Strains belonging to the Gram-positive coryneform soil bacteria were screened genotypically by temperature-gradient gel electrophoresis (TGGE). This method allows the sequence-specific separation of amplified fragments of 16S rRNA genes. A total of 115 reference strains representing the majority of the species of the genera Aeromicrobium, Agromyces, Arthrobacter, Aureobacterium, Cellulomonas, Curtobacterium, Nocardioides and Terrabacter were characterized. Depending on the genus investigated, the resolution limit of the technique appeared to be at the species or genus level or intermediate between the two. Aberrant TGGE profiles of strains within particular taxa revealed genomic heterogeneity and generic misclassification of nine strains studied. Beyond that, indications of 16S rRNA gene heterogeneity were found within the genomes of three Curtobacterium strains. The misclassifications revealed by TGGE were confirmed using whole-cell fatty acid methyl ester analysis and subsequent comparison with a database. TGGE has been demonstrated to be a useful tool in bacterial taxonomy.
Members of the family Pseudonocardiaceae are difficult to identify on the basis of their micromorphology only. The biochemical characterization of each new isolate is a painstaking and time-consuming task which cannot always be undertaken when handling large numbers of strains as is the case in natural product screening programmes. In this study, two sets of genus-specific oligonucleotides were designed which allow rapid detection of members of the genera Pseudonocardia and Saccharopolyspora by means of PCR-specific amplification. The genus specificity of these primers was validated on a wide range of collection strains and the primers were subsequently used to study group of 106 wild-type isolates that possessed morphological characteristcs of the family. Out of this group, 51 strains could be identified as members of the genus Pseudonocardia and only nine isolates could be assigned to the genus Saccharopolyspora. The diversity indicated by whole-cell fatty acid profiles of both wild-type and reference strains was compared with that identified using the oligonucleotide primers. The partial 16S rDNA sequencig of representative wild-type strains was used to validate their genus assignment by PCR-specific amplification. This study shows the industrial usefulness of the application of these direct identification tools as well as the complementary use of two sources of data, PCR-specific amplification results and fatty acid composition, to assess the diversity of a microbial population.
Lactococcus lactis strains were examined for their ability to produce Γ-aminobutyric acid (GABA). Results showed that strains of L lactis subsp. lactis were able to produce this acid, whereas L. lactis subsp. cremoris were not. GABA production thus represents another effective characteristic for distinguishing L. lactis subsp. lactis from L. lactis subsp. cremoris.
Polyamine patterns of coryne-and nocardioform representatives of the class Actinobacteria with ll-diaminopimelic acid in the peptidoglycan, comprising strains of the genera Aeromicrobium, Nocardioides, Intrasporangium, Terrabacter, Terracoccus, Propioniferax, Friedmanniella, Microlunatus, Luteococcus and Sporichthya, were analysed. The different polyamine patterns were in good agreement with the phylogenetic heterogeneity within this group of actinomycetes. Strains of the closely related genera Nocardioides and Aeromicrobium were characterized by the presence of cadaverine. The second cluster, consisting of the type strains of the species Friedmanniella antarctica, Propioniferax innocua, Microlunatus phosphovorus and Luteococcus japonicus displayed as a common feature the presence of the two predominant compounds spermidine and spermine. The presence of putrescine was common to the type strains of the species Intrasporangium calvum, Terrabacter tumescens and Terracoccus luteus. Sporichthya polymorpha, which is a representative of a separate line of descent, displayed spermidine as the predominant polyamine. These data indicate that polyamine patterns are suitable for the classification of actinomycetes with ll-diaminopimelic acid in the peptidoglycan.
The identification and classification of yeasts have traditionally been based morphological, physiological and biochemical traits. Various kits have been developed as rapid systems for yeast identification, but mostly for clinical diagnosis. In recent years, different molecular biology techniques have been developed for yeast identification, but there is no available database to identify a large number of species. In the present study, the restriction patterns generated from the region spanning the internal transcribed spacer (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of 132 yeast species belonging to 25 different genera, including teleomorphic and anamorphic ascomycetous and basidiomycetous yeasts. In many cases, the size of the PCR products and the restriction patterns obtained with endonucleases Cfol, Haelll and Hinfl yielded a unique profile for each species. Accordingly, the use of this molecular approach is proposed as a new rapid and easy method of routine yeast identification.