Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 2, Num. 2, 1991

DNA relatedness in Streptomyces species groups

DAVID P. LABEDA

Microbial Properties Research, National Center for Agricultural Utilization Research, Agricultural Research Service, USDA, Peona, Illinois 61604, U.S.A.

 
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Taxonomic descriptions of Streptomyces species using traditional morphological and physiological criteria have resulted {n the proliferation of valid and invalid species names in the literature, totaling nearly 3,000 by 1970 (Trejo, 1970)(1). The Approved Lists of Bacterial Names (Skerman et al., 1980)(2) and taxa subsequently validly published now total 346 species for the genus Streptomyces. A study of these named taxa using numerical taxonomic analysis of data for 159 unit characters by Williams et al. (1983)(3) reduced the genus Streptomyces to 77 cluster groups, which is currently reflected in the recently published Volume 4 of Bergey's Manual of Systematic Bacteriology (Williams et al., 1989)(4) Some of these groups, such as the Streptomyces cyaneus cluster, are rather heterogeneous in terms of mor- phological characteristics. Other clusters, such as the Streptomyces violaceus-niger group, are relatively homogeneous morphologically, consisting of gray-spored strains. In order to clarify the genetic relationships of the strains in these two dusters, DNA hybridization methodology was used to measure genetic relatedness among strains in each cluster group. Since many of the International Streptomyces Project (ISP) strains originated from the ARS Culture Collection, and all of the ISP collection of strains was accessioned into the ARS Culture Collection upon Prof. Shirling's retirement, it was very easy to compare the same strains as used in the numerical taxonomic study of Williams et al. (1983)(3).

Chromosomal DNA was isolated and purified from strains using previously described methods (Labeda and Lyons, 1991)(5), and DNA relatedness among strains was determined from Coto.5 values in 5X SSC and 20% DMSO at 50 C (Tm-23 C) by the method of Seidler et al. (1975)(6) and Seidler and Mandel (1971)(7) as modified by Kurtzman et al. (1980)(8). Data analysis was performed using SAS for Personal Computers, Release 6.04 (SAS Institute Inc., Cary, NC) on a DTK Datatek KEEN-16 microcomputer. The DNA relatedness data were clustered by the unweighted pair group average algorithm and also analyzed by principal component analysis.

Evaluation of DNA relatedness among strains of the S.cyaneus cluster revealed that S.cyaneus NR RL B-16305, which is equivalent to the strain used in the Williams et al. study, is one of the most genetically distant strains in the cluster group, averaging approximately 25% DNA complementarity to the other strains in the cluster. Two major DNA relatedness groups were observed for the S.cyaneus cluster at >70% DNA homology: the S. coeruleorubidus group and the S.purpurascens group. DNA relatedness greater than 70% has been proposed as the level to define bacterial species by the ad hoc ICSB Committee on Reconciliation of Approaches to Bacterial Systematics (Wayne et al., 1987)(9). The strains composing the S.coeruleorubidus group are: S.coeruleorubidus NRRL B-2569, S.coeruleorubidus NRRL 3045, S. bellus NRRL B- 2575, and S. curacoi NRRL B-2901. It is interesting to note that all of these strains are morphologically very similar, i.e., they all have blue, spiny spores borne on spiral sporophores, and the color of their substrate mycelium is similar. The strains belonging to the S. purpurascens group are: S.purpurascens NRRL B12230, S.afghaniensis NRRL B-5621, S.janthinus NRRL B-3365, and S. violatus NRRL B-2867. Interestingly, these strains are also morphologically similar, i.e., they all have red, spiny spores borne on spiral sporophores and have similar substrate mycelium pigmentation and produce reddish diffusible pigments. The emended descriptions of these species, including the DNA-related species as subjective synonyms, will be published shortly (Labeda and Lyons, 1991)(5).

Evaluation of DNA relatedness among 13 strains belonging to the S.violaceusniger resulted in similarly interesting data. The eight strains of S. violaceusniger evaluated separated into 7 different DNA relatedness groups with 70% DNA homology defining the minimum for "species" relatedness. Four of the relatedness groups contained only a single strain. It was found that two of the S. violaceusniger strains, NRRL B-1476 and NRRL B-1477, and S. endus NRRL 2339, the type strain for this species, demonstrate species level DNA relatedness (>70%) with S.hygroscopicus NRRL 2387, the type strain of this species. An exchange of strain numbers between S. violaceusniger strains NRRL B-1476 and NRRL B-1468 during lyophilization in 1958 was discovered when DNA from these strains was hybridized with that from ISP 5563, which was received from Dr. Shirling's collection. This strain was provided to him originally from the ARS Culture Collection as NRRL B-1476. This mistake in the ARS Culture Collection had been unknowingly corrected when the strains were relyophilized in 1976 from pre1958 ampules. The actual identity of ISP 5563 was found to be NRRL B-1478 rather than NRRL B1476, and these strains are not the same species, i.e., they exhibit only approximately 40% DNA related- ness. Strain NRRL B-1478 exhibits >97% DNA relatedness with the type strains for S. violaceusniger held in other world culture collections, including ATCC 27477, DSM 40563m and JCM 4850. Thus, it is proposed in a manuscript to be published elsewhere that the species description of S. violaceusniger be designated on NRRL B1478 as the type strain, rather than NRRL B-1476. The S. violaceusniger strains NRRL B1476 and NRRL B-1477 are actually strains of the species S. hygroscopicus, as is the type strain of S. endus, NRRL 2339. Based on DNA relatedness data, it appears that S. sparsogenes NRRL 2940 and S. melanosporofaciens NRRL B12234 are distinct species, and S. hygroscopicus subspecies geldanus NRRL 3602 probably should be elevated to a separate species.

A preliminary evaluation (unpublished data) of the correlation of data from analysis of banding patterns from single-dimension polyacrylamide gel electrophoresis of radio- labelled whole cell proteins with DNA relatedness data was encouraging, but it appeared that more studies were needed to ascertain that cells in exactly the same life stage were being labelled, since some anomalies were observed.

The study of DNA relatedness in the S. cyaneus phenotypic cluster reestablished the validity of morphological characteristics, such as spore color, spore surface, and sporophore morphology, for the description of streptomycete taxa. Both of the emended taxa S. coeruleorubidus and S. purpurascens can be described in terms of morphological and physiological properties that could be determined in laboratories not capable of performing molecular systematic studies. The utility of DNA relatedness studies in establishing the pedigree of strains was further illustrated by the detection of the exchange of strain numbers for S. violaceusniger strain during relyophilization in 1958. If the unusual hybridization data had not been observed, an examination of the older lyophilization records would not have been done.

References

1) Trejo, W.H.: An evaluation of some concepts and criteria used in the speciation of streptomycetes. Trans. N.Y. Acad. Sci. Ser. II, 32: 989-997, 1970.

2) Skerman, V.B.D., V.McGowan & P.H.A. Sneath: Approved lists of bacterial names. Int. J. Syst. Bacteriol., 30: 225420, 1980.

3) Williams, S.T., M.Goodfellow, G. Alderson, E.M.H.Wellington, P.H.A.Sneath & M.J. Sakin: Numerical classification of Streptomyces and related genera. J. gen. Microbiol., 129: 1743-1813, 1983.

4) Williams, S.T., M.Goodfellow & G. Alderson: Genus Streptomyces Waksman and Henrici, 1943, 339^AL. In "Bergey's Manual of Systematic Bacteriology, Volume 4" (eds. S.T. Williams & M.E. Sharpe), pp. 2452-2492. Williams and Wilkins, Baltimore, 1989.

5) Labeda, D.P. & A.J.Lyons: Deoxyribonucleic acid relatedness among species of the "Streptomyces cyaneus" cluster. Syst. Appl. Microbiol., 14: 1991, in press.

6) Seidler, R.J., M.D.Knittel & C.Brown: Potential pathogens in the environment: cultural reactions and nucleic acid studies on Klebsiella pneumoniae from chemical and environmental sources. Appl. Microbiol., 29: 819- 825, 1975.

7) Seidler, R.J. & M.Mandel: Quantitative aspects of deoxyribonucleic acid renaturation: base composition, state of chromosome replication, and polynucleotide homologies. J. Bacteriol., 106: 608-614, 1971.

8) Kurtzman, C.P., M.J .Smiley, C .J. Johnson, L.J.Wickerhan & G.B.Fuson: Two new and closely related heterothallic species, Pichia amylophila and Pichia mississippiensis: Characterization by hybridization and deoxyribonucleic acid reassociation. Int. J. Syst. Bacteriol., 30: 208-216, 1980.

9) Wayne, L.G., D.J.Brenner, R.R.Colwell, P.A.D.Grimont, O.Kandler, M.I.Krichevsky, L.H.Moore, W.E.C.Moore, R.G.E.Murray, E. Stackebrandt, M.P.Starr & H.G.Truper: Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int. J. Syst. Bacteriol., 37: 463-464, 1987.

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