Ecological factors governing the occurrence and persistence of anthrax reservoirs in the environment remain obscure. A long-held belief that the growing, or vegetative, form of B. anthracis does not survive outside its animal host and must immediately differentiate into a dormant endospore is poorly supported by direct evidence. In our studies, we have discovered a far more dynamic lifestyle for B. anthracis in which exposure to environmental bacteriophage profoundly alters the long-term survival capacities of both vegetative and spore forms. Using a collection of novel bacteriophages isolated from a variety of environments, we showed that stable lysogens of B. anthracis undergo a process of lysogenic conversion that is associated with major changes in their capacity to sporulate, produce exopolysaccharide, form biofilms, survive in the soil, and colonize the earthworm gut. Thus, for B. anthracis, bacteriophages enable alternatives to the bleak prospect of sporulation and indicate an important environmental phase between outbreak cycles. Here, we seek to expand our analysis of lysogenic conversion with B. anthracis to understand the mechanism by which bacteriophages induce these changes. Preliminary evidence suggests that lysogenic conversion in B. anthracis occurs by a novel mechanism in which phage-encoded sigma factors drive the expression of bacterial-encoded phenotypes. To pursue these findings, we will first use a series of genetic methods to identify the lysogen-converting factors encoded by six known environmental phages and by constituents of B. anthracis phage-enhanced metagenomic libraries. We will also identify the phage-induced, B. anthracis- encoded effectors of at least two lysogen phenotypes - biofilm formation and earthworm colonization - through a variety of genetic techniques, transcription studies, and mutant constructions. In this manner we intend to study the mechanism by which prophages of B. anthracis can drive the elaboration of novel phenotypes related to environmental survival. As part of this work, we will also determine whether lysogeny alters the virulence of B. anthracis. Finally, we will determine how phages exist in B. anthracis (as plasmidial or integrated prophage forms) and how their presence affects virulence plasmid maintenance and horizontal-transfer into and out of this pathogen. The implications of these findings with respect to the B. anthracis lifecycle and its ability to evolve, maintain and transfer its pathogenic phenotype, and respond to environments other than an infected animal are important if we are to devise strategies to prevent infection by this pathogen. Ultimately, if we can understand how viruses help pathogens adapt to life outside their host, then we may be able to use these mechanisms to control not only B. anthracis virulence, but that of other Category A biological agents with extended soil phases, like Yersinia pestis and Francisella tularensis, which also have extensive environmental phage systems.
We have discovered for the first time that the anthrax agent Bacillus anthracis can survive outside the infected host in long-lived, non-spore forms and in novel niches as a result of being infected by environmental bacteriophages. This new information will now enable us to examine similar occurrences in other Category A biological agents with extended soil phases, like Yersinia pestis (the plague agent) and Francisella tularensis (the tularemia agent). This latest information changes our thinking about the versatility of these pathogens, allowing us to devise innovative strategies to control them both in the environment and during infection.
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