Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 3, Num. 3, 1992

ANALYSES OF DNA HOMOLOGY AND rDNA RESTRICTION PATTERNS OF SOME SPECIES IN THE GENUS NOCARDIOPSIS

Z.LIU, J.RUAN, J.ZAKRZEWSKA-CZERWINWKA* and M.MORDARSKI*

Institute of Microbiology, Academia Sinica, Beijing, China
and
*Institute of Immunology and Experimental Therapy, Wroclaw, Poland

Code Number: AC92011
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ABSTRACT.

Eleven species of the Nocardiopsis genus were investigated by DNA:DNA homology and RFLP analysis. The results of DNA:DNA pairing show that the type species Nocardiopsis dassonvillei shares 18-25% homology with N. dassonvillei subsp. prasina and that RFLP patterns (BamI-H, KpnI and SalI) are markedly different. The subspecies is therefore considered to represent a different species of the genus Nocardiopsis. Similarly other species attributed to the taxon have no relationship with the type species, either in terms of DNA homology or with reference to RFLP patterns. Results obtained point out the relevance of molecular techniques in the determination of taxonomic validity of the generic taxon.

The genus Nocardiopsis was proposed as a new taxon of Actino- mycetales by Meyer in 1976. The type species Nocardiopsis dassonvillei was separated from the genus Actinomadura on the basis of its cell wall chemotype III/C (i.e., presence of meso-- DAP and lack of other characteristic amino acids and of madurose). Subsequent studies showed that the type species lacks mycolates, it contains large amounts of iso- and anteisobranched chain fatty acids, type MK-10 (H4, H6) menaquinone, type III phospholipids and a DNA G+C mol % content of 71 to 76 (Fischer et al., 1983; Goodfellow and Cross, 1984).

Chemotaxonomical studies on 11 species of Nocardiopsis showed their difference, either in phospholipids type or in menaquinone patterns, from the type species and among themselves (Liu and Ruan, 1990), partly in agreement with previous reports (Shimazu et al., 1989; Grund and Kroppenstedt, 1990). Some of the species were transferred to other genera (Poschner et al., 1985; Grund and Kroppenstedt, 1990).

Considering that chemotaxonomic characters represent confusing taxonomic criteria for this genus, nucleic acid techniques were employed in this study aimed at establishing the status of Nocardiopsis species.

MATERIALS and METHODS

Organisms and growth conditions. Strains, listed in Table 1, were grown in submerged culture on modified (Mordarska et al., 1972) Sauton's medium (peptone 10 g, yeast extract 2 g, casein 2 g, NaCl 6 g, glucose 10 g, water 1 l, pH 7.2) at 28 C for 4 to 8 dd. Cultures were checked for purity and the biomass harvested.

Table 1. Source of strains. (IMRU=Institute of Microbiology, Rutgers University; CCCCM=China Committee of Culture Collection of Microorganisms; IFO=Institute for Fermentation, Osaka; JCM--Japan Collection of Microorganisms. Species transferred to the genera Actinomadura^1 and Saccharothrix^2).

----------------------------------------------------------------------
      Organism                  Strain No.                Source 
----------------------------------------------------------------------
Nocardiopsis dassonvillei       IMRU LL-Hi                IMRU, USA 
N. atra                         IFO 14198                 IFO, Japan 
N. africana^1                   JCM6420                   JCM, Japan
N. coeruleofusca^2              JCM A-0313 (IFO 14520)    JCM, Japan 
N. dassonvillei subsp.          JCM 3237 (IFO 13908)      JCM, Japan 
     dassonvillei 
N. dassonvillei subsp. prasina  JCM 3336 (IFO 14423)      JCM, Japan 
N. flava^2                      JCM 3296 (IFO 14521)      JCM, Japan 
N. longispora^2                 JCM 3314 (IFO 14522)      JCM, Japan 
N. streptospora                 AS 4.1176 (IFO 14362)     CCCCM, China 
N. mutabilis^2                  JCM 3380 (IFO 14475)      JCM, Japan 
N. trehalosea                   AS 4.1174 (IFO 14201)     CCCCM, China 
Actinoalloteichus cyanogriseus  AS 4.1159 (IFO 14455,     CCCCM, China 
                                NRRL B-16252, JCM  6059)  
Streptomyces griseus            AS 4.139                  CCCCM, China
------------------------------------------------------------------------

Extraction of DNA. High molecular DNA was prepared according to Chater et al. (1982).

DNA molecular size. 1.0% agarose gel electrophoresis was used to analysing the molecular size of DNA samples. DNA l/Hind III was used as standard (Maniatis et al., 1982).

Labelling of reference DNA. The DNA in vitro-nick translation method (Maniatis et al., 1982) was used to label reference DNA from N. dassonvillei LL-H1, N. dassonvillei subsp. prasina 3336, Streptomyces griseus AS 4.139 and Actinoalloteichus cyanogriseus AS 4.1159.

Preparation of rRNA Probes. A chemoluminescent digoxigenin (DIG) labelled plasmid PUC18 (from Streptomyces lividans) rDNA with 5S, 16S and 23S rRNA sequences was used as hybridisation probe (Zakrzewska- Czerwinska et al., 1988.

Analysis of DNA base composition. The thermal denaturation method of Marmur and Doty (1962) was used to determine the guanine (G) plus cytosine (C) content of the DNA extracted from the test organisms. DNA was obtained from Streptomyces griseus AS 4.139. The mol % G+C was calculated after Mandel and Marmur (1968).

DNA:DNA hybridisation. Levels of DNA relatedness between the test strains were obtained using a nitrocellulose membrane filter technique (Mordarski et al., 1986; Zakrzewska-Czerwinska et al., 1988). Filters were examined for bound radioactivity according to Bray (1960).

RFLP Analysis. Gel electrophoresis of endonuclease (BamHI, KpnI, Pst I and SalI) digested DNA was carried out (Maniatis et al., 1982; Zakrzewska-Czerwinska et al., 1988). Southern hybridisation of DNA fragments (Maniatis et al., 1982) was performed at 68 C overnight (18 hr).

RESULTS and DISCUSSION

The DNA base composition of the Nocardiopsis strains ranges from 70- 74 mol % G+ C (Table 2) which are within the range of previously published data. Similarly the DNA base composition of the reference strains S. griseus (71.2 mol%) and A. cyanogriseus (71.2 mol%) is in line with earlier studies (Mordarski et al., 1986).

Table 2. DNA base composition and DNA relatedness of test strains in Nocardiopsis (* In the homologous systems 21-33% pairing (3700-4500 c.p.m./sample) was found and taken as 100%).

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                                             DNA        DNA binding % 
    Organism                       Strain    G+C   ------------------------
                                             mol%  LL-HI  3336 4.1159 4.139

---------------------------------------------------------------------------
Nocardiopsis dassonvillei           LL-H1    70.0   100     25     4     4 
N. atra                             14198    70.0     2      4     5     3 
N. africana                          6420    70.0    12     20     3     3 
N. coeruleofusca                   A-0313    73.9     4      4     7     4 
N. dassonvillei subsp. dassonvillei  3237    72.2     4      5     5     5 
N. dassonvillei subsp. prasina       3336    70.0    18    100     5     4 
N. flava                             3296    70.0     4      4     7     4 
N. longispora                        3314    71.5 
N. streptospora                     4.1176   74.4     3      4     4     5 
N. mutabilis                         3380    72.2     3      4     4     3 
N. trehalosea                       4.1174   72.2     5      6     4     3 
Actinoalloteichus cyanogriseus      4.1159   72.2     4      7   100     6 
Streptomyces griseus                4.139    71.2     2      6     6   100
---------------------------------------------------------------------------

The results of DNA:DNA pairing are given in Table 2. The type species N. dassonvillei shares 18-25% homology with N. dassonvillei subsp. prasina. All the other organisms tested show a much lower homology value with the type species.

According to the present experimental data, and in agreement with previously proposed criteria, genus attribution by DNA:DNA hybridisation homology values could be defined at a level above 15% (Stackebrandt. et al., 1981).

We therefore propose that N. dassonvillei subsp. prasina should be considered as a genospecies at the same level as the type species N. dassonvillei and that it should be kept in the genus Nocardiopsis.

In addition N. dassonvillei subsp. prasina differs in chemical markers (menaquinone pattern MK-9 (H4), phospholipid type II) from the type species. Chemotaxonomic criteria defining the genus Nocardiopsis do not appear to be informative. Some studies, either using DNA homology or chemotaxonomic features, have shown the lack of relationship of other Nocardiopsis species with N. dassonvillei.

The question arises now as to whether some species attributed to Nocardiopsis actually belong to the taxon.

Number and mapping of rRNA genes of eubacteria differ widely (King et al., 1986). The rRNA restriction patterns examined by gene probe have been shown to be a useful criterion for distinguishing various taxa with low DNA:DNA homology.

Three strains were chosen for RFLP analysis. Results of their restriction patterns (BamHI, KpnI and Sail) are shown in Fig. 1.

Figure 1. SalI (lanes 1-3), Pst I (4-6), KpnI (7-9) and BamHI (10-12) digested DNA patterns (Southern blot) after hybridisation with DIG-labelled rDNA probes from N. dassonvillei subsp. prasina, 3336, N. africana, 6420 and Nocardiopsis dassonvillei, LL-H1, respectively. Markers: Kb(l/Hind III).

ACKNOWLEDGEMENTS. The authors wish to thank Ms. A. Gaszewska- Mastalarz, Ms. Barbara Lis and Isaac Kaszen for technical assistance. The collaboration of Drs. Akira Yokota (IFO, Japan) and Takuji Kado (JCM, Japan), who provided cultures of Nocardiopsis, is gratefully acknowledged.

REFERENCES

Bray, G.A. (1960). A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Anal.Biochem., 1: 279-285

Chater, K.F., D.A.Hopwood, T.Kieser & C.J. Thompson (1982). Gene cloning in Streptomyces. Curr. Topics Microb. Immunol., 96:69-95

Fischer, A., R.M.Kroppenstedt & E.Stackebrandt (1983). Molecular, genetic and chemotaxonomic studies on Actinomadura and Nocardiopsis. J. Gen. Microbiol., 129:3433-3446

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Mordarski, M., M.Goodfellow, S.T.Williams & P.H.A. Sneath (1986). Evaluation of species groups in the genus Streptomyces. In: G.Szabo, S.Biro & M.Goodfellow (eds.) Biological, Biochemical and Biomedical Aspects of Actinomoycetes. Akademiai Kiado Budapest, pp. 517-525

Poschner, J., R.M.Kroppenstedt, A.Fischer & E.Stackebrandt (1985). DNA-DNA reassociation and chemotaxonomic studies on Actinomadura, Microbispora, Microtetraspora, Micropolyspora and Nocardiopsis. Syst.Appl. Micro- biol., 6:264-270

Shimazu, A., A. Takahashi, H.Seto & Y. Okami (1989). Chemotaxonomic diversity of the genus Nocardiopsis and the relation to other genera. In: Y.Koyama (ed.). Trends in Actinomycetology in Japan. Society for Actinomycetes, Japan, pp. 13-16

Stackebrandt, E., B. Wunner-Fussel, V.J. Fowler & K.H. Schleifer (1981). Deoxyribonucleic acid homologies and ribosomal ribonucleic acid similarities among spore-forming members of the order Actinomycetales. Int. J. Syst. Bacteriol., 31:420-431

Zakrzewska-Czerwinska, J., M.Mordarski & M.Goodfellow (1988). DNA base composition and homology values in the classification of some Rhodococcus species. J. Gen. Microbiol., 134: 2807-2813.

Copyright 1992 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia

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