Sensor domains encoded in Bacillus anthracis virulence plasmids prevent sporulation by hijacking a sporulation sensor histidine kinase

Abstract

Anthrax toxin and capsule, determinants for successful infection by Bacillus anthracis, are encoded on the virulence plasmids pXO1 and pXO2, respectively. Each of these plasmids also encodes proteins that are highly homologous to the signal sensor domain of a chromosomally encoded major sporulation sensor histidine kinase (BA2291) in this organism. B. anthracis Sterne overexpressing the plasmid pXO2-61-encoded signal sensor domain exhibited a significant decrease in sporulation that was suppressed by the deletion of the BA2291 gene. Expression of the sensor domains from the pXO1-118 and pXO2-61 genes in Bacillus subtilis strains carrying the B. anthracis sporulation sensor kinase BA2291 gene resulted in BA2291-dependent inhibition of sporulation. These results indicate that sporulation sensor kinase BA2291 is converted from an activator to an inhibitor of sporulation in its native host by the virulence plasmid-encoded signal sensor domains. We speculate that activation of these signal sensor domains contributes to the initiation of B. anthracis sporulation in the bloodstream of its infected host, a salient characteristic in the virulence of this organism, and provides an additional role for the virulence plasmids in anthrax pathogenesis.

FIG. 1.

Amino acid sequence alignment of the B. anthracis sensor domains encoded by pXO1-118 and pXO2-61 and the BA2291 sensor domain (residues 1 to 161). Sequences were aligned by the ClustalW program. Asterisks indicate identical residues in all three sequences; colons denote conserved substitutions. Paired scores resulted in 34% identity between the pXO2-61 and BA2291 sensor domains, 29% identity between the pXO1-118 and BA2291 sensor domains, and 62% identity between the pXO1-118 and pXO2-61 sensor domains.

FIG. 2.

Sporulation phenotypes of B. anthracis parental 34F2, ΔBA4223, and ΔBA2291 strains harboring sensor domains encoded by pX01-118 (pHT315-118) and pXO2-61 (pHT315-61) expressed from their native promoters on multicopy plasmid pHT315. Cultures of each strain were grown in 5 ml of Schaeffer's sporulation medium (24) with the appropriate antibiotics for 17 h at 37°C with shaking.

FIG. 3.

Effects of overexpression of the sensor domains encoded by pX01-118 and pX02-61 on the sporulation phenotypes of B. subtilis wild-type, ΔkinA mutant, and ΔkinA ΔkinB mutant strains with (+2291) and without (−2291) the B. anthracis sporulation sensor kinase BA2291 integrated in a single copy on the chromosome. Strains were streaked on Schaeffer's sporulation medium agar (24) and incubated at 37°C for 48 h. Opaque sporulating strains appear white, and nonsporulating strains appear black/gray. The streak numbers correspond to the numbers in column 1 of Table 1.

FIG. 4.

In vitro activity of the BA2291 histidine sensor kinase. (A) Autophosphorylation and phosphoryl transfer activity assays of BA2291 purified from E. coli were carried out as described in Materials and Methods. Autophosphorylation and phosphoryl transfer activities of purified B. anthracis BA2291 (5 μM) were compared to those observed for B. subtilis proteins KinA (0.2 μM) and Spo0F (2 μM) in the presence of γ-³²P-labeled ATP at 0 and 60 min. The samples were run on 15% SDS-PAGE gels. (B) Schematic representation of the phosphorelay signal transduction system for sporulation initiation (7). Emphasized is the role of BA2291 in inducing sporulation (in the presence of activating signal) or inhibiting sporulation (in the absence of activating signal or in the presence of pXO1-118 and pXO2-61) by removing phosphoryl groups from Spo0F∼P.