The cytochrome oxidase reaction in bacillus species
Bacteria of the genus Bacillus were surveyed for cytochrome oxidase activity using a colorimetric type tetramethyl-p-phenylenediamine (TMPC) oxidase assay. This assay allows for the calculation of specific activities and provides a quantitative method for using cytochrome oxidase measurements for the taxonomic evaluation of the organisms of the genus Bacillus. The qualitative Kovacs filter-paper spot test using purified TMPD as well as the Marion oxidase reagent, and the PathoTec oxidase test, were also performed along with the colorimetric TMPD oxidase assay. The results of the quantitative oxidase assay correlated well with the results obtained for the qualitative Kovacs, Marion, and the PathoTec oxidase tests. The 89 Bacillus strains examined could be subdivided into three groups, according to the quantitative TMPD oxidase test, (a) weakly oxidase active Group-I, (b) moderately active oxidase Group-II, and (c) the highly active oxidase Group-Ill. These findings indicated again that the organisms of genus Bacillus are a highly heterogeneous group, taxonomically. A high coincident relationship exists between the various Bacillus strains analyzed by the quantitative colorimetric oxidase test and those whose classification studies are based on the comparative fingerprinting analyses of 16S rRNA. Results of the quantitative TMPD oxidase test, show that this simple assay can be of great practical value in distinguishing and classifying different Bacilli species. One representative from each of the 3 oxidase groups was selected for further study. The respiratory membranes of Bacillus polymyxa (a highly active Group-Ill oxidase strain). Bacillus megaterium ATCC 9885 (a weakly positive Oxidase Group-II), and Bacillus pumilus (an Oxidase Group-I negative strain) were analyzed spectrally for cytochrome content and types. The data obtained for these three species indicated that the lack of TMPD oxidase activity can be attributed to the absence of c-type cytochrome; all 3 Bacilli spp. contained the terminal cytochrome oxidase, cytochrome o. Type a cytochrome oxidases were only detected in B. megaterium and B. pumilus membrane, and was absent in the highly active B. polymyxa.