ABSTRACT
All Bacillus spores are encased in macromolecular shells. One of these is a proteinacious shell called the coat that, in Bacillus subtilis, provides critical protective functions. The Bacillus anthracis spore is the infectious particle for the disease anthrax. Therefore, the coat is of particular interest because it may provide essential protective functions required for the appearance of anthrax. Here, we analyse a protein component of the spore outer layers that was previously designated BxpA. Our data indicate that a significant amount of BxpA is located below the spore coat and associated with the cortex. By SDS-PAGE, BxpA migrates as a 9 kDa species when extracted from Sterne strain spores, and as 11 and 14 kDa species from Ames strain spores, even though it has predicted masses of 27 and 29 kDa, respectively, in these two strains. We investigated the possibility that BxpA is subject to post-translational processing as previously suggested. In B. subtilis, a subset of coat proteins is proteolysed or cross-linked by the spore proteins YabG or Tgl, respectively. To investigate the possibility that similar processing occurs in B. anthracis, we generated mutations in the yabG or tgl genes in the Sterne and Ames strains and analysed the consequences for BxpA assembly by SDS-PAGE. We found that in a tgl mutant of B. anthracis, the apparent mass of BxpA increased. This is consistent with the possibility that Tgl directs the cross-linking of BxpA into a form that normally does not enter the gel. Unexpectedly, the apparent mass of BxpA also increased in a yabG mutant, suggesting a relatively complex role for proteolysis in spore protein maturation in B. anthracis. These data reveal a previously unobserved event in spore protein maturation in B. anthracis. We speculate that proteolysis and cross-linking are ubiquitous spore assembly mechanisms throughout the genus Bacillus.
Subject(s)
Bacillus anthracis/genetics , Bacterial Proteins/metabolism , Animals , Bacillus anthracis/metabolism , Bacterial Proteins/genetics , Female , Gene Expression Regulation, Bacterial , Guinea Pigs , Mice , Mice, Inbred BALB C , Mutation , Protein Structure, Quaternary , Spores, Bacterial/genetics , Spores, Bacterial/metabolismABSTRACT
A substantial proportion of bacteria from five Alexandrium cultures originally isolated from various countries produced sodium channel blocking (SCB) toxins, as ascertained by mouse neuroblastoma assay. The quantities of SCB toxins produced by bacteria and dinoflagellates were noted, and the limitations in comparing the toxicities of these two organisms are discussed. The chemical nature of the SCB toxins in selected bacterial isolates was determined as paralytic shellfish toxins by pre- and postcolumn high-performance liquid chromatography, capillary electrophoresis-mass spectrometry, and enzyme immunoassay.
Subject(s)
Bacteria/metabolism , Bacterial Toxins/biosynthesis , Dinoflagellida/metabolism , Dinoflagellida/microbiology , Neurotoxins/biosynthesis , Shellfish/microbiology , Shellfish/parasitology , Animals , Bacteria/isolation & purification , Bacteria/pathogenicity , Bacterial Toxins/chemistry , Bacterial Toxins/isolation & purification , Chromatography, High Pressure Liquid , Dinoflagellida/pathogenicity , Enzyme-Linked Immunosorbent Assay , Food Contamination , Mass Spectrometry , Molecular Structure , Neurotoxins/chemistry , Neurotoxins/isolation & purification , Shellfish PoisoningABSTRACT
Fumonisin B1, a mycotoxin produced by a common fungal contaminant of corn, Fusarium moniliforme, was analyzed by capillary electrophoresis-electrospray ionization mass spectrometry. System performance was maximal with uncoated columns. System efficiencies of approximately 44,000 plates/m and reproducible analysis times of about 13 min were obtained. System efficiency with methyl-coated columns was approximately 24,000 plates/m. Reproducible analysis times of about 3.5 min were obtained with these columns. With uncoated columns, the concentration limit of detection was 156 ppb with a s/n ratio of approximately 10. The estimated injected mass at 156 ppb was 1.1 pg. Repeated injections of extracts containing constant fumonisin B1 concentrations showed that peak areas were slightly inconsistent, although generally similar to variations encountered with liquid chromatography-electrospray ionization mass spectrometry. The source of this inconsistency was traced to sample solubility, errors inherent in electrophoresis injections, and electrospray instability. Minimizing these problem areas will produce a technique with peak area reproducibilities comparable to liquid chromatography-electrospray ionization mass spectrometry, but with potentially greater resolving power.
