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

Use of actinophage for selective isolation purposes:
Current problems

D.I.KURTBOKE* and S.T.WILLIAMS

The University of Liverpool, Department of Genetics and Microbiology, P.O. Box 147, Liverpool L69 3BX, U.K. (*Present address: The University of Western Australia, School of Agriculture, Soil Science and Plant Nutrition, Nedlands, Perth, W.A. 6009, Australia)

Code Number: AC91007
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Until recently, inaccurate classification of many actinomycete taxa has hindered not only the development of efficient selective isolation procedures (Goodfellow and Williams, 1986, Goodfellow, 1988) but also the establishment of actinophage typing systems, for reliable identification of actinomycete species or genera (Prauser, 1984). Consequently the use of actinophage as a natural tool has not been accepted as an effective and inexpensive aid to taxonomy of many novel genera and could not be exploited for selective isolation purposes.

When host range studies of actinophage. started (Bradley and Anderson, 1958; Bradley et al., 1961) the importance. of differences in cell wall composition had not been realised or adequately understood. Consequently, the taxonomic position of strains used could not be checked with respect to this character, thereby risking misclassification. Thus, actinophage were reported to be equally active against strains of pairs of genera differing in their cell wall chemotype such as Streptomyces and Nocardia, Streptomyces and Micromonospora and Streptomyces and Actinomadura (Prauser, 1976). As a result for many years actinophage susceptibility has not been accepted as a criterion in the taxonomy of actinomycetes. This continued until the misclassified positions became clarified (Prauser, 1984). Cell wall composition is now a generally accepted criterion in the classification of actinomycetes (Goodfellow, 1989). Actinophage specificity relies upon adsorption to receptors of cell wall, that are usually present in only a very limited number of closely related bacterial strains. Since phage susceptibility is in full correspondence with cell wall composition it again becomes an accepted criterion in actinomycete taxonomy and could be used for the classification of many novel genera e.g. Amycolata and Amycolatopsis (Prauser, 1984, Lechevalier et al., 1986).

Wellington and Williams (1981) in conjunction with an-extensive numerical phenetic study of the genus Streptomyces and related genera compared the limits and relationships of the Streptomyces genus as defined by phage activity spectra. Generic host ranges of polyvalent phage isolated to a variety of Streptomyces species were studied and host ranges of polyvalent phage isolated to genera sharing the wall chemotype I with Streptomyces and genera with different chemotypes were also investi- gated. Streptomyces (cell wall chemotype I) was clearly distinguished from a range of genera with chemotypes II, III, IV and VI according to their phage typing results. Common phage susceptibility was understood as one of the distinctive characters of the genera Streptomyces and Streptoverticillium of the family Streptomycetaceae (Prauser, 1970; Wellington and Williams, 1981).

Recently these substantial amounts of data about Streptomyces phage was used for the selective isolation of nonstreptomycete actinomycetes from soil (Kurtboke, 1990). Phage susceptibility of streptomycetes provided a selective means of reducing streptomycetes on isolation plates and hence increased the chances of other non-streptomycete genera developing (Kurtboke, 1990) on agar.

Before use of actinophage in selective isolation can be extended for other genera, more information is required on the specificity of actinophage activity. Almost no phage is virulent for all strains of the taxon under study (Prauser, 1970), therefore number of the members of each taxon studied and the number and selection of phage applied is important to obtain valid statements on actinophage specificity. For most actinomycete genera actinophage specificity requires further study and isolation of more phage banks. For some established genera such as, those within the sporoactihomycetes of cell wall chemotype III, no phage has been located despite repeated attempts (Prauser, 1984). For some species, such as for Arthrobacter globiformis, phage are rarely detected unless soil is nutritionally amended and incubated (Casida and Liu, 1974). Monospecificity of some genera such as Sporichthya and Kineosporia, hinders the isolation of their phage and consequently the availability of adequate knowledge about the activity of the latter at taxonomic level (Prauser, 1984). Also the relatively small number of strains of Nocardioides, Oerskovia and Promicromonospora isolated in different laboratories may indicate their rareness, and compound the difficulties of detecting their phage in soil. Prauser (1984) suggested that either, isolation techniques have been inappropriate for these actinomycetes or, the taxonomic description of these hosts found in the literature does not accurately reflect the forms that occur in soil.

For example, for many years the susceptibility of Pimelobacter (Arthrobacter) simplex to Nocardioides phage and susceptibility of Nocardioides to Pimelobacter (Arthrobacter) simplex phage were not taken into consideration as a result of the view that the inclusion of morphologically different organisms in one genus might render the practical use of the classification more difficult (Prauser, 1984). However, now it is known that N. albus and A. simplex are closely related organisms. The two species share the peptidoglycan type (Prauser, 1976), they have similar levels of DNA base composition (Suzuki and Komagata, 1983), a similar pattern of fatty acids (Collins et al., 1983) and identical predominating menaquinones (O'Donnell et al., 1982).

Similarly, clearing effects caused by soil-isolated polyvalent Streptomyces phage on Nocardiopsis dassonvillei gave rise to some speculation about the relationships of these two taxa (Prauser, 1984), but information about the taxon specificity of four Nocardiopsis phage isolated by Prauser (1981) is still lacking. The current situation of this genus requires further comparative biochemical and genetic studies of all members of the genus together with the genus Saccharothrix (Labeda et al., 1984), all of which share many morphological and biochemical properties (Meyer, 1989). Promicromonospora was found to be susceptible to taxon-specific phage but not susceptible to phage of various sets that attack oerskoviae and other nocardioform organisms (Prauser, 1984). Cellulomonads have also been found to be resistant to Oerskovia phage and vice versa which may be used as additional evidence (Prauser, 1984) against the case for combining Promicromonospora with Oerskovia and uniting them with Cellulomonas (Stackebrandt et al., 1982).

Despite many novel advances in chemical and molecular biological techniques (Goodfellow et al., 1988), argu- ments and classification problems still exist and many genera may be referred to as "in search of a family" (e.g. Promicromonospora, Kalakoutskii et al., 1989; Intrasporangium, Kalakoutskii, 1989; Sporichthya, Lechevalier and Lechevalier, 1989). This exemplifies the problems encountered in obtaining a wider, accurate picture of actinophage specificity, which is needed for the extension of the principle of using phage for selective isolation purposes.

In the latest edition of Bergey's Manual of Systematic Bacteriology (Williams et al., 1989) the family Streptomycetaceae includes four genera: Streptomyces, Streptoverticillium, Kineosporia and Sporichthya. Unlike Streptomyces, species of the genera Kineosporia and Sporichthya have not been commonly isolated (Prauser, 1984). These two genera are monospecific and only five strains of Sporichthya poly- morpha and one strain of Kineosporia aurantiaca have been identified (Prauser, 1984). Monospecificity of these genera hindered the isolation of their phage. A phage isolated for a strain of Sporichthya polymorpha by Prauser (1984) was found to be ineffective against several strains of Streptomyces. Although the cell wall composition of this genus resembled that of other members of the family Streptomycetaceae, because of its unusual morphology and difficulties of determining the chemistry of its phospholipids, menaquinones and DNA, it has been referred to as "a genus in search of a family" (Lechevalier and Lechevalier, 1981 ).

Another genus having cell wall chemotype I Intrasporangium was found to be susceptible to the phage isolated to Streptomyces (Prauser and Falta, 1968; Wellington and Williams, 1981a). However, extensive numerical taxonomic studies by Williams et al. (1983a) pointed to a distinct and separate position of Intrasporangium compared to streptomycetes and to other actinomycetes. Kutzner (1981) suggested that Intrasporangium might not belong to the order Actinomycetales. The other genus, sharing same cell wall chemotype with the members of the family Streptomycetaceae, Nocardioides has been readily distinguished from them as it is resistant to the phage isolated for other family members (Williams et al., 1980; Prauser, 1984).

For the determination of the natural relationships of genera, the members of a host specific actinophage set must be selected from a variety of phage after intensive cross-infection studies including as many strains and phage as possible. This requires considerable knowledge about the host and actinophage ecology and information concerning the isola- tion of more phage for potential hosts. Although recent advances in isolation techniques provides a basis for more de- tailed studies of actinophage-host interactions in their natural habitats (Williams et al., 1987), it is impossible to provide optimal conditions for the isolation of all actinophage present in a given sample, since phage-host systems differ considerably in their susceptibility to in- activation by a range of environmental conditions (Lanning and Williams, 1982), such as the adsorption of phage to soil colloids (Sykes and Williams, 1978), pH (Sykes et al., 1981) and temperature (Williams and Lanning, 1984).

It has become clear that the concept of selective isolation is no longer an application of a single method. It is a combi- nation of adequate knowledge in eco-taxonomic disciplines. Further detailed studies in actinophage ecology and phage-host interactions in their natural substrates may aid to obtain more information about actinophage specificity which in turn may encourage the use of actinophage for selective isolation purposes.

References

Bradley, S.G. & D.L.Anderson (1958). Taxonomic implication of actinophage host-range. Science, 128:413-414

Bradley, S.G., D.L.Anderson & L.A.Jones (1961). Phylogeny of actinomycetes as revealed by susceptibility to actinophage. Dev. Ind. Microbiol., 2:223-237

Casida, L.E. & K.C.Liu (1974). Arthrobacter globiformis and its bacteriophage in soil. Appl. Microbiol., 28, 951-959

Collins, M.D., R.M.Keddie & R.M.Kroppenstedt (1983). Lipid composition of Arthrobacter simplex, Arthrobacter tumescens and the possibly related taxa. System.Appl. Microbiol., 10, 897-903

Goodfellow, M. (1988). Numerical taxonomy and selective isolation of industrially important actinomycetes. Actinomycetologica, 2:13-29

Goodfellow, M. (1989). Suprageneric classification of actinomycetes. In: S.T.Williams, M.E. Sharpe and J.G.Holt (eds.) Bergey's Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore, Vol. 4, pp. 2333-2339

Goodfellow, M. & E.Williams (1986). New strategies for the selective isolation of industrially important bacteria. Biotech. Gen.Eng. Revs, 4, 213-262

Goodfellow, M., E.Stackebrandt & R.M. Kroppenstedt (1988). Chemotaxonomy and actinomycete systematics. In: Y. Okami, T. Beppu and H. Ogawara (eds.) Biology of Actinomycetes' 88. Japan Sci. Soc. Press, Tokyo, pp. 288-293

Kalakoutskii, L.V. (1989). Genus Intrasporangium Kalakoutskii, Kirillova and Krasil'nikov 1967, 79 AL. In: S.T.Williams, M.E. Sharpe and J.G.Holt (eds.) Bergey's Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore, Vol. 4,, pp. 2313-2316

Kalakoutskii, L.V. N.S.Agre, H.Prauser & L.L.Evtushenko (1989). Genus Promicromonospora Krasil'nikov, Kalakoutskii and Kifillova 1961a, 107 AL. In: S.T.Williams, M.E. Sharpe and J.G.Holt (eds.) Bergey's Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore, Vol. 4, pp. 2392-2395

Kurtboke, D.I. (1990). Ph.D Thesis. New approaches to the isolation of non-streptomycete actinomycetes from soil. The University of Liverpool, U.K.

Labeda, D.P., R.T.Testa, M.P.Lechevalier & H.A.Lechevalier (1984). Saccharothrix, a new genus of the Actinomycetales. Int. J.Syst. Bacteriol., 34, 426-431

Lanning, S. & S.T.Williams (1982). Methods for the direct isolation and enumeration of actinophages in soil. J.gen.Microbiol., 135, 121-133

Lechevalier, M.P. & H.A.Lechevalier (1989). Genus Sporichthya Lechevalier, Lechevalier and Holbert 1968, 279 AL. In: S.T.Williams, M.E. Sharpe and J.G.Holt (eds.) Bergey's Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore, Vol. 4, pp. 2507-2508

Lechevalier, M.P., H.Prauser, D.P.Labeda & J.S.Ruan (1986). Two new genera of nocardioform actinomycetes: Arnycolata gen. nov. and Amycolatopsis gert. nov. Int. J. Syst. Bacteriol., 3:29-37

Meyer, J. (1989). Genus Nocardiopsis Meyer 1976, 487AL. In: S.T.Williams, M.E. Sharpe and J.G.Holt (eds.) Bergey's Manual of Systematic Bacteriology. Williams & Wilkins, Baltimore, Vol. 4, pp. 2562-2563

O'Donnell, A.G.M.Goodfellow & D.E.Minnikin (1982). Lipids in the classification of Nocardioides: Reclassification of Arthrobacter simplex (Jensen) Lochhead in the genus Nocardioides (Prauser) emend. O'Donnell et al., as Nocardioides simplex comb.nov. Arch. Microbid., 133, 323-329

Prauser, H. (1970). Characters and genera arrangement in the Actinomycetales. In: H. Prauser (ed.) The Actinomycetales. G.Fisher Verlag, Jena, pp. 407-418

Prauser, H. (1976). Nocardioides, a new genus of the order Actinomycetales. Int. J. Syst. Bacteriol., 26, 58-65

Prauser, H. (1981). Nocardioform organisms: General characterisation and taxonomic relationships. Zbl. Bakt. Mikrobtol. Hyg. Suppl., 11, 17-24

Prauser, H. (1984). Phage host ranges in the classification and identification of gram-positive branched and related bacteria. In: L.OrtizOrtiz, J.F.Bojalil and V.Yakoleff, (eds.) Biological, Biochemical and Biomedical Aspects of Actinomycetes. Academic Press, Orlando, pp. 617-633

Stackebrandt, E., H.Seiler & K.-H. Schleifer (1982). Molecular systematics of actinomycetes and related organisms. Zbl. Bakt. Mikrobiol. Hyg. Abt.l. Orig. C, 3, 403-409

Suzuki, K. & K.Komagata (1983). Pimelobacter gen.nov. - a new genus of coryneform bacteria with LL-diaminopimelic acid in the cell wall. J. Gen Appl. Microbiol. 29, 59-71

Sykes, I.K. & S.T.Williams (1978). Interaction of actinophage and clays. J.gen. Microbiol. 108, 97-102

Sykes, I.K., S.Lanning, & Williams, S.T. (1981). The effect of pH on soil actinophage. J. gen.Microbiol. 122, 271-280

Wellington, E.M.H. & S.T.Williams (1981). Host ranges phages isolated to Streptomyces and other genera. Zbl. Bakt. Mikrobiol. Hyg., Suppl., 11, 7-16

Williams, S.T. & S.Lanning (1984). Studies on the ecology of streptomycete phage in soil. In: L.Ortiz-Ortiz, J.F.Bojalil and V.Yakoleff, (eds.) Biological, Biochemical and Biomedical Aspects of Actinomycetes. Academic Press, Orlando, pp. 473-483

Williams, S.T., A.M.Mortimer & L. Manchester (1987). Ecology of bacteriophages. In: S.M.Goyal, C.P.Gerba and G.Bitton (eds.) Phage Ecology. J.Wiley & Sons, Inc., Chichester, pp. 157-179

Williams, S.T., M.Goodfellow, Gjlderson, E.M.H.Wellington, P.H.A.Sneath & M.J. Sackin (1983a). Numerical classification of Streptomyces and related taxa. J. gen. Micro- biol., 129:1743-1813

Williams, S.T. M.Goodfellow, E.M.H. Wellington, J.C.Vickers, G.Alderson, P.H.A. Sneath, M.J.Sackin & A.M.Mortimer (1983b). A probability matrix for identification of streptomycetes. J.gen. Microbiol., 129:1815-1830

Williams, S.T., E.M.H.Wellington, M.Goodfellow, G.Alderson, M.J.Sackin, & P.H.A. Sneath (1981a). The genus Streptomyces - a taxonomic enigma. Zbl. Bakt. Mikrobiol.Hyg., Suppl., 11, 47-57.

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