Development of new classification system for pathogenic Nocardia based on whole genome sequences and microarray analysis

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We reported the genomic sequence of Nocardia farcinica IFM 10152 in 2004. Based on the sequence, we found and analyzed genes which were specific to this species. DNA of size 238 bp specific to N. farcinica was selected for further studies. A section of this DNA partially encoded a hypothetical protein (nfa 29510), and the remaining was a non-coding region located next to a membrane protein (nfa 29520) gene. From these genetic analyses, a specific PCR primer for N. farcinica strains was designed, and their specificity was confirmed. In addition, the primer was found to be useful for the detection of N. farcinica in the blood of mice with experimental nocardiosis. The primer was also found to be useful for RealTime PCR.Based on the sequence of gyrB gene from N. farcinica IFM 10152, a modified PCr primer for the efficient amplification of the gene in Nocardia was designed. The gyrB gene sequences of all 60 Nocardia type species were analyzed, and a phylogenetic tree was constructed. This allowed the Nocardia species to be clearly differentiated from Mycobacterium and Rhodococcus, with homogeneity values of 82% and 76%, respectively.In bacterial classification, sequence of 16S rDNA is most commonly used, with no exception in Nocardia. As for the width of difference among the 60 Nocardia species, their values are distributed from 94.5 to 100.0%. However, in gyrB which we analyzed, this difference markedly increased to 82.4 to 99.9%. Usefulness of gyrB sequence for the classification of these species was confirmed, and we proposed it as a new classification system n Nocardia. Although 1,200 bp sequence of gyrB was used in the present study, further work is in progress for the development of a system using less than 600 bp from variation domains in the gene.