Immunodiffusion Analyses of Phenetically Defined Strains of Streptomyces, Streptoverticillium and Nocardiopsis

System. Appl. Microbiol. 8, 24-27 (1986)

Immunodiffusion Analyses of Phenetically Defined Strains of Streptomyces, Streptoverticillium and Nocardiopsis MALIN RIDELL\ GUN WALLERSTROM 1 and S. T. WILLIAMS 2 1 2

Department of Medical Microbiology, University of Goteborg, 413 46 Goteborg, Sweden Department of Botany, University of Liverpool, Liverpool L69 3BX, United Kingdom Received October 10, 1985

Summary Seventeen strains of Streptomyces, 13 of Streptoverticillium and two of Nocardiopsis dassonvillei were studied by the immunodiffusion technique and the results were compared with those of a previous numerical phenetic study. Generally there was good agreement between the serological and phenetic data, and the former supported the view that there is much synonymy among the described species of Streptomyces and Streptoverticillium. Thus, the number of species of these genera can be substantially reduced. The immunological results also underlined the close relationships between these genera, this being particularly marked between streptoverticillia and the Streptomyces lavendulae cluster. The relationships of Nocardiopsis dassonvillei were ambiguous as one strain showed clear serological affinities with streptomycetes but the other did not.

Key words: Streptomyces - Streptoverticillium - Nocardiopsis - Taxonomy - Immundiffusion

Introduction Immunological analyses have provided valuable taxonomic information for many bacteria, including some actinomycete genera. However, there have been relatively few serotaxonomic studies of streptomycetes, and related genera since Cross and Spooner (1963) demonstrated the potential taxonomic value of immunodiffusion techniques. Assessment of the serologiCal relationships within the genus Streptomyces and between it and allied genera have been impaired by lack of an objective classification scheme. The numerical classification of streptomycetes and related genera (Williams et al., 1983) provided comprehensive data for assessing the congruence between phenetic and serological similarity. Thus Ridell and Williams (1983) found that strains of Streptomyces and Streptoverticillium which shared a high number of precipitinogens, as determined by the immunodiffusion technique, also belonged to the same phenetic cluster, while strains less serologically related usually fell into different clusters. Having established the value of immunological analysis in this area of taxonomy, it was decided to apply it to obtain further information on the relationships between the genera Streptomyces, Streptoverticillium and Nocardiopsis, which are still somewhat unclear. Streptoverticillia can be distinguished from streptomycetes by their pro-

duction of sporophores in verticils or whorls on the aerial mycelium (Baldacci et aI., 1966; Locci et aI., 1969). The numerical phenetic data of Williams et al. (1983) indicated quite high similarity between strains of Streptoverticillium and the Streptomyces lavendulae cluster, but the continued recognition of the genus was supported. However, Streptoverticillium and Streptomyces have generally been regarded as closely related genera (Goodfellow and Cross, 1984). Nocardiopsis dassonvillei, the type strain of the genus (Meyer, 1976), also shares many phenetical characters with streptomycetes (Gordon and Horan, 1968; Williams et aL, 1983) but differs in wall (Lechevalier and Lechevalier, 1970 a) and lipid composition (Lechevalier et aI., 1981) and in sensitivity to phages (Wellington and Williams, 1981).

Materials and Methods

Test strains. Seventeen strains of Streptomyces, 13 of Streptoverticillium and two of Nocardiopsis dassonvillei were studied (Table 1). They represented 11 of the major clusters defined by Williams et al. (1983) namely Streptomyces anulatus (sub-cluster IB), S. halstedii (sub-cluster lC), Nocardiopsis dassonvillei (c1us-

25

Immunodiffusion Analyses Streptomyces Table 1. Number of precipitogens identified by comparative immunodiffusion analysis using seven reference systems (only combinations giving 4 or more precipitates are included)

No. of precipitinogens using reference system: b

~ ~

"

.~ v;

Strain

Cluster'

Streptomyces griseus ISP 5236'

IB

S. griseinus ISP 5047

IB

S.olivaceus ISP 5072

1C

Nocardiopsis dassonvillei NCTC 10488'

4

N. dassonvillei NCTC 10489

4

Streptomyces diastaticus lSP 5496'

19

Streptoverticillium cinnamoneum ISP 5005'

55

Stv.luteoverticillatum ISP 5038

55

Stv. mashuense lSP 5221

55

Stv. f/avopersicum ISP 5093'

56

Stv. neutropsis ISP 5259'

56

Stv. eurocidicum ISP 5604

56

Streptomyces avidinii lSP 5526

56

Stv.hiroshimense lSP 5037

57

Stv. blastomyceticum lSP 5029

58

Stv.griseoverticillatum ISP 5507

58

Stv.orinoci lSP 5571

58

Stv.biverticillatum ATCC 23615

59

·Stv.griseostramineum' lSP 5161

60

·Stv. kurssanovi' lSP 5162

60

S. goshikiensis ISP 5190'

61

S. colombiensis ISP 5558

61

S.f/avotricini ISP 5152 S. katrae ISP 5550

61 61

S.lavendulae ISP 5069

61

S.lavendulocolor ISP 5216

61

!!

.~

i

""v;

4

19

!:

"!:

.:l

""c

"

.~

~

~

~

"c

'"

'"

g " ;i" '"

55

56

56

"" '"";;

""

<;:,

;i

.~

"

.~

~

~

eo v; 61

10d (100) 6 (81)

4 (71)

9d (100) 5 (93)

8d (100)

4 (66) 8d (100) 6 (80)

5 (78)

4 (79) 4 (82) 5 (83) 4 (78) 4 (77)

4 (79)

8d (100) 7 (86) 6 (81)

4 (75) 4 (79)

6 (78) 5 (78) 5 (75) 6 (86) 9d (100) 5 (74) 4 (78) 4 (73) 4 (73) 4 (78) 4 (73) 5 (71)

4 (80)

61

S. racemochromogenes ISP 5194

61

S. subrutifus ISP 5445

61

S. toxytricini ISP 5178

61

S. virginiae ISP 5094

61

4 (85)

9d (100)

4 4

(91)

5

(74)

(80)

(72)

(79) 6 (80)

4

4 (73)

S.polychromogenes ISP 5316

S. xanthophaeus ISP 5134

IB

""c ~

~

.~

!:

6

5

(77) 4 (81)

5

(81)

5

4 (72)

(83) 5 (81)

(73)

(80)

4

6

61

Strain sources: ISP, Inrernational Streptomyces Project; ATCC, American Type Culture Collection, Rockville, Md., USA; NCTC, National Collection of Type Culture, Central Public Health Laboratory, Colindale Ave., London, UK. , Allocation to clusters defined at the 77.5% SSM similariry level from Williams et al. (1983). Subclusters IB and IC were defined at the 81 % SSM similarity level. b The numbers and parenrheses are SSM similarity values from Williams et al. (1983). , Reference strain. d Total number of identifiable precipitates in the reference system. Bionomials in inverted commas are not on the Approved Lists of Bacterial Names (Skerman et aI., 1980).

26

M. Ridell, G. Wallerstrom, and S. T. Williams

ter 4}, S. diastaticus, (cluster 19), Streptouerticillium griseocarneum (cluster 55), Stu. netropsis (cluster 56), Stu. hiroshimense (cluster 57), Stu. blastomyceticum (cluster 58), Stu. biuertici/latum (cluster 59), 'Stu. griseostramineum' (cluster 60) and Streptomyces lauendulae (cluster 61). Antigen preparation. Strains were grown in glucose (1 % w/v)-

nutrient broth (Difco) and incubated on a rotary shaker at 33 0c. Mycelium was harvested, washed, disintegrated and prepared as earlier described by Ridell and Williams (1983). Antisera preparation. Seven of the test strains were used to produce antisera. These were: S. griseus ISP 5236 (subcluster 1B), N. dassonuillei NCTC 10488 (cluster 4), S. diastaticus ISP 5496 (cluster 19), Stu. cinnamoneum ISP 5005 (cluster 55), Stu. f/auopersicum ISP 5093 (cluster 56), Stv. netropsis ISP 5259 (cluster 56), and S. goshikiensis ISP 5190 (cluster 61). The antisera were prepared in rabbits as earlier described by Ridell and Williams (1983). Reference precipitation systems. These were established according to the principles given by Lind (1961) and Ridell (1975) and represented each of the 7 strains selected for antiserum production. The reference system for S. griseus consisted of 10 precipitates, those for N. dassonvillei, Stv. netropsis and S. goshikiensis of 9 each, and those for S. diastaticus, Stv. cinnamoneum and Stv. f/avopersicum of 8 precipititates each. Immunodiffusion technique. The analyses were carried out using a micromodification of the immunodiffusion technique (Wadsworth, 1962) of Ouchter/ony (1958, 1962). Only precipitinogens which could be identified by the reference systems were recorded (Ridell, 1975) although additional precipitates frequently occurred.

Results and Discussion A total of 224 test combinations (7 reference X 32 test strains) were examined, of which only 41 failed to yield any identifiable precipitates. Of the latter, 36 were accounted for by the N. dassonvillei NCTC 10488 reference strain. The majority of combinations (132) produced from one to three identified precipitates. The remaining 51 combinations yielded four or more identifiable precipitates and these were selected as an indication of close serological relationships (Table I). The S. griseus reference serum formed most precipitates with S. griseinus, the other representative of sub-cluster 1B, most other combinations producing only one or two reactions. However, it gave four precipitates with N. dassonvillei (NTCC 10489) to which it is phenetically closely related. Surprisingly, the phentically similar Nocardiopsis reference strains (NCTC 10488) showed no crossreactions with S. griseus, nor with any of the other reference or test strains except for strain NCTC 10489. The latter, in contrast, shared some (1-5) precipitinogens with each of the reference strains. Streptomyces diastaticus, the sole representative of cluster 19, did not share an appreciable number (::: 4) of precipitates with any of the test strains. The Stv. cinnamoneum reference system shared six precipitinogens with Stv. luteoverticillatum but only three with Stv. mashuense, the other representative of cluster 55. It shared four or more precipitinogens with seven of the twelve streptoverticillia tested. Crossreactions between the two reference strains from cluster 56, Stv. f/avopersicum and Stv. netropsis, revealed

seven and six common precipitinogens respectively, and the other representative of this cluster, Stv. eurocidicum, shared several with both reference strains. All of the streptoverticillia test strains, with the exception of those in cluster 60, shared four or more precipitinogens with the Stv. netropsis reference strain. This strain also had four precipitinogens in common with six of the twelve strains representing cluster 61 (S. lavendulae). The reference strain from cluster 61, S. goshikiensis, shared four or more antigens with all other members of this cluster (S. lavendulae) with the exception of S. xanthophaeus with which it shared 3 precipitinogens. It also shared four precipitinogens with S. avidinii which was recovered in cluster 56 (Stv. netropsis) by Williams et al. (1983). This was one of the few streptomycetes to fall into the streptoverticillia clusters; it lacks the typical verticillate spore chains and its likely relationship with S. lavendulae has been suggested (Shirling and Gottlieb, 1972). Thus the results indicate that there is considerable congruence between serological and phenetic data for streptomycetes and related genera, as suggested by Ridell and Williams (1983). The previously reported phenetic similarity between S. griseus and N. dassonvillei (Gordon and Horan, 1968; Williams et al., 1983) was reflected by the serological relationships of one Nocardiopsis strain, but the reactions of the other were inexplicably different despite the high similarity (93% SSM) between them (Williams et ai., 1983). This raises yet another problem in classifying this enigmatic genus which, despite their phenetic similarities, can be differentiated from Streptomyces by its wall chemotype (Lechevalier and Lechevalier, 1970 a), lipid composition (Minnikin and Goodfellow, 1978), 16S rRNA similarity (Stackebrandt and Woese, 1981) and phage sensitivity (Wellington and Williams, 1981). The integrity of the phenetically defined Streptomyces and Streptoverticillium clusters was generally supported by the serology, although the close relationships between the latter were underlined. The results for the S. lavendulae cluster (61) provide a good example of the undoubted synonymy which exists among the described species of streptomycetes, as can be similarly concluded for the streptoverticillia. The number of species may thus be substantially reduced. The genera Streptomyces and Streptoverticillium are undoubtedly closely related, as they have a wall chemotype I (Lechevalier and Lechevalier, 1970 b), share high DNA homology values (Kroppenstedt et al., 1981), are lysed by the same phages (Prauser, 1976; Wellington and Williams, 1981) and contain similar lipids (Lechevalier et aI., 1981; Minnikin and O'Donnell, 1984). However, numerical phenetic data lend support for the continued recognition of Streptoverticillium and a study of rRNA-DNA duplexes (Gladek et ai., 1985) indicated that while the two genera were phylogenetically close, they could be distinguished on some similarity maps. The results here and those from the more limited study of Ridell and Williams (1983) show that these genera are serologically close, although distinction is possible between some of their species. The close phenetic similarity between streptoverticillia and S. lavendulae, which joined the same cluster-group at the 70.1 %

Immunodiffusion Analyses Streptomyces

Ssm similarity level (Williams et aI., 1983) was reflected by the serological cross reactions between them. Acknowledgement. M. R. and G.W. are grateful for support from the Ellen, Walter and Lennart Hesselman Fondation (Sweden). S.T.W. is grateful for support from the Science and Engineering Research Council (Great Britian). The skilful technical assistance of Susanne Johansson and Vivianne Sundaeus is gratefully acknowledged.

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Dr. Malin Ridell, Department of Medical Microbiology, University of G6teborg, Guldhedsgatan 10, S-413 46 G6teborg, Sweden