Negative regulation of Bacillus anthracis sporulation by the Spo0E family of phosphatases

Abstract

The initiation of sporulation in Bacillus species is controlled by the phosphorelay signal transduction system. Multiple regulatory elements act on the phosphorelay to modulate the level of protein phosphorylation in response to cellular, environmental, and metabolic signals. In Bacillus anthracis nine possible histidine sensor kinases can positively activate the system, while two response regulator aspartyl phosphate phosphatases of the Rap family negatively impact the pathway by dephosphorylating the Spo0F intermediate response regulator. In this study, we have characterized the B. anthracis members of the Spo0E family of phosphatases that specifically dephosphorylate the Spo0A response regulator of the phosphorelay and master regulator of sporulation. The products of four genes were able to promote the dephosphorylation of Spo0A approximately P in vitro. The overexpression of two of these B. anthracis Spo0E-like proteins from a multicopy vector consistently resulted in a sporulation-deficient phenotype. A third gene was found to be not transcribed in vivo. A fourth gene encoded a prematurely truncated protein due to a base pair deletion that nevertheless was subject to translational frameshift repair in an Escherichia coli protein expression system. A fifth Spo0E-like protein has been structurally and functionally characterized as a phosphatase of Spo0A approximately P by R. N. Grenha et al. (J. Biol. Chem. 281:37993-38003, 2006). We propose that these proteins may contribute to maintain B. anthracis in the transition phase of growth during an active infection and therefore contribute to the virulence of this organism.

FIG. 1.

The B. anthracis Spo0E-like family. A. Amino acid sequence alignment of the B. anthracis Spo0E-like proteins. Proteins were aligned with the ClustalW program. Asterisks indicate identical residues in all five sequences; colons denote conserved substitutions. The four B. anthracis (Ba) proteins analyzed in this study are aligned against the B. subtilis Spo0E protein (35). The proteins resulting from the frameshift mutation in the BAYisI gene are indicated with the numbers 1 and 2. B. Schematic representation of the chromosomal regions containing the B. anthracis spo0E-like genes. Open reading frames and their orientations are shown by arrows. The regions forming stem-loop structures are indicated by the Ω-like symbol. The lines represent the fragments cloned in the pJM115 and pTCVlac vectors generating lacZ transcriptional fusion plasmids. The dotted bar represents the 51 codons possibly extending the BA0046 gene at the 5′ end.

FIG. 2.

Sporulation phenotypes of B. anthracis (A) and B. subtilis (B) strains expressing the Spo0E-like proteins from the multicopy plasmid pHT315. Streaks in panel A are strains carrying the following plasmids: sector 1, pHT315; sector 2, pHT315-YisI; sector 3, pHT315-0046; sector 4, pHT315-1877; sector 5, pHT315-2416; sector 6, pHT315. Strains in panel B carry the following plasmids: sector 1, pHT315; sector 2, pHT315-1877; sector 3, pHT315-2416; sector 4, pHT315-YisI; sector 5, pHT315-0046. Sporulation on agar plates results in a level of opacity of the streak that is associated with the efficiency of spore formation. The more opaque the streak is, the whiter it appears in a black-and-white picture. Sporulation-deficient, and thus transparent, streaks, as in sector 5 in panel A and sector 2 in panel B, have a gray appearance. Strains were streaked on SM plates and incubated at 37°C for 48 h.

FIG. 3.

Phenotype of the B. anthracis BA1877 deletion strain. The parental strain 34F2 (A and C) and the 34F2ΔBA1877 deletion strain (B and D) were grown on SM agar plates for 48 h (A and B) or in SM liquid medium for 9 h (C and D) at 37°C. (A and B) Plates were photographed with an AlphaInnotech imaging system. Two representative papillae are indicated by the arrows. (C and D) Samples were analyzed with a phase-contrast microscope. The images shown are representative of multiple images taken for both strains. Phase-bright spores (some indicated by the arrows) were found at a higher frequency (at least 20-fold) in fields of 34F2ΔBA1877 cells than in fields of 34F2 cells.

FIG. 4.

Phosphorelay dephosphorylation assay. Purified Spo0E-like proteins of B. anthracis were tested in vitro for their ability to dephosphorylate the B. subtilis phosphorelay. KinA (0.2 μM), Spo0F (2.5 μM), Spo0B (0.2 μM), and Spo0A (2.5 μM) were incubated in the presence of [γ-³²P]ATP for 30 min. The reaction mixture was then divided in two aliquots: one was incubated with the corresponding Spo0E protein buffer (−), and the other was incubated with buffer plus the Spo0E protein (+). Aliquots were withdrawn at the indicated times. BA1877 (A), BA0046 (B), BAYisI (C), and BA2416 (D) proteins purified from E. coli were used at a 5 μM final concentration. The position of the phosphorylated proteins is indicated by the arrows.

FIG. 5.

Transcriptional analysis of B. anthracis spo0E-like genes in B. anthracis (A) and B. subtilis (B). Cultures were grown in SM liquid medium. Symbols: •, BA0046; ▪, BAYisI; ▴, BA1877; ⧫, BA2416; ▾, pTCVlac vector. The growth curve of strains carrying the BA0046 construct is shown as representative of all growth curves (○).