Three computer software packages were written in the C++ language for the analysis of numerical phenetic, 16S rRNA sequence and pyrolysis mass spectrometric data. The X program, which provides routines for editing binary data, for calculating test error, for estimating cluster overlap and for selecting diagnostic and selective tests, was evaluated using phenotypic data held on streptomycetes. The AL16S program has routines for editing 16S rRNA sequences, for determining secondary structure, for finding signature nucleotides and for comparative sequence analysis; it was used to analyse 16S rRNA sequences of mycolic acid-containing actinomycetes. The ANN program was used to generate backpropagation-artificial neural networks using pyrolysis mass spectra as input data. Almost complete 1 6S rDNA sequences of the type strains of all of the validly described species of the genera Nocardia and Tsukamurel!a were determined following isolation and cloning of the amplified genes. The resultant nucleotide sequences were aligned with those of representatives of the genera Corynebacterium, Gordona, Mycobacterium, Rhodococcus and Turicella and phylogenetic trees inferred by using the neighbor-joining, least squares, maximum likelihood and maximum parsimony methods. The mycolic acid-containing actinomycetes formed a monophyletic line within the evolutionary radiation encompassing actinomycetes. The "mycolic acid" lineage was divided into two clades which were equated with the families Coiynebacteriaceae and Mycobacteriaceae. The family Coiynebacteriaceae contained the genera Cotynebacterium, Dietzia and Turicella and the family Mycobacteriaceae the genera Gordona, Mycobacterium, Nocardia, Rhodococcus and Tsukamurella. It was clear from the 1 6S rDNA sequence data that Nocardia pinensis was misclassified in the genus Nocardia and that TsukamurelIa wratislaviensis belonged to the genus Rhodococcus. The genus Nocardia formed a distinct dade that was clearly associated with the genus Rhodococcus. Two sublines were recognised within the Nocardia dade; one consisted of Nocardia asteroides and related taxa and the other of Nocardia otitidiscaviarum and allied species. The two sublines are distinguished by nucleotide differences in helix 37-1. The type strains of all of the Nocardia species contained hexahydrogenated menaquinones with eight isoprene units in which the two end units were cyclised. Actinomycetes selectively isolated from an activated sludge plant showing extensive foaming were the subject of a polyphasic taxonomic study. The sludge isolates, which clearly belong to the genus Tsukamurella on the basis of 1 6S rRNA data, contained highly unsaturated long chain mycolic acids and unsaturated menaquinones with nine isoprene units, properties consistent with their classification in the genus Tsukamurella. Six representative isolates and marker strains of Tsukamurella paurometabola were the subject of a numerical phenetic taxonomic study. The test strains were assigned to four groups in the simple matching coefficient, unweighted pair group method with arithmetic averages analysis. The sludge isolates formed a homogeneous cluster with the three remaining clusters composed of Tsukamurella paurometabola strains. Excellent congruence was found between these numerical taxonomic data and results derived from corresponding studies based on Curie point pyrolysis mass spectrometric and whole-organism protein electrophoretic analyses. The combined data suggest that the sludge isolates form the nucleus of a new species of the genus Tsukamurella and that Tsukamure!!a paurometabola is a heterogeneous taxon. Representatives of three putatively novel streptomycete species isolated from soil were used to develop and evaluate an identification system based on Curie point pyrolysis mass spectromety and artificial neural network analysis. The test strains consisted of sixteen target Streptomyces strains and one hundred and thirty-eight actinomycetes belonging to the genera Actinomadura, Mycobacterium, Nocardia, Nocardiopsis, Saccharomonospora and Streptosporangium. It was clear from the identification results that artificial neural network analysis was superior to conventional procedure based on principal component and canonical variate statistics. The problem of misidentification of some of the non-target strains was solved by the development of a neural network topology which contained an output neuron designed to detect non-target pyrolysis mass spectrometric patterns. The pyrolysis mass spectrometry-artificial neural network system was evaluated using thirteen fresh streptomycete isolates and found to be capable of long-term identification of the target strains.

Molecular systematic methods were applied in a series of studies designed to resolve the taxonomic relationships of thermophilic actinomycetes known to be difficult to classify using standard taxonomic procedures. The test strains included representatives of clusters defined in an extensiven umerical phenetic survey of thermophilic streptomycetesa nd twelve marker strains. The resultant genotypic data together with the results of corresponding phenotypic studies were used to highlight novel taxa and to improve the circumscription of validly described species. The most comprehensive study was undertaken to clarify relationships within and between representative alkalitolerant, thermophilic and neutrophilic, thermophilic streptomycetes isolated from soil and appropriate marker strains. The resultant data, notably those from DNA: DNA relatedness studies, supported the taxonomic integrity of the validly described species Streptomyces thermodiastaticus, Streptomyces thermoviolaceus and Streptomyces thermovulgaris. However, the genotypic and phenotypic data clearly show that Streptomyces thermonitrificans Desai and Dhala 1967 and Streptomyces thermovulgaris (Henssen 1957) Goodfellow et al. 1987 represent a single species. On the basis of the priority, Streptomyces thermonitrificans is a later subjective synonym of Streptomyces thermovulgaris. Similarly, eight out of eleven representative alkalitolerant, thermophilic isolates and three out of sixteen representative neutrophilic, thermophilic isolates had a combination of properties consistent with their classification as Streptomyces thermovulgaris. One of the remaining alkalitolerant, thermophilic isolate, Streptomyces strain TA56, merited species status. The name Streptomyces thermoalcalitolerans sp. nov. is proposed for this strain. A neutrophilic, thermophilic isolate, Streptomyces strain NAR85, was identified as Streptomyces thermodiastaticus. Four other neutrophilic thermophilic isolates assigned to a numerical phenetic cluster and a thermophilic isolates from poultry faeces were also considered to warrant species status; the names Streptomyces eurythermophilus sp. nov. and Streptomyces thermocoprophilus sp. nov. are proposed to accommodate these strains. It was also concluded that additional comparative taxonomic studies are required to clarify the relationships between additional thermophilic streptomycete strains included in the present investigation. A corresponding polyphasic approach was used to clarify the taxonomy of six thermophilic isolates provisionally assigned to either the genera Amycolatopsis or Excellospora. Two of the isolates, strain NT202 and NT303, had properties consistent with their classification in the genus Amycolatopsis. However, the genotypic and phenotypic data also showed that these strains formed a new centre of taxonomic variation for which the name Amycolatopsis eurythermus sp. nov. is proposed. Similarly, the four remaining strains formed two new centre of taxonomic variation within the genus Excellospora. It is proposed that isolates TA113 and TA114 be designated Excellospora alcalithermophilus sp. nov. Similarly, the name Excellospora thermoalcalitolerans sp. nov. is proposed for strains TA86 and TA111. An emended description is also given for the genus Excellospora.