Selective isolation and taxonomic analysis of the genus Micromonospora

Micromonosporae were isolated from a diverse range of environmental habitats and geographic locations. The dispersion and differential centrifugation

technique, coupled with a phenol pre-treatment and the use of colloidal chitin and M3 agars as selective media proved to be highly efficient for the recovery of micromonosporae from environmental samples. The dispersion and centrifugation technique yielded recoveries of up to 10,000 fold more micromonosporae than the traditional vortex technique. Counts varied between 0 and 9 * 104 colony forming units/g for the terrestrial samples and from 6 to 5 * 102 colony forming units/g for the marine samples. The highest counts of micromonosporae (9 * 104 colony forming units/g) were recovered from a Namibian sample. A number of alkali tolerant micromonosporae were isolated from the Indonesian samples using the same procedure but with the medium adjusted to pH 10. Counts of alkalitolerant micromonosporae ranged from 0 to 5 * 103 colony forming units/g of sample, with the highest counts being recovered from a rice paddy field. Representative alkalitolerant micromonosporae grew well on media at pH 7 to 8. Pyrolysis mass spectrometry (PyMS) was used to rapidly screen representatives

of Micromonospora isolates from all environmental habitats and geographical locations sampled. PyMS revealed a number of pyro-groups containing isolates distinct from the Type strains suggesting that these isolates may represent novel

taxa. There was some evidence of isolates forming pyro-groups based on their sample location. One hundred and eighty representatives of the genus

micromonospora and 15 duplicated strains were examined for 134 unit characters and the resultant data analysed using the Jaccard (SJ) and simple matching SSM coefficients. Clustering was achieved using the unweighted pair group method with arithmetic averages algorithm (UPGMA). Good congruence was found between the classifications based on the SJ and SSM, UPGMA analyses. The numerical phenetic data were difficult to interpret but a measure of confidence can be placed in the numerical classification based on the SJ, UPGMA analysis given the acceptable cophenetic correlation and test error values and the congruence observed with the results from the SSM, UPGMA analysis. The test strains were assigned to major (4 or more strains), minor (2-3 strains) and single membered clusters defined at the 69% similarity level in the S], UPGMA classification. There was good agreement between the pyrogroups formed in the PyMS analyses and the clusters generated from the numerical taxonomy analysis. The PyMS and numerical taxonomic analyses revealed that there is some evidence for Micromonospora isolates existing as geovars as both techniques revealed some evidence for isolates grouping according to their sample location. Attempts to sequence the 16S gene of Micromonospora isolates were unsuccessful due to difficulties in obtaining high quantities of pure DNA and because questions have been raised over whether this genus contains more than one rRNA operon. The single strand conformation polymorphism (SSCP) technique demonstrated that differences in 16S rONA sequences between isolates could be detected and individual profiles for the isolates could be generated. The SSCP technique also highlighted the discrepancies that may exist between supposedly identical type strains in culture collections.