Transcriptional regulation of pagA in Bacillus anthracis

M. Persuh, E. Bader, D.G. Bazemore and M.J. Blaser

Departments of Medicine and Microbiology, NYU School of Medicine, New York, NY

Background: B. anthracis major virulence factors, antiphagocytic capsule and toxins are encoded on two large plasmids. CO₂ activates transcription of genes for protective antigen (pagA), lethal factor (lef), and edema factor (cya) on pXO1. Protective antigen plays a major role in B. anthracis pathogenesis, binding to a cell receptor and translocating lethal and edema factors, and is also a major immunogen. atxA is necessary for transcription of pagA, lef and cya in the presence of CO₂. Studies on pagA regulation are needed to better understand B. anthracis pathogenesis and for prevention and treatment strategies. Some B. anthracis paralogs of regulators in B. subtilis may have a role in pagA transcription. Methods: We constructed a transcriptional pagA-lacZ fusion in single copy in the B. subtilis chromosome and the resulting strain was transformed with a multicopy atxA plasmid. Next, we separately introduced knock-outs in five regulatory genes: spo0A, spo0H, abrB, codY, and sinR. To determine the effects in different backgrounds, we measured Beta-galactosidase activities. In B. anthracis, we constructed a transcriptional pagA-gfp fusion on a shuttle plasmid, and examined the effects of growth conditions on fluorescence intensity. Results: pagA-lacZ had low basal activity in single copy in the heterologous host B. subtilis; this activity was not significantly different in the presence of the specified regulatory gene mutations. atxA strongly stimulated pagA transcription in B. subtilis. In this genetic background, introduction of a null mutation in spo0A significantly decreased pagA transcription. Using the pagA-gfp fusion in B. anthracis we demonstrated induction of pagA transcription with either increased CO₂ or low nutrient medium; the combination of both resulted in maximum pagA-gfp expression. Conclusions: Our results suggest a role of spo0A in regulation of pagA transcription. We did not observe an abrB effect, so spo0A, which represses abrB, might have an additional role in pagA regulation. With the pagA-gfp fusion, we showed that medium composition and CO₂ have separate and additive effects on pagA transcription.