Transcriptional profiling of Bacillus anthracis during infection of host macrophages.

The interaction between Bacillus anthracis and the mammalian phagocyte is one of the central stages in the progression of inhalational anthrax, and it is commonly believed that the host cell plays a key role in facilitating germination and dissemination of inhaled B. anthracis spores. Given this, a detailed definition of the survival strategies used by B. anthracis within the phagocyte is critical for our understanding of anthrax. In this study, we report the first genome-wide analysis of B. anthracis gene expression during infection of host phagocytes. We developed a technique for specific isolation of bacterial RNA from within infected murine macrophages, and we used custom B. anthracis microarrays to characterize the expression patterns occurring within intracellular bacteria throughout infection of the host phagocyte. We found that B. anthracis adapts very quickly to the intracellular environment, and our analyses identified metabolic pathways that appear to be important to the bacterium during intracellular growth, as well as individual genes that show significant induction in vivo. We used quantitative reverse transcription-PCR to verify that the expression trends that we observed by microarray analysis were valid, and we chose one gene (GBAA1941, encoding a putative transcriptional regulator) for further characterization. A deletion strain missing this gene showed no phenotype in vitro but was significantly attenuated in a mouse model of inhalational anthrax, suggesting that the microarray data described here provide not only the first comprehensive view of how B. anthracis survives within the host cell but also a number of promising leads for further research in anthrax.

Mutagenesis of basic residues R151 and R161 in manganese-stabilizing protein of photosystem II causes inefficient binding of chloride to the oxygen-evolving complex.

Manganese-stabilizing protein of photosystem II, an intrinsically disordered polypeptide, contains a high ratio of charged to hydrophobic amino acid residues. Arg151 and Arg161 are conserved in all known MSP sequences. To examine the role of these basic residues in MSP structure and function, three mutants of spinach MSP, R151G, R151D, and R161G, were produced. Here, we present evidence that replacement of Arg151 or Arg161 yields proteins that have lower PSII binding affinity, and are functionally deficient even though about 2 mol of mutant MSP/mol PSII can be rebound to MSP depleted PSII membranes. R161G reconstitutes O(2) evolution activity to 40% of the control, while R151G and R151D reconstitute only 20% of the control activity. Spectroscopic and biochemical techniques fail to detect significant changes in solution structure. More extensive O(2) evolution assays revealed that the Mn cluster is stable in samples reconstituted with each mutated MSP, and that all three Arg mutants have the same ability to retain Ca(2+) as the wild-type protein. Activity assays exploring the effect of these mutations on retention of Cl(-), however, showed that the R151G, R151D, and R161G MSPs are defective in Cl(-) binding to the OEC. The mutants have Cl(-) K(M) values that are about four (R161G) or six times (R151G and R151D) higher than the value for the wild-type protein. The results reported here suggest that conserved positive charges on the manganese-stabilizing protein play a role in proper functional assembly of the protein into PSII, and, consequently, in retention of Cl(-) by the O(2)-evolving complex.

Transcriptional profiling of the Bacillus anthracis life cycle in vitro and an implied model for regulation of spore formation.

The life cycle of Bacillus anthracis includes both vegetative and endospore morphologies which alternate based on nutrient availability, and there is considerable evidence indicating that the ability of this organism to cause anthrax depends on its ability to progress through this life cycle in a regulated manner. Here we report the use of a custom B. anthracis GeneChip in defining the gene expression patterns that occur throughout the entire life cycle in vitro. Nearly 5,000 genes were expressed in five distinct waves of transcription as the bacteria progressed from germination through sporulation, and we identified a specific set of functions represented within each wave. We also used these data to define the temporal expression of the spore proteome, and in doing so we have demonstrated that much of the spore's protein content is not synthesized de novo during sporulation but rather is packaged from preexisting stocks. We explored several potential mechanisms by which the cell could control which proteins are packaged into the developing spore, and our analyses were most consistent with a model in which B. anthracis regulates the composition of the spore proteome based on protein stability. This study is by far the most comprehensive survey yet of the B. anthracis life cycle and serves as a useful resource in defining the growth-phase-dependent expression patterns of each gene. Additionally, the data and accompanying bioinformatics analyses suggest a model for sporulation that has broad implications for B. anthracis biology and offer new possibilities for microbial forensics and detection.