Bacillus anthracis is the causative agent of anthrax disease. Improvement of existing anthrax vaccines, which are currently based on the administration of Protective Antigen (the highly immunogenic nontoxic subunit of the bacterial toxin) may entail other bacterial immunogenic elements, part of which are predicted to reside on the surface of bacterial cells. In the present study, membranal proteins extracted from a stationary-phase culture of a nonvirulent B. anthracis strain, devoid of the native virulence plasmids pXO1 and pXO2, were separated by two-dimensional electrophoresis (2-DE) and a characteristic protein map was defined. The proteomic analysis allowed matrix-assisted laser desorption/ionization-time of flight mass spectrometry-assisted identification of 86 protein spots which represent the product of 30 individual open reading frames (ORF). Among these, a prevalent class of proteins was the S-layer proteins (which were found to represent more than 75% of the B. anthracis membranal fraction) and proteins containing S-layer homology (SLH)-membranal localization domains. Five novel SLH proteins, previously inferred only from bioinformatic ORF analysis (draft genome sequence), were identified and one was shown to be a highly abundant membranal protein. Western blots of the 2-DE gels were probed with sera from convalescent rabbits and guinea pigs infected with virulent B. anthracis (Vollum strain). This analysis revealed that B. anthracis immune animals exhibit antibodies against at least 14 distinct membranal proteins present in the 2-DE map, establishing that these proteins are expressed in vivo and are able to elicit an immune response. The identification of the protein components of the B. anthracis membranal fraction, as well as the establishment of their potential immunogenicity, underscore the strength of the proteomic approach for identifying molecules which may serve for further analysis of immune and protective abilities.

The functional R6K alpha origin is composed of two DNA elements, one of 580 bp carrying the alpha origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previously identified as also required for gamma and beta origin activity. These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for alpha origin activity. The function of the alpha origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K. Activation of the alpha origin depends on the R6K replication initiation protein pi. Within the 580 bp of the alpha origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the beta replicon. Deletion of the 96 bp or 98 bp results in inactivation of the alpha and the beta origins respectively. These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the alpha and the beta origins of R6K. DNA homology analysis performed on alpha, beta and gamma origin sequences, also reveals 10-23 bp sequences in the alpha and the beta origins that are related to the family of 22 bp direct repeats in the gamma origin which were shown previously to be binding sites for the pi protein.

SUMMARYWhen Escherichia cali organisms were suspended in distilled water and freezedried the maximum loss of viability did not occur during the drying process proper, but during the time of contact of the dried organisms with air between the primary and the secondary drying periods. By substituting other gases for air a t this stage, it was proven that oxygen was the active agent involved. The dried organisms which were exposed to different pressures of air and oxygen at different temperatures proved to be extremely sensitive to traces of oxygen, even at very low temperatures. The implications of this oxygen effect in connexion with existing freezedrying procedures, as well as some preliminary kinetic experiments concerning the shape of the survival curve, are discussed.

SUMMARYVarious substances protect dry Escherichia coli against oxygen. The concentration of these substances in the bacterial suspension, necessary to achieve a given degree of protection, is a function of the concentration of the bacteria in the suspension. The protector seems to act in the dry state. The viability of freeze-dried bacteria, unexposed to oxygen, may also depend on the concentration of the bacterial suspension. A common mechanism is suggested to explain the dependence of killing on population density, both during freeze-drying and during exposure of dried organisms to oxygen. The viability of bacteria during freeze-drying and of dried bacteria exposed to oxygen are both markedly affected by the presence of certain substances such as serum albumin or Bacto-protone.

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