Rap phosphatase of virulence plasmid pXO1 inhibits Bacillus anthracis sporulation

Abstract

This study shows that the Bacillus anthracis pXO1 virulence plasmid carries a Rap-Phr system, BXA0205, which regulates sporulation initiation in this organism. The BXA0205Rap protein was shown to dephosphorylate the Spo0F response regulator intermediate of the phosphorelay signal transduction system that regulates the initiation of the developmental pathway in response to environmental, metabolic, and cell cycle signals. The activity of the Rap protein was shown to be inhibited by the carboxy-terminal pentapeptide generated through an export-import processing pathway from the associated BXA0205Phr protein. Deregulation of the Rap activity by either overexpression or lack of the Phr pentapeptide resulted in severe inhibition of sporulation. Five additional Rap-Phr encoding systems were identified on the chromosome of B. anthracis, one of which, BA3790-3791, also affected sporulation initiation. The results suggest that the plasmid-borne Rap-Phr system may provide a selective advantage to the virulence of B. anthracis.

FIG.1.

Amino acid sequence alignment of the Rap and Phr proteins of B. anthracis. (A) The Rap proteins of B. anthracis were aligned against the RapA protein of B. subtilis. The six TPR domains in RapA are underlined. The gray box in the BA3760 sequence indicates a duplication in the nucleotide sequence of the 34F2 strain used in this study that is not present in the database at accession number NC_003997. The dash replacing residue 226 in the sequence of BA4060 indicates the position of the frame shift that inactivates this gene product. The alignment was obtained by the ClustalW program. (B) Amino acid sequences of the Phr proteins of B. anthracis and the PhrA protein of B. subtilis. Gray boxes in the BsPhrA and BXA0205phr sequences indicate the sequence of the active pentapeptide inhibitor. The gray box in the BA3791 sequence indicates the region presumably containing the pentapeptide inhibitor. The amino-terminal positively charged domain (N), the hydrophobic domain (H), and the putative signal peptidase cleavage domain (C) of PhrA are indicated. The dashes indicate the putative signal peptidase cleavage site identified by the SignalP program (30). , identical residue; :, conserved residue.

FIG. 2.

In vitro activity assay of B. anthracis Rap proteins against the B. subtilis phosphorelay components. Purified B. anthracis Rap proteins were assayed in a reaction containing KinA (0.1 μM), Spo0F (2.5 μM), and [γ-³²P]ATP. Each Rap protein was added at 5 μM final concentration. Time course experiments were carried out and aliquots withdrawn at the indicated time points. The positions of the KinA and Spo0F proteins in each gel are indicated by the bars. The labeled band denoted by the asterisk is a dimer form of Spo0F.

FIG. 3.

Dephosphorylation of B. subtilis Spo0F∼P by B. anthracis Rap proteins. Purified Spo0F∼P (2.5 μM) was incubated in the absence or presence of the BA3790 (A) or BXA0205 (B) proteins (5 μM) and aliquots were withdrawn at the indicated time points. The positions of the Spo0F protein and of the inorganic phosphate (Pi) are indicated by the bars.

FIG. 4.

Inhibition of BXA0205 phosphatase activity by the BXA0205phr pentapeptide GHTGG in vitro. The B. subtilis KinA (0.1 μM) and Spo0F (2.5 μM) proteins were incubated with or without the B. anthracis BXA0205 Rap protein, at the concentrations indicated, in the presence of [γ-³²P]ATP and increasing concentrations of the synthetic BXA0205phr pentapeptide. The reaction was carried out for 15 min prior to analysis by SDS-PAGE.

FIG. 5.

Transcription analysis of the B. anthracis rap promoters. β-Galactosidase assays were carried out on B. subtilis strains carrying rap-promoter fusions to the E. coli lacZ gene. Each fusion was integrated at the amyE locus via a double-crossover event. (A) β-Galactosidase activity of the BXA0205 promoter (•). (B) β-galactosidase activity of the BA4060 (▴), BA3016 (▪), BA3790 (♦), BA1582 (▾), BA3760 (•), and BXA0205phr (□) promoters. Cultures were carried out in Schaeffer's sporulation medium. The time zero on the x axis represents the time of transition from exponential to stationary phase. The data are representative of multiple independent experiments.

FIG. 6.

Transcription analysis of the abrB and atxA promoters in B. anthracis BXA0205 and BXA0205phr mutant strains. (A) β-Galactosidase analysis of an abrB promoter-lacZ fusion construct in the parental strain 34F2 (▴), in the BXA0205 mutant (•), and in the BXA0205phr mutant (♦). (B) β-Galactosidase analysis of the atxA promoter-lacZ fusion construct. Strains and symbols are as for panel A. Strains were grown in Schaeffer's sporulation medium. The time zero on the x axis indicates the time of transition from exponential to stationary phase. The growth curve of one representative strain for each panel is shown by the open triangles. OD₅₂₅, optical density at 525 nm.