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Application of sucrose-gradient centrifugation for selectiveisolation of Nocardiaspp. from soil

H. Yamamura, M. Hayakawa and Y. IimuraDepartment 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 WA A N D Y . I I M U R A . 2 0 0 3 .

Aims: To devise and evaluate a method forselective 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 gfor 30 min. The 20% sucrose layer, which is enriched withNocardia

spp., is then diluted and plated on humic acidvitamin agar supplemented with antibacterial agents. The proposed

method consistently achieved selective isolation ofNocardia 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 efficiently isolate soilNocardiapopulations that are

suppressed by conventional dilution plating approaches.

Significance 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 Nocardiaare 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 anddifferentiate into arthrospores. The genus belongs to the

suborderCorynebacterineae(Stackebrandtet al.1997), which

currently encompasses the mycolic acid group of genera,

Corynebacterium, Dietzia, Gordonia, Mycobacterium, Nocar-

dia, Rhodococcus, Skermania, Tsukamurella, Turicella and

Williamsia (Chun et al. 1996; Goodfellow et al. 1998). A

combination of biochemical, chemical and morphological

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,Nocardiaspp. appear to play a significant roleas 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 anti-

biotics. 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

Correspondence to: M. Hayakawa, Department of Applied Chemistry and Biotech-

nology, Faculty of Engineering, Yamanashi University, Takeda-4, Kofu 400-8511,

Japan (e-mail: [email protected]).

2003 The Society for Applied Microbiology

Journal of Applied Microbiology2003, 95, 677685 doi:10.1046/j.1365-2672.2003.02025.x


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they may present, and to identify useful strains that produce

novel bioactive metabolites.

Nocardiaspp. 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 efficientrecovery. Modifications of the paraffin-baiting technique of

Sohnghen (1913) have been used to isolate Nocardia spp.

from soil (Portaels 1976; Schaal and Bickenbach 1978).

Nocardiaspp. in soil do attack paraffin 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 ofNocardiaspp. (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 combi-nations of antibacterial antibiotics including demethylchlor-

tetracycline, methacycline and chlortetracycline. Use of this

modified 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] supplementedwith chlortetracycline.

MATERIALS AND METHODS

Strains and culture conditions

NineNocardiastrains (refer to Table 3) and fiveStreptomyces

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 screw-

cap 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. Purified spore suspensions of the test

actinomycete strains were prepared using the filtration

technique (Hayakawa and Nonomura 1989). For synchron-

ization, 1 ml of a spore suspension was layered on the top of

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 dilutedsuspension were plated in triplicate on HV agar. Plates were

incubated at 30C for 10 days before counting the colonies.

As a control, purified 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 Nocardiaspp. 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 chlortetra-cycline (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 fields

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 des-cribed 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 40long working distance objective (modelULWDCD-Plan; Olympus, Tokyo, Japan) and tentatively

identified up to the genus rank based on morphological

criteria (Labeda 1987; Cross 1989; Lechevalier 1989;

Goodfellow 1992). Nocardia strains were identified as those

isolates that form thin, flat colonies with sparse to abundant,

white aerial hyphae. Microscopic observation subsequently

confirmed formation of branching substrate hyphae, frag-

menting into rod to coccoid-shaped elements, and relatively

short aerial hyphae with chains of arthrospores (Goodfellow

and Lechevalier 1989).

678 H . Y A M A M U R A ET AL.

2003 The Society for Applied Microbiology, Journal of Applied Microbiology, 95, 677685, doi:10.1046/j.1365-2672.2003.02025.x


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Taxonomic analyses

Strains. Twenty-eight representative isolates with mor-phology typical of the genus Nocardiawere subcultured and

their taxonomic properties examined in greater detail. For

the comparative studies, the following known Nocardiastrains 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. flavorosea 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 previouslydescribed 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 deter-

mined using the freeze-dried filter paper disc method of

Goodfellow and Orchard (1974).

Chemotaxonomic analysis. Analysis of whole-cellsugars, polar lipids, isoprenoid quinones, mycolic acids

and diaminopimelic acid isomers was performed as previ-

ously described (Otoguro et al. 2001).

RFLP analysis. Genomic DNA was prepared as describedby Torres et al. (1996). A portion (1 kb) of 16S ribosomal

DNA (rDNA) was amplified 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 infor-

mation was available. Primers were 5-CGA-ACG-CTG-

GCG-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-ACC-ACA-AGG-GGG-3 (10491023). PCR products were

subjected to the digestion using HhaI (TaKaRa), NdeI

(Wako) andBstEII (Wako), all of which were recommended

for use in identification ofNocardiaspp. based on restriction

fragment length polymorphism (RFLP) analysis of 16S

rDNA (Convilleet al. 2000). All digestions were performed

under conditions defined by the manufacturer.

Restriction fragment patterns of nine reference Nocardia

cultures (five 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 (Steingrubeet 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 16SrDNA (15 kb; positions 281524) was amplified by PCR

following the procedure described previously (Otoguro et al.

2001). The amplified products were purified using aSUPRECTM PCR Kit (TaKaRa) and cycle sequenced using

a Beckman CEQ DTCS Sequencing Kit (Beckman-Coulter,

Fullerton, CA, USA) and previously described oligonucleo-

tide primers (Otoguroet 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/Gen-

Bank/DDBJ databases. A phylogenetic tree was inferred

using neighbour-joining tree algorithms (Saitou and Nei

1987). The program CLUSTAL W (Thompsonet al.1994)

was used to calculate evolutionary distances and similarityvalues. Topography of the constructed tree was evaluated by

bootstrap analysis with 1000 replicates (Felsenstein 1985).

Nucleotide sequence accession numbers. The 16SrDNA 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 andGram-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. coliIFO 3044. The fungi examined were

Aspergillus nigerATCC (American Type Culture Collection,

Rockville, MD, USA) 9642 and Saccharomyces cerevisiae

IFO 10217. Spot-inoculated, 10-day-old colonies on Ben-

netts agar plates supplemented with humic acid (05 g l)1)

(Hayakawa et al. 1995) were inverted over 15 ml chloro-

form for 40 min. Killed colonies were overlaid with 5 ml of

I S O L A T I O N O F N O C A R D I A 679



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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 aftercentrifugation

Purified 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 ofN. asteroides JCM 3384T were concen-

trated in the 20% sucrose layer. For the other Nocardia strains

tested, including N. carnea IFO 14403T and N. flavorosea

14341

T

, the majority (>70%) of each spore population wasalso identified in the 20% sucrose layer. In contrast, the

largest number ofS. griseus IFO 13849T spores was encoun-

tered at the 30% sucrose layer (Fig. 1). Similar results were

observed in other Streptomyces strains tested, including

S. cyaneus ISP 5108 (IFO 13346T), S. corchorusiiISP 5340

(IFO 13032T), S. gannmycicus ISP 5572 (IFO 13467T) and

S. hygroscopicussubsp.hygroscopicusIFO 13346T.

Enrichment and selective isolation of

Nocardiaspp.

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% sucroselayers. Micromonospora spp. were detected in the 20 and

30% sucrose layers but in relatively low numbers. Actino-

planes spp., motile actinomycetes forming flagellated zoo-

spores, 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 nonfil-

amentous bacteria. Furthermore, a major actinomycete

group recovered from the agar plate was Streptomyces spp.,

which tended to prevent detection and pure-culture isolationof 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 significant decrease in the number of both

Streptomyces spp. and nonfilamentous bacteria occurring on

the plate. Although a slight decrease in colony forming units

(CFU g)1 soil) ofNocardia 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

0

1

2

3

4

10 20 30 40 50

Sucrose layer (%)

CFU

105l

ayer1

Fig. 1 Sucrose-gradient fractionation of spore populations from

known actinomycete cultures. Purified arthrospore suspension of

Nocardia asteroidesJCM 3384T (d) orStreptomyces 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

0

1

2

3

10 20 30 40 50

Sucrose layer (%)

CFU

105

layer1

Fig. 2 Fractionation of soil actinomycete population using sucrose-

gradient 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 gfor 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 unidentified actinomycetes

(j). Bars represent standard deviations of triplicate experiments




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further decreased the occurrence of Streptomyces spp. and

unicellular bacteria. Thus, the combined use of sucrose-

gradient centrifugation with HV agar supplemented with

antifungal and antibacterial antibiotics resulted in selective

isolation ofNocardia spp., which accounted for 56% of the

total population recovered.

The efficiency of the combined method for Nocardiaspp.

isolation was confirmed when applied to 14 different soil

samples (Table 2). From all samples, the combined method

selectively isolatedNocardiaspp., which constituted 589%

of the total microbial population recovered. The population

(CFU) ofNocardia spp. per gram of dried soil ranged from

13 103 to 39 105. Among the samples examined, the

paddy-field soil, which was less well drained and lower inpH than the nonpaddy cultivated soil, showed a relatively

low incidence ofNocardia spp.

Taxonomic evaluation

Confirmation of generic identification. In order to valid-ate presumptive generic identification according to light

microscopy, 28 putative Nocardia isolates from 14 samples

were the subject of further confirmatory tests including

antibiotic sensitivity, scanning electron microscopy (SEM)

and chemical analysis. In addition, two representative

isolates were characterized by 16S rDNA sequence analysisto establish their phylogenetic positions.

The SEM confirmed that all 28 test isolates showed

distinct morphological features typical of the genusNocardia

(Goodfellow et al. 1998, 1999). All 28 test strains were

resistant to lincomycin hydrochloride (100 mg l)1), neomy-

cin sulphate (50 mg l)1), penicillin-G (10 i.u. ml)1), rif-

ampicin (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

sensuLechevalieret al.1981). In addition, thin layer chroma-

tography revealed that the strains all contained methyl

mycolates equivalent in mobility to nocardomycolic acid.

These physiological and chemical properties are consistentwith classification 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 sub-

order Corynebacterineae (Stackebrandt et al. 1997). The

phylogenetic dendrogram derived from evolutionary distan-

ces 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 confidence level of 88%. The 16SrDNA similarity values of YU 011-1 and YU 019-2 to the

Table 1 Effect of centrifugation and antibacterial agents on isolation ofNocardia spp. from a lowland forest soil using density gradient

centrifugation*

Soil

treatment

Antibacterial

agents in HV

agar

CFU per g of air-dried soil

Nocardia/total

countStreptomyces Nocardia Micromonospora

Other

actinomycetes

Other

bacteria

None None 35 (04) 106a 17 (04) 105a 17 (08) 105a 95 (03) 105a 27 (04) 106a 001

SGC None 15 (05) 105b 12 (03) 105a 67 (29) 104b 33 (29) 104b 27 (06) 105b 018

SGC NA + CT 35 (05) 104c 12 (03) 105a ND ND 60 (20) 104c 056

*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 significantly 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.

Table 2 Mean CFU g)1 soil and percentage

of total population ofNocardia spp. from

various soil samples using density gradient

centrifugationSoil

source

No. of samples

examined

Sample

pH

Mean CFU per g of air-dried sample

Nocardia

Other

actinomycetes

Other

bacteria

Cultivated field 10 69 66 104 (280%) 80 104 44 104

Paddy field 2 64 32 103 (201%) 18 104 14 104

Forest 2 68 11 105 (500%) 79 104 34 104




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most closely related species N. uniformis and N. asteroides

were 989 and 977%, respectively. It was also confirmed

that the two isolates showed nucleotide sequence signatures

typical of members of the genus Nocardia (Stackebrandt

et al. 1997).

Presumptive specific identification based on RFLP

analysis. The 28 test isolates together with nine referenceNocardia cultures (five species) were evaluated by amplifi-

cation and restriction endonuclease analysis of a portion

(1 kb) of 16S rDNA. Digestion with a combination ofHhaI,NdeI and BstEII produced RFLP patterns that can distin-

guish between all test Nocardiaspecies (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 differ-

entiated from those of the testNocardiareference strains and

from those predicted based on the NocardiaDNA 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 five (18%) were active against Staph. aureus and five

againstE. 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 environ-

ment, 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 (Mayfield et al. 1972; Kalakoutski and

Streptomyces ambofaciensATCC23877T (M27245)

Rhodococcus percolatusMBS1T(X92114)

Rhodococcus opacus DSM43205T(X80630)

Nocardia uniformis JCM 3224TZ46752)Nocardiasp. YU011-1

Nocardia asteroides ATCC19247T(Z36934)Nocardia sp. YU019-2

Gordonia bronchialis DSM 43247T(X79287)Gordonia amarae DSM43392T(X80635)Skermania piniformisIFO 15059T(Z35435)

Williamsia murale DSMZ 44343T (Y17384)

Dietzia marisDSM 46102T(X79290)

Tsukamurella paurometabola ATCC8368T(AF283280)

Tsukamurella inchonensisATCC700082T(AF283281)Mycobacterium tuberculosis (X52917)Mycobacterium bovis ATCC19210T(X55589)

Corynebacterim diphtheriae NCTC 11397T(X84248)

Corynebacterim glutamicum NCIB 10025T(X84257)

Turicella otitidisDSM8821T(X73976)

Dietzia natronolimnaea CBS 10795T(X92175)

999796

1000

1000

933

537

1000

761

1000

999

612

001Knuc

1000

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 Corynebacteri-

neae. Bar, 001 nucleotide substitutions per

site. Numbers on branches are confidence

limits estimated by bootstrap analysis of 1000

replicates

Table 3 Scheme for presumptive identification ofNocardia spp. by RFLP pattern analysis of an amplified portion of 16S rDNA

Sizes of fragments (bp) after digestion with:

Reference strains No. of isolatesHhaI NdeII BstEII

420, 350, 225 700, 200, 90 Uncut N. asteroidesJCM 3384T, IFO

3384, IFO 3423 and IFO 3424

7

420, 350, 225 700, 200, 90 725, 265 N. uniformis IFO 13702T 10

420, 350, 225 900, 90 725, 265 N. salmonicida IFO 13393T 5

645, 350 450, 250, 200, 90 725, 265 N. vinacea IFO 16497T 0

645, 350 450, 250, 200, 90 Uncut N. carneaIFO 14403T 0

645, 350 390, 250, 200, 90, 60 Uncut N. flavorosea IFO 14341T 0

430, 200, 140, 80, 50 520, 230, 180, 70, 50 Uncut 2

430, 200, 140, 80, 50 450, 250, 200, 90 Uncut 4




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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 efficient isolation.

Various methods that have been developed for isolating

particular genera of nonstreptomycete actinomycetes relyupon 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 actino-

mycete 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 actino-

mycetes occurring in soil, although he failed to isolate

Nocardia spp. In the present study, investigation of

arthrospore suspensions isolated from pure actinomycetecultures suggested that simple, sucrose-gradient centrifu-

gation 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; Kurtboke 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 ofStreptomyces 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 aselective medium. HV agar, which contained humic acid

as the sole source of carbon and nitrogen, was used, because

it reportedly enables efficient recovery of a diverse range of

nonstreptomycete actinomycetes in soil while suppressing

the growth of nonfilamentous 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

Nocardiaspp. 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 bioactiv-ity (Goodfellow 1992; Williams et al. 1993). Although the

proposed isolation method cannot be used to estimate actual

Nocardia population in soil, it has already confirmed 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 pheneticand 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.

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