Transcriptional Profiling of the Bacillus anthracis Life Cycle In Vitro and an Implied Model for Regulation of Spore Formation

Abstract

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.The members of the Bacillus genus are spore-forming bacteria that switch between two morphologies-the dormant endospore and the vegetative cell-depending on whether the local environment will facilitate growth. In spore form, the bacterium is resistant to a variety of physical extremes, including heat, desiccation, UV and ␥-irradiation, and oxidation, and the ability to switch between this cell type and the rapidly dividing vegetative form provides the bacilli with a highly effective strategy for persistence in the environment (28). Given the different functions of each, it is unsurprising that the two morphologies are microscopically and biochemically distinct, and the processes by which one form converts to the other, termed germination and sporulation, are known to be highly complex (10,30,35).In several Bacillus species, the two forms of the bacterium play very different roles in the establishment and progression of disease. The most prominent example of this is Bacillus anthracis, the causative agent of anthrax; in this case, the spore morphology allows the bacterium to evade host defenses and establish infection, and the disease state is then perpetuated by growth of (and toxin/capsule production by) vegetative cells within the body (9). Because the two morphologies each play a distinct role, the transitions between germination and sporulation represent key phases in the pathogenesis of this organism, and it is clear t...

The Exosporium Layer of Bacterial Spores: a Connection to the Environment and the Infected Host

“…Although the location of this protein has not been mapped precisely, it was reported to likely lie below the BclA nap layer on the exosporium, based on increased exposure to antibodies in the absence of BclA (68). The bas5303 determinant is monocistronic and expressed at a very late stage of sporulation (73). The open reading frame is preceded by two putative K promoter elements, consistent with this expression profile.…”

“…There are two proteins annotated as arginases encoded in the B. anthracis Sterne genome (BAS0155 and BAS2260). Of these, only BAS0155 has an expression profile consistent with a potential spore-associated protein (73), and the open reading frame is preceded by a putative K promoter sequence.…”

Structure of the Type III Pantothenate Kinase from Bacillus anthracis at 2.0 Å Resolution: Implications for Coenzyme A-Dependent Redox Biology,

“…The coaX gene encoding BaPanK is up-regulated in wave II, as is the coaD locus encoding phosphopantetheine adenylyltransferase; the coaBC and coaE genes corresponding to the bifunctional phosphopantothenoylcysteine synthetase/decarboxylase and dephospho-CoASH kinase, respectively, are up-regulated in waves I and III. Bergman et al (61) have suggested, that since waves I and II are the phases of the B. anthracis life cycle that occur within the infected host, genes up-regulated specifically within this temporal frame may be particularly useful as therapeutic targets. The B. anthracis CoADR (BA1263) and CoADR-rhodanese homology domain (BA0774) proteins were expressed in waves III and V, respectively, and the presence of CoADR in the fractured spore was confirmed by proteomics analysis (62).…”

Bacillus anthracis -derived nitric oxide is essential for pathogen virulence and survival in macrophages

“…anthracis. Upon germination, bNOS, which has been accumulated in the spore during the sporulation phase (32,33), generates NO that instantly protects the pathogen from H 2O2 toxicity by a dual mechanism. NO interrupts the production of damaging hydroxyl radicals from the Fenton reaction and directly activates catalase (Kat) (8), which has already been shown to be a part of the exosporium (34).…”