Actinomycetes University of Udine, Mycology Department ISSN: 0732-0574 Vol. 7, Num. 1, 1996

Present Role Of Phage Typing In Reliable

Actinomycete Identification Schemes

D. I. KURTBOKE

Murdoch University, Department of Biological and Environmental Sciences, Perth, WA 6150, Australia.

Present address: AMRAD Natural Products Pty. Ltd., 576 Swan Street, Richmond, Melbourne, Victoria 3121, Australia

Code Number: AC96006
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ABSTRACT. Phage sensitivity of actinomycetes belonging to different genera is discussed at species, genus and family levels. Current information on phage specificity at taxonomic level indicates that results are in full correspondence with data obtained from numerical, chemical and molecular biological studies. The value of phage susceptibility as an aid to reliable actinomycete identification schemes is emphasised.

"Phages are better taxonomists than us" (S.T. Williams, personal communication, 1990).

Until recently, inaccurate classification of actinomycete taxa and associated problems with the identification of actinophage susceptibility has hindered the use of actinophage typing systems for a reliable identification of actinomycete species or genera. Consequently actinophage typing has not been widely accepted as an effective and inexpensive aid to the taxonomy of actinomycetes (Prauser, 1978; Prauser, 1984; Williams et al., 1980; Wellington and Williams, 1981; Kurtboke and Williams, 1991).

When host range studies of actinophage started, the importance of differences in cell wall composition had not been adequately studied. Consequently, the taxonomic position of actinomycete strains could not be checked, thereby risking misclassification (Prauser, 1984; Williams et al., 1980). Thus, actinophage were initially reported to be equally effective for distinguishing between pairs of genera differing in their cell wall chemotype such as Streptomyces and Nocardia, Micromonospora and Actinomadura (Prauser, 1984; Williams et al., 1980). Following the recognition of the relationship between cell wall composition and phage susceptibility, actinophage typing has become an accepted criterion in actinomycete taxonomy and could be used as an additional criterion for the classification of many novel genera, e.g. Amycolata and Amycolatopsis (Prauser, 1984; Lechevalier et al., 1986). However although phage sensitivity may help to elucidate relationships between taxa, it is still not possible to identify unknown taxa by this method (Prauser, 1978).

To determine the place of phage typing in reliable actinomycete identification schemes still more information is required on the specificity of actinophage activity. No phage is virulent for all strains of a taxon and therefore the number and selection of phage applied vital in order to ascertain actinophage specificity using as many host strains and phage as possible (Prauser, 1984). This requires knowledge of the host and actinophage ecology and information concerning the isolation of alternative phage (Prauser, 1984; Kurtboke and Williams, 1991).

RECENT PROGRESS

a. Species Specific Phage. As a consequence of recent advances in isolation techniques more information has become available on actinophage-host interactions in their natural habitats and on actinophage specificity (Kurtboke and Williams, 1991). For example, the isolation of specific phage has contributed to the identification of Clavibacter toxicus, which played an important role in the identification of the "annual ryegrass toxicity" bacterium (Riley and Gooden, 1991; Ophel et al., 1993, Kurtboke, 1994) which is responsible for $16-20 million of livestock losses/annum in Western Australia.

Phage typing is also currently used for the identification of Dermatophilus congolensis, agent in Western Australia of the "lumpy wool" of sheep (Patten et al., 1995)

Isolation of species specific phage to some Streptomyces species, e.g., S.albus (Prauser, 1978) has also provided useful taxonomic information and led to a better identification of synonyms, e.g., in S.vio- laceoruber (Korn-Wendish and Schneider, 1992).

b. Genus Specific Phage. Detection of Actinomadura phage from soils collected from the Philippines (Long et al., 1991) was an encouraging step which led to question previous failures in the isolation of Actinomadura phage. Further detection of the Actinomadura phage in organic mulches used for avocado plantations in Western Australia (Kurtboke et al., 1993) revealed the heterogeneity of the genus Actinomadura as revealed by chemotaxonomic and molecular biological studies. The lysis of Nocardiopsis flava by the phage isolated to Actinomadura verrucosospora and Actinomadura pusilla (Kurtboke et al., 1993) may give rise to some speculation about the relationship between these species and the transfer of former Actinomadura flava to the genus Nocardiopsis (Preobrazhenskaya et al., 1982) and later to the genus Saccharothrix (Grund and Kroppenstedt, 1989).

Long et al., (1993) also isolated two phage that attack species of the genera Microtetraspora and Microbispora.

c. Family Specific Phage. Korn-Wendish and Schneider (1992) reported that in Pseudonocardiaceae phage were clearly limited to the family genera, e.g., Amycolatopsis, Saccharomonospora and Saccharopolyspora.

Genus Thermoactinomyces, which was placed in the family Bacillaceae (Stackebrandt and Woese, 1980) was not susceptible to the phage isolated to actinomycete species (Prauser, 1984; Kempf et al., 1987; Kurtboke and Sivasithamparam, 1993), however, it was found to be susceptible to the phage isolated to the members of the of the family Bacillaceae from a composted eucalyptus bark in Western Australia (Kurtboke and Sivasithamparam, 1993). This provided further evidence on the transfer of the genus into the family Bacillaceae.

FUTURE WORK

Despite many novel advances in chemical and molecular biological techniques, classification problems still exist and many genera may be referred to as "in search of a family" (e.g., Promicromonospora, Intrasporangium and Sporichthya). Monospecificity of certain actinomycete genera, e.g., Kineosporia also hinders the isolation of their phage (Prauser, 1984). In addition for some genera phage susceptibility does not correspond to data obtained from numerical and other taxonomic techniques, e.g., the genus Intrasporangium is susceptible to Streptomy- ces phage (Prauser, 1984). These facts exemplify the problems encountered in obtaining more reliable information on actinophage specificity, which is crucial for the acceptance of phage typing as a taxonomic criterion.

A number of points still remain:

- Why is one strain of a given taxon resistant to a given phage while other strains of the same taxon are suitable hosts for phage reproduction (Prauser, 1978)?.

- What is the taxonspecificity of lytic actinophage that do not multiply in the cells affected (Prauser, 1981)?.

- Why the thermophilic streptomycetes are not sensitive to Streptomyces phage which are active against mesophilic strains and vice versa (Prauser, 1978)?.

- Taxonomic classification of actinomycetes is greatly facilitated by virulent mutants of the temperate phage (Rautenstein, 1970). Would the sensitivity of cultures belonging to different species to one and the same specific phage and their ability to be lysogenized indicate that such cultures may have some other features in common (Rautenstein, 1970)?

- Dowding (1973) reported that actinophage differ from other bacteriophage only in the complex, mycelial nature of its host. He noted that, since germination of the actinomycete spores does not occur synchronously, by the time a high enough proportion of the spores have germinated, some of them will be large clumps of mycelium and they can adsorb a great many phage particles yet continue to plate as single colony-forming units. Therefore he concluded that the concept of multiplicity of infection will thus remain meaningless in this system until a means is found of causing synchronous and uniform spore germination. Following these observations can we also relate the difficulties encountered during the isolation of phage for cell wall chemotype III actinomycetes to the complex growth nature of these organisms and why the isolated phage have so far proven to be poorly lytic (Long et al., 1993) and differences were noted between the patterns obtained using the routine test dilution (Williams et al., 1993).

- Irregular distribution of phage morphotypes may reflect evolution or in some cases, characteristic phage morphology may also reveal bacterial relationships (Francki et al., 1991). Although most actinophage detected for actinomycetes belong to the B1 morphotype and no special features (Ackermann, 1985) it may be noteworthy to investigate why phage with contractile tails are rare in actinomycetes (H.W.Ackermann, personal communication).

Until such points are clarified phage may only help to elucidate relationships between taxa (Prauser, 1978). Further studies in host-phage interactions and actinophage ecology will contribute important information on actinophage specificity at a taxonomic level which will encourage the use of phage typing systems in reliable actinomycete identification schemes.

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Copyright 1996 C.E.T.A., The International Centre for Theoretical and Applied Ecology, Gorizia