Advances in carbohydrate and nucleic acid analysis applied to the taxonomy and physiology of bacilli

Differentiation between members of the B. cereus group (B. cereus, B. anthracis and B. thuringiensis) is a difficult taxonomic problem because of their high degree of phenotypic and genotypic similarity. Therefore this group was the focus of a taxonomic study using carbohydrate profiling and comparison of common genomic regions. B. subtilis was included as a more distantly related Bacillus. B. thuringiensis was found to be quite similar to B. cereus in carbohydrate composition for both vegetative and spore forms. A change in carbohydrate composition on transition from vegetative cell to spore was noted for the B. cereus group and B. subtilis. It was unclear whether the carbohydrate transition observed on sporulation was related to the teichoic/teichuronic acid transition (described by Ellwood and Tempest) where bacilli change their carbohydrate composition when grown under conditions of phosphate deprivation. An existing gas chromatography mass spectrometry procedure was available for studying neutral and amino sugar components of these cells. However, in order to analyze glucuronic acid (an acidic sugar marker for teichuronic acid) it was necessary to develop a liquid chromatography electrospray ionization tandem mass spectrometry procedure. This allowed quantitative profiling of neutral and acidic sugar monomers in a complex microbial matrix. Vegetative cells, grown under conditions of phosphate deprivation, contained large amounts of both glucuronic acid and galactosamine (teichuronic acid markers) but not quinovose (a spore component). The results prove that there are three distinct polysaccharides in B. subtilis produced under different physiological growth conditions. PCR amplification was used to examine the 16S/23S rRNA spacer region of each species. The study of this variable genomic region allowed B. subtilis to be differentiated from the B. cereus group of organisms. However, the three B. cereus species produced PCR products that could not be distinguished by low resolution gel electrophoresis. Electrospray mass spectrometry was investigated as a means to obtain precise molecular weight measurements of PCR products. Single base substitutions, additions or deletions could be recognized. It proved vital to remove primers, deoxynucleotides, DNA polymerase and metal ions from the PCR reaction mixture. These reaction components reduced sensitivity and metal ion adduction adversely affected the accuracy of mass measurements. PCR products of 89 base pairs from the B. cereus group were readily differentiated from 114 base pairs products of B. subtilis.