In the year of 2014, we finished a study characterizing the functionality of the Bacillus anthracis atxA gene. Using Next Generation sequencing, we found 15 pXO1-encoded genes and 3 chromosomal genes that were strongly regulated by the separate or synergistic actions of AtxA and carbon dioxide. The majority of the regulated genes responded to both AtxA and carbon dioxide rather than to just one of these factors. Interestingly, we identified two previously unrecognized small RNAs that are highly expressed under physiological carbon dioxide concentrations in an AtxA-dependent manner. Expression levels of the two small RNAs were found to be higher than that of any other gene differentially expressed in response to these conditions. Secondary structure and small RNA-mRNA binding predictions for the two small RNAs suggest that they may perform important functions in regulating B. anthracis virulence. We used our gene targeting tools to produce mutants for both small RNAs. A double sRNAs mutant was prepared for global transcriptomic analysis by RNA sequencing and for protein analysis by mass spectrometry. The components of the AtxA regulatory pathway that we identified and their interactions with CO2 represent molecular targets for specific inhibitory interventions aimed at decreasing the pathogenesis of B. anthracis. Also, using newly developed technology for DNA replacement within the genome of B. anthracis, we consecutively inserted several copies of atxA gene into the chromosomal DNA of the multi-protease mutant strain BH490, a new approach that may provide a basis for a new B. anthracis host/vector system. In a second project executed in 2014, we analyzed genes and sequences that are needed for maintenance of the key virulence plasmid pXO1, which encodes all three anthrax toxin proteins. In previous work we found that a B. anthracis pXO1 minireplicon (MR) plasmid consisting of ORFs GBAA_pXO1_0020 - GBAA_pXO1_0023 is not stably maintained in B. anthracis, whereas the full-size parent pXO1 plasmid (having 181,677 bp and 217 ORFs) is extremely stable under the same growth conditions. Two genetic tools developed for DNA manipulation in B. anthracis (Cre/loxP and Flp/FRT systems) were used to identify pXO1 regions required for plasmid stability. We localized a large segment of pXO1 that enables stable plasmid maintenance during vegetative growth. Further genetic analysis identified three genes that are necessary for pXO1 maintenance: amsP (GBAA_pXO1_0069), minP (GBAA_pXO1_0082), and sojP (GBAA_pXO1_0084). Analysis of conserved domains in the corresponding proteins indicated that only AmsP (Activator of Maintenance System of pXO1) is predicted to bind double-stranded DNA, due to its strong helix-turn-helix domain. Two conserved domains were found in the MinP protein (Min from pXO1): an N-terminal domain having some similarity to the B. anthracis septum site-determining proteins MinD and MinC, and a C-terminal domain that resembles a baculovirus single-strand DNA-binding protein. The SojP protein (Soj from pXO1) contains putative Walker-box motifs and belongs to the ParA family of ATPases. No sequences encoding other components of Type I plasmid partition systems, namely cis-acting centromere parS and its binding ParB protein, were identified within the pXO1 genome. A model describing the role of the MinP protein in pXO1 distribution between mother and daughter cells was proposed. This knowledge will provide targets for anti-infective agents - those that do not directly kill the pathogen but instead render it less virulent, thereby allowing host immune responses to effectively combat it. During 2014 we also participated in clarifying the role of the clpC operon in B. anthracis. We generated knockout strains of the four clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC on essential processes of B. anthracis. Growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defects in these strains were pronounced at elevated temperatures. The growth defects were restored on complementation of mcsB and clpC in the respective mutants. Electron microscopic examination that was performed by our co-authors revealed that mcsB and clpC deletion also causes defects in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. These results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and open up further interest in this operon, which might be of importance to success of B. anthracis as pathogen.
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