Characterization of the operon encoding the alternative sigma(B) factor from Bacillus anthracis and its role in virulence

Abstract

The operon encoding the general stress transcription factor sigma(B) and two proteins of its regulatory network, RsbV and RsbW, was cloned from the gram-positive bacterium Bacillus anthracis by PCR amplification of chromosomal DNA with degenerate primers, by inverse PCR, and by direct cloning. The gene cluster was very similar to the Bacillus subtilis sigB operon both in the primary sequences of the gene products and in the order of its three genes. However, the deduced products of sequences upstream and downstream from this operon showed no similarity to other proteins encoded by the B. subtilis sigB operon. Therefore, the B. anthracis sigB operon contains three genes rather than eight as in B. subtilis. The B. anthracis operon is preceded by a sigma(B)-like promoter sequence, the expression of which depends on an intact sigma(B) transcription factor in B. subtilis. It is followed by another open reading frame that is also preceded by a promoter sequence similarly dependent on B. subtilis sigma(B). We found that in B. anthracis, both these promoters were induced during the stationary phase and induction required an intact sigB gene. The sigB operon was induced by heat shock. Mutants from which sigB was deleted were constructed in a toxinogenic and a plasmidless strain. These mutants differed from the parental strains in terms of morphology. The toxinogenic sigB mutant strain was also less virulent than the parental strain in the mouse model. B. anthracis sigma(B) may therefore be a minor virulence factor.

FIG. 1

Organization of the B. subtilis sigB operon and current model of ς^B regulation. (A) Schematic diagram of the B. subtilis sigB region. P_A is the promoter for the eight-gene operon, and P_B is an internal, ς^B-dependent promoter. (B) Schematic diagram of the partner-switching modules. For the function of each protein, see main text and references therein. Arrows indicate activation, and T-headed arrows indicate inhibition. P and K stand for phosphatase and kinase, respectively, and U-act stands for RsbU activator.

FIG. 2

Schematic diagram of the B. anthracis sigB region. (A) The sigB operon and the following ORF are represented by long arrows indicating the size and direction of transcription of the genes identified from sequence data. The arrowheads at the ends of the dashed lines indicate the position and orientation of binding of the oligonucleotides used for the cloning experiments described in this work. For the sake of clarity, they have been aligned and sometimes duplicated to indicate the fragments obtained using the various pairs. The 1.8 kb 5′ and 0.3 kb 3′ to the four indicated genes, which were cloned and sequenced from pOSB10, are not represented. (B) Schematic representation of the B. anthracis chromosomal fragments cloned in different vectors during this work. The bgaB and erm cassettes are also represented. The only restriction sites indicated are those used for chromosome walking by inverse PCR: E, EcoRI; C, ClaI.

FIG. 3

Expression of β-galactosidase from pgsiB-bgaB (A), prsbV-bgaB (B), and pORF4-bgaB (C) fusions in parental (squares) and ΔsigB (triangles) B. subtilis strains. Samples were assayed at the times indicated for growth (continuous lines) and for β-galactosidase activity (dotted lines). The bacteria were cultured in 121J medium (open symbols) and 121J medium from which glucose was depleted at the time indicated by the arrows (solid symbols). OD600, optical density at 600 nm.

FIG. 4

Expression of β-galactosidase from a sigB-bgaB fusion in B. anthracis GSB1 in the stationary phase and in response to heat shock. Samples were assayed at the times indicated for growth (continuous lines) and β-galactosidase activity (dotted lines). Bacteria were cultured in L broth at 37°C (open symbols) or subjected to heat shock (arrow) and then cultured further at 44°C (solid symbols). OD600, optical density at 600 nm.

FIG. 5

Expression of β-galactosidase from a porf4-bgaB fusion in parental (squares) and ΔsigB (triangles) B. anthracis strains SSB3 and SSB13, respectively, during growth. Samples were assayed at the times indicated for growth (continuous lines) and β-galactosidase activity (dotted lines). Bacteria were cultured in L broth. OD600, optical density at 600 nm.

FIG. 6

Virulence of B. anthracis SSB10 (triangles) and 7702 (squares) strains. Swiss mice were inoculated subcutaneously with 10⁵ spores per mouse (groups of 10 mice). Mortality was recorded daily and plotted as the cumulative number of deaths.