar supplemented with antibacterial agents. All plates were incubated at 30C for 23 weeks before counting actinomycete colonies, and all experiments were performed in triplicate. Actinomycete colonies growing on the isolation plates were examined directly under a light microscope equipped with a 40 long working distance objective (model ULWDCD-Plan; Olympus, Tokyo, Japan) and tentatively identied up to the genus rank based on morphological criteria (Labeda 1987; Cross 1989; Lechevalier 1989; Goodfellow 1992). Nocardia strains were identied as those isolates that form thin, at colonies with sparse to abundant, white aerial hyphae. Microscopic observation subsequently conrmed formation of branching substrate hyphae, fragmenting into rod to coccoid-shaped elements, and relatively short aerial hyphae with chains of arthrospores (Goodfellow and Lechevalier 1989).

M A T E R I A LS A N D M E T H O D S Strains and culture conditions Nine Nocardia strains (refer to Table 3) and ve Streptomyces strains were obtained from the Institute for Fermentation, Osaka (IFO) and the Japan Collection of Micro-organisms (JCM; RIKEN, Saitama). These strains were stored on slopes of otmeal agar supplemented with yeast extract, glucose and glycerol (YGG) (Hayakawa et al. 1982). Recovery of actinomycetes after centrifugation A discontinuous sucrose gradient was prepared in a screwcap centrifuge tube (165 105 mm) by carefully layering 1 ml each of 10, 20, 30, 40 and 50% (w/v) sucrose in order of decreasing density. Puried spore suspensions of the test actinomycete strains were prepared using the ltration technique (Hayakawa and Nonomura 1989). For synchronization, 1 ml of a spore suspension was layered on the top of

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Taxonomic analyses Strains. Twenty-eight representative isolates with morphology typical of the genus Nocardia were subcultured and their taxonomic properties examined in greater detail. For the comparative studies, the following known Nocardia strains were simultaneously examined: N. asteroides JCM 3384T (IFO 15531T; T, type strain), N. asteroides IFO 3384, N. asteroides IFO 3423, N. asteroides IFO 3424, N. carnea IFO 14403T, N. avorosea 14341T, N. salmonicida IFO 13393T, N. uniformis IFO 13702T and N. vinacea IFO 16497T. Distilled water suspensions of spores and hyphae, prepared from stock culture slants, were used as inocula (Shirling and Gottlieb 1966). All cultural preparations were incubated at 30C for 14 days unless otherwise stated. Phenotypic characterization. Morphology was observed by light and scanning electron microscopy under previously described conditions (Hayakawa et al. 1996b). Resistance to lysozyme (005%, w/v) was determined using yeast extract glucose broth (Hayakawa et al. 1996b). Resistance to lincomycin hydrochloride (100 mg l)1; Wako), neomycin sulphate (50 mg l)1; Wako), penicillin-G (10 IU ml)1; Wako) and rifampicin (50 mg l)1; Wako) was determined using the freeze-dried lter paper disc method of Goodfellow and Orchard (1974). Chemotaxonomic analysis. Analysis of whole-cell sugars, polar lipids, isoprenoid quinones, mycolic acids and diaminopimelic acid isomers was performed as previously described (Otoguro et al. 2001). RFLP analysis. Genomic DNA was prepared as described by Torres et al. (1996). A portion (1 kb) of 16S ribosomal DNA (rDNA) was amplied by PCR using TaKaRa Taq polymerase (Takara Shuzo, Kyoto, Japan) and a set of three primers designed by Conville et al. (2000) in order to amplify all species of Nocardia for which sequence information was available. Primers were 5-CGA-ACG-CTGGCG-GCG-TGC-TTA-AC-3 (positions 3052, according to the Escherichia coli numbering system of Brosius et al. 1978), 5-CCT-GTA-CAC-CGA-CCA-CAA-GGG-GG-3 (positions 10481023) and 5-ACC-TGT-ACA-CCA-ACCACA-AGG-GGG-3 (10491023). PCR products were subjected to the digestion using HhaI (TaKaRa), NdeI (Wako) and BstEII (Wako), all of which were recommended for use in identication of Nocardia spp. based on restriction fragment length polymorphism (RFLP) analysis of 16S rDNA (Conville et al. 2000). All digestions were performed under conditions dened by the manufacturer. Restriction fragment patterns of nine reference Nocardia cultures (ve species) and 28 isolates were analysed by gel electrophoresis of each restriction mixture in a 2%

MetaPhor agarose minigel (FMC Bioproducts, Rockland, MN, USA) containing 05 mg l)1 of ethidium bromide. Fragment band sizes were estimated by Software Tools for Fragment Analysis and Databasing, Diversity DatabaseTM ( pdi Inc., New York, NY, USA) and expressed as the number of nucleotide base pairs rounded to the nearest 5 bp. A 100-bp ladder was used as DNA molecular weight marker (TaKaRa). Restriction fragments smaller than 50 bp were not considered (Steingrube et al. 1995). For validly described Nocardia spp. that were not used in the present study, restriction fragment patterns were predicted from DNA sequencing data, available from the EMBM/GenBank/ DDBJ databases, using Webcutter 20 (Heiman 1997). 16S rDNA sequence analysis. Almost complete 16S rDNA (15 kb; positions 281524) was amplied by PCR following the procedure described previously (Otoguro et al. 2001). The amplied products were puried using a SUPRECTM PCR Kit (TaKaRa) and cycle sequenced using a Beckman CEQ DTCS Sequencing Kit (Beckman-Coulter, Fullerton, CA, USA) and previously described oligonucleotide primers (Otoguro et al. 2001). A Beckman CEQ2000XL (Beckman-Coulter) DNA sequencer was used to conduct electrophoresis of the sequencing reaction mixtures. The 16S rDNA sequences obtained in the present study were manually aligned with the published sequences of the validly described species available from the EMBM/GenBank/DDBJ databases. A phylogenetic tree was inferred using neighbour-joining tree algorithms (Saitou and Nei 1987). The program CLUSTAL W (Thompson et al. 1994) was used to calculate evolutionary distances and similarity values. Topography of the constructed tree was evaluated by bootstrap analysis with 1000 replicates (Felsenstein 1985). Nucleotide sequence accession numbers. The 16S rDNA sequences of strains YU 011-1 and YU 019-2 are available from the EMBL/GenBank/DDJB databases under the accession numbers AB092437 and AB092438, respectively. Determination of antimicrobial activity The ability to inhibit the growth of Gram-positive and Gram-negative bacteria, and fungi was determined by as previously described (Williams et al. 1983; Otoguro et al. 2001). The tested bacteria included Staphylococcus aureus IFO 3061 and E. coli IFO 3044. The fungi examined were Aspergillus niger ATCC (American Type Culture Collection, Rockville, MD, USA) 9642 and Saccharomyces cerevisiae IFO 10217. Spot-inoculated, 10-day-old colonies on Bennetts agar plates supplemented with humic acid (05 g l)1) (Hayakawa et al. 1995) were inverted over 15 ml chloroform for 40 min. Killed colonies were overlaid with 5 ml of

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sloppy Bennetts agar inoculated with the test organisms. Zones of inhibition around the colonies were recorded after 24 h at 30C.

RESULTS Recovery of actinomycete spores after centrifugation Puried arthrospore suspensions of several Nocardia and Streptomyces strains were centrifuged through layers of 10, 20, 30, 40 and 50% sucrose at 240 g for 30 min. After centrifugation, the number of spores present in each sucrose layer was estimated using the plate culture method. Figure 1 shows that spores of N. asteroides JCM 3384T were concentrated in the 20% sucrose layer. For the other Nocardia strains tested, including N. carnea IFO 14403T and N. avorosea 14341T, the majority (>70%) of each spore population was also identied in the 20% sucrose layer. In contrast, the largest number of S. griseus IFO 13849T spores was encountered at the 30% sucrose layer (Fig. 1). Similar results were observed in other Streptomyces strains tested, including S. cyaneus ISP 5108 (IFO 13346T), S. corchorusii ISP 5340 (IFO 13032T), S. gannmycicus ISP 5572 (IFO 13467T) and S. hygroscopicus subsp. hygroscopicus IFO 13346T. Enrichment and selective isolation of Nocardia spp. A water suspension of forest soil sample no. 030 was applied to the discontinuous gradient of 10, 20, 30, 40 and 50% sucrose. After centrifugation (240 g, 30 min), the number

and type of actinomycetes present in each sucrose layer was determined by the plate culture technique using HV agar with the antifungal antibiotic cycloheximide (Fig. 2). Most of the soil Nocardia population was found to be present in the 20% sucrose layer. In contrast, Streptomyces spp. were observed in larger numbers in the 30, 40 and 50% sucrose layers. Micromonospora spp. were detected in the 20 and 30% sucrose layers but in relatively low numbers. Actinoplanes spp., motile actinomycetes forming agellated zoospores, were only recovered from the 10% sucrose layer. Three methods were used to isolate Nocardia spp. from soil sample no. 033 (Table 1). Colonies of Nocardia spp. were recovered by directly plating and incubating a water suspension of the sample on HV agar with cycloheximide, but these were largely outnumbered by colonies of nonlamentous bacteria. Furthermore, a major actinomycete group recovered from the agar plate was Streptomyces spp., which tended to prevent detection and pure-culture isolation of Nocardia spp. We therefore attempted to perform selective recovery of this less-abundant actinomycete group. Use of sucrose-gradient centrifugation followed by plating the 20% sucrose layer on HV agar with cycloheximide resulted in a signicant decrease in the number of both Streptomyces spp. and nonlamentous bacteria occurring on the plate. Although a slight decrease in colony forming units (CFU g)1 soil) of Nocardia spp. was observed, recovery of Nocardia spp. as a percentage of total population was markedly increased. Incorporation of two antibacterial agents, chlortetracycline and nalidixic acid, into HV agar

4
CFU 105 layer 1

CFU 105 layer 1

1
0 10 20 30 Sucrose layer (%) 40 50

0 10 20 30 40 50 Sucrose layer (%)

Fig. 1 Sucrose-gradient fractionation of spore populations from known actinomycete cultures. Puried arthrospore suspension of Nocardia asteroides JCM 3384T (d) or Streptomyces griseus IFO 13849T (s) was subjected to discontinuous sucrose-gradient fractionation (10, 20, 30, 40 and 50% sucrose, 240 g, 30 min) and each sucrose layer was plated on HV agar. Bars represent standard deviations of triplicate experiments

Fig. 2 Fractionation of soil actinomycete population using sucrosegradient centrifugation. A water suspension of soil no. 030 (forest, pH 63) was centrifuged through a gradient of 10, 20, 30, 40 and 50% sucrose at 240 g for 30 min. The number and type of actinomycetes present in each sucrose layer was examined using the dilution plating method with HV agar. Actinoplanes (n), Micromonospora (m), Nocardia (d), Streptomyces (s) and other genera and unidentied actinomycetes (j). Bars represent standard deviations of triplicate experiments

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Table 1 Effect of centrifugation and antibacterial agents on isolation of Nocardia spp. from a lowland forest soil using density gradient centrifugation* Antibacterial agents in HV agar None None NA + CT CFU per g of air-dried soil Other actinomycetes 95 (03) 105a 33 (29) 104b ND Other bacteria 27 (04) 106a 27 (06) 105b 60 (20) 104c Nocardia/total count 001 018 056

Soil treatment None SGC SGC

Streptomyces 35 (04) 106a 15 (05) 105b 35 (05) 104c

Nocardia 17 (04) 105a 12 (03) 105a 12 (03) 105a

Micromonospora 17 (08) 105a 67 (29) 104b ND

*Soil sample no. 033 (pH 68). Colony forming units. Counts represent mean of triplicate experiments (triplicate plate counts for each experiment) with standard deviations given in parentheses. Within each column, means having the same superscript were not signicantly different (P < 005) by Duncans multiple range test. SGC, sucrose-gradient centrifugation. NA, nalidixic acid (10 mg l)1); CT, chlortetracycline (50 mg l)1). ND, None detected.

further decreased the occurrence of Streptomyces spp. and unicellular bacteria. Thus, the combined use of sucrosegradient centrifugation with HV agar supplemented with antifungal and antibacterial antibiotics resulted in selective isolation of Nocardia spp., which accounted for 56% of the total population recovered. The efciency of the combined method for Nocardia spp. isolation was conrmed when applied to 14 different soil samples (Table 2). From all samples, the combined method selectively isolated Nocardia spp., which constituted 589% of the total microbial population recovered. The population (CFU) of Nocardia spp. per gram of dried soil ranged from 13 103 to 39 105. Among the samples examined, the paddy-eld soil, which was less well drained and lower in pH than the nonpaddy cultivated soil, showed a relatively low incidence of Nocardia spp. Taxonomic evaluation Conrmation of generic identication. In order to validate presumptive generic identication according to light microscopy, 28 putative Nocardia isolates from 14 samples were the subject of further conrmatory tests including antibiotic sensitivity, scanning electron microscopy (SEM) and chemical analysis. In addition, two representative isolates were characterized by 16S rDNA sequence analysis to establish their phylogenetic positions.
Table 2 Mean CFU g)1 soil and percentage of total population of Nocardia spp. from various soil samples using density gradient centrifugation

The SEM conrmed that all 28 test isolates showed distinct morphological features typical of the genus Nocardia (Goodfellow et al. 1998, 1999). All 28 test strains were resistant to lincomycin hydrochloride (100 mg l)1), neomycin sulphate (50 mg l)1), penicillin-G (10 i.u. ml)1), rifampicin (50 mg l)1) and lysozyme (0005%, w/v). The 28 strains contained meso-diaminopimelic acid, arabinose and galactose in whole-cell hydrolysates. Their predominant menaquinone component was MK-8(H4), and they also contained phosphatidylethanolamine (phospholipid type PII sensu Lechevalier et al. 1981). In addition, thin layer chromatography revealed that the strains all contained methyl mycolates equivalent in mobility to nocardomycolic acid. These physiological and chemical properties are consistent with classication of the 28 test isolates into Nocardia. Nearly complete 16S rDNA sequences of the isolates YU 011-1 and YU 019-2 were determined and compared with corresponding sequences of selected members of the suborder Corynebacterineae (Stackebrandt et al. 1997). The phylogenetic dendrogram derived from evolutionary distances by the neighbour-joining method is shown in Fig. 3. A total of 1365 nucleotides were used for this analysis. Isolates YU 011-1 and YU 019-2 were closely related to N. uniformis JCM 3224T and N. asteroides ATCC 19247T, and these four strains formed a coherent cluster supported by bootstrap analysis at a condence level of 88%. The 16S rDNA similarity values of YU 011-1 and YU 019-2 to the

Mean CFU per g of air-dried sample Soil source Cultivated eld Paddy eld Forest No. of samples examined 10 2 2 Sample pH 69 64 68 Other actinomycetes 80 104 18 104 79 104 Other bacteria 44 104 14 104 34 104

Nocardia 66 104 (280%) 32 103 (201%) 11 105 (500%)

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001Knuc

Turicella otitidis DSM8821T (X73976) Corynebacterim glutamicum NCIB 10025T (X84257) Corynebacterim diphtheriae NCTC 11397T (X84248) 1000 Mycobacterium bovis ATCC19210T (X55589) Mycobacterium tuberculosis (X52917) T 1000 Tsukamurella inchonensis ATCC700082 (AF283281) Tsukamurella paurometabola ATCC8368T (AF283280) 612 Dietzia natronolimnaea CBS 10795T (X92175) 1000 Dietzia maris DSM 46102T (X79290) Nocardia sp. YU019-2 933 Nocardia asteroides ATCC19247T (Z36934) Nocardia sp. YU011-1 1000 537 Nocardia uniformis JCM 3224T Z46752) T 1000 Rhodococcus opacus DSM43205 (X80630) Rhodococcus percolatus MBS1T (X92114) Skermania piniformis IFO 15059T (Z35435) 796 Gordonia amarae DSM43392T (X80635) 999 Gordonia bronchialis DSM 43247T (X79287) Williamsia murale DSMZ 44343T (Y17384) Streptomyces ambofaciens ATCC23877T (M27245)
999 761

Fig. 3 Neighbour-joining tree based on 16S rDNA gene sequences, showing relationships among strains YU011-1 and YU019-2 and representatives of the suborder Corynebacterineae. Bar, 001 nucleotide substitutions per site. Numbers on branches are condence limits estimated by bootstrap analysis of 1000 replicates

Table 3 Scheme for presumptive identication of Nocardia spp. by RFLP pattern analysis of an amplied portion of 16S rDNA Sizes of fragments (bp) after digestion with: HhaI 420, 350, 225 420, 420, 645, 645, 645, 430, 430, 350, 350, 350 350 350 200, 200, 225 225 NdeII 700, 200, 90 700, 900, 450, 450, 390, 520, 450, 200, 90 250, 250, 250, 230, 250, 90 200, 200, 200, 180, 200, 90 90 90, 60 70, 50 90 BstEII Uncut 725, 265 725, 265 725, 265 Uncut Uncut Uncut Uncut Reference strains N. asteroides JCM 3384T, IFO 3384, IFO 3423 and IFO 3424 N. uniformis IFO 13702T N. salmonicida IFO 13393T N. vinacea IFO 16497T N. carnea IFO 14403T N. avorosea IFO 14341T No. of isolates 7 10 5 0 0 0 2 4

140, 80, 50 140, 80, 50

most closely related species N. uniformis and N. asteroides were 989 and 977%, respectively. It was also conrmed that the two isolates showed nucleotide sequence signatures typical of members of the genus Nocardia (Stackebrandt et al. 1997). Presumptive specic identication based on RFLP analysis. The 28 test isolates together with nine reference Nocardia cultures (ve species) were evaluated by amplication and restriction endonuclease analysis of a portion (1 kb) of 16S rDNA. Digestion with a combination of HhaI, NdeI and BstEII produced RFLP patterns that can distinguish between all test Nocardia species (Table 3). Of the 28 test isolates, 7, 10 and 5 strains showed RFLP patterns identical to those of N. asteroides (JCM 3384T, IFO 3384, IFO 3423, IFO 3424), N. uniformis (IFO 13702T) and N. salmonicida (IFO 13393T), respectively. The remaining six isolates showed unique RFLP patterns that can be differentiated from those of the test Nocardia reference strains and from those predicted based on the Nocardia DNA sequences listed in GenBank.

Antimicrobial activity The Nocardia isolates were tested for antimicrobial activity using an overlay method (Williams et al. 1983). Of the 28 strains tested, 20 (71%) showed inhibitory activity. Most of this activity was directed against Aspergillus niger and S. cerevisiae, which were inhibited by 16 (57%) and nine (32%) of the isolates, respectively. Of the all isolates tested, only ve (18%) were active against Staph. aureus and ve against E. coli. DISCUSSION Actinomycetes are characterized by their reproductive strategies, which lead to formation of a variety of sporing structures such as arthrospores, aleuriospores and motile zoospores (Cross 1970; Ensign 1978). In the soil environment, where supplies of available nutrients are often limited, actinomycetes appear to be largely present in these spore forms, and this may promote their survival and dispersal (Mayeld et al. 1972; Kalakoutski and

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Pouzharitskaja 1973). Therefore, it is advisable to consider the differential properties of spores from the target actinomycete genera and exploit such properties to facilitate efcient isolation. Various methods that have been developed for isolating particular genera of nonstreptomycete actinomycetes rely upon the superior ability of their spores to withstand heating, desiccation or treatment with sporicidal chemicals (Cross 1982; Williams and Wellington 1982; McCarthy 1985; Goodfellow and ODonnel 1989; Labeda and Shearer 1990). Differences in buoyant density of actinomycete spores could also be utilized for selective isolation of desired genera. By using cesium chloride density gradient ultracentrifugation, Karwowski (1986) was able to isolate a Micromonospora population from other actinomycetes occurring in soil, although he failed to isolate Nocardia spp. In the present study, investigation of arthrospore suspensions isolated from pure actinomycete cultures suggested that simple, sucrose-gradient centrifugation at low-speed can be used to differentiate Nocardia spp. from Streptomyces spp. (Fig. 1), which are the numerically predominant actinomycetes in most soils and tend to grow quickly on any isolation media, often impeding growth of nonstreptomycete colonies (Williams and Vickers 1988; Kurtbo ke et al. 1992). A differential centrifugation stage was therefore incorporated into our isolation method and shown to be effective in separating a Nocardia population from other actinomycetes in a soil suspension prior to inoculation (Fig. 2). In Fig. 1 very few Streptomyces spores were observed at 40 and 50% sucrose layers using known strains, but larger numbers of Streptomyces spp. in a soil suspension occurred at 40 and 50% sucrose layers (Fig. 2). Although the rationale behind this difference remains to be studied, Ruddick and Williams (1972) have pointed out that adsorption of streptomycete spores to soil particles is likely to occur in the soil environment. Sucrose-gradient centrifugation at low-speed has been successfully used for separating E. coli mini-cells from intact cells (Yoda 1992). However, it has not been applied to the isolation of particular actinomycete genera. Successful isolation of Nocardia spp. depends on the combined use of sucrose-gradient centrifugation and a selective medium. HV agar, which contained humic acid as the sole source of carbon and nitrogen, was used, because it reportedly enables efcient recovery of a diverse range of nonstreptomycete actinomycetes in soil while suppressing the growth of nonlamentous bacteria (Hayakawa and Nonomura 1987). Therefore, use of this medium may also confer a selective advantage to Nocardia spp. Selective reduction in the number of contaminating bacteria was further achieved by supplementing HV agar with a mixture of nalidixic acid and chlortetracycline. The insensitivity of

Nocardia spp. to these antibacterial agents has already been thoroughly demonstrated by Goodfellow and Orchard (1974) and Hayakawa et al. (1996a). The development and application of new methods to isolate the less studied actinomycetes has improved our knowledge about their distribution, taxonomy and bioactivity (Goodfellow 1992; Williams et al. 1993). Although the proposed isolation method cannot be used to estimate actual Nocardia population in soil, it has already conrmed their widespread distribution in soils. Taxonomic characterization based on RFLP analysis of 16S rDNA, on the other hand, has revealed that numerous Nocardia isolates, obtained by the proposed isolation method, may belong to one of the three recognized species, N. asteroides, N. salmonicida and N. uniformis. More importantly, this isolation method also provided several Nocardia isolates that were differentiated from any of known species in terms of RFLP patterns. Further detailed investigation, such as numerical phenetic and molecular systematic analyses, could reveal their exact taxonomic status at the species level. Nocardia isolates are also worthy of further investigation as potential producers of valuable antibiotics and/or other bioactive secondary metabolites, because many of the test strains exhibited antimicrobial activity. REFERENCES
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