Minor Differences in the Proteome of Bacillus subtilis and Bacillus mojavensis Based upon High Abundance/ Conserved Protein Mass Spectra; Implications for Rapid, Improved Identification of Two Pathogen Genetically Closely Related

The genus Bacillus comprises a complex group of species, many of which cannot be readily differentiated by phenotypic and genotypic methods. Here, we utilised two species that are genetically very closely related, Bacillus subtilis and Bacillus mojavensis as a model to ascertain the potential of a linear MALDITOF MS to differentiate them against a background of their sporulation cycle and ultrastructural changes. Previous studies indicated that MALDI-TOF MS ionises the high abundance/conserved intracellular proteins but also produces additional lower mass ions that, with the appropriate software, may help to delineate such closely related taxa. Cells were grown in liquid culture over 24 hours and samples taken intermittently to monitor growth and the presence of spores. Harvested cells were studied by electron microscopy to detect potential changes in ultra structure and its potential effect on reliable and reproducible mass spectra. Sporulation was clearly evident by 24 hours and correlated inversely with reliable identification scores obtained using MALDI-TOF MS. Thus, cells cultured for 4, 6, 8 and 24 hours had identification scores of 2.157, 2.204, 2.295 and < 2 respectively. Contrary to previous findings, these results demonstrated unequivocally that Bacillus species may be reliably identified using this approach prior to sporulation. Furthermore, the software ClinProTools 3.0 was used to tease out minor components of the mass spectrum that enabled unambiguous separation of both groups of strains.