Bacillus anthracis sin locus and regulation of secreted proteases

Abstract

Bacillus anthracis shares many regulatory loci with the nonpathogenic Bacillus species Bacillus subtilis. One such locus is sinIR, which in B. subtilis controls sporulation, biofilm formation, motility, and competency. As B. anthracis is not known to be motile, to be naturally competent, or to readily form biofilms, we hypothesized that the B. anthracis sinIR regulon is distinct from that of B. subtilis. A genome-wide expression microarray analysis of B. anthracis parental and sinR mutant strains indicated limited convergence of the B. anthracis and B. subtilis SinR regulons. The B. anthracis regulon includes homologues of some B. subtilis SinR-regulated genes, including the signal peptidase gene sipW near the sinIR locus and the sporulation gene spoIIE. The B. anthracis SinR protein also negatively regulates transcription of genes adjacent to the sinIR locus that are unique to the Bacillus cereus group species. These include calY and inhA1, structural genes for the metalloproteases camelysin and immune inhibitor A1 (InhA1), which have been suggested to be associated with virulence in B. cereus and B. anthracis, respectively. Electrophoretic mobility shift assays revealed direct binding of B. anthracis SinR to promoter DNA from strongly regulated genes, such as calY and sipW, but not to the weakly regulated inhA1 gene. Assessment of camelysin and InhA1 levels in culture supernates from sinR-, inhA1-, and calY-null mutants showed that the concentration of InhA1 in the culture supernatant is inversely proportional to the concentration of camelysin. Our data are consistent with a model in which InhA1 protease levels are controlled at the transcriptional level by SinR and at the posttranslational level by camelysin.

FIG. 1.

Schematic representation of the sin loci of B. subtilis and B. anthracis. Open reading frames are represented by block arrows.

FIG. 2.

sinR-controlled transcriptome of B. anthracis. (A) Scatter plot of ORFs differentially regulated in the sinR mutant (UTA21) relative to the parent strain (Ames). Data from microarray experiments were analyzed using EXCEL to subtract background and to determine the averages of results from three replicates per strain per time point (see Materials and Methods). Data are presented as log₂ fold changes. (B and C) Genes exhibiting 2-fold or greater differences in regulation between the parent and sinR strains when microarray data were assessed using three independent analysis programs were designated sinR regulated. (B) sinR-regulated genes during exponential growth phase grouped by annotated function. (C) sinR-regulated genes during stationary growth phase grouped by annotated function.

FIG. 3.

Comparison of the sinR regulons of B. anthracis and B. subtilis. sinR-regulated genes are indicated (exponential and stationary growth phases) from B. anthracis transcriptional profiling experiments (this study) and from B. subtilis literature (3, 15, 16, 35, 37, 40, 42, 45, 47, 48, 65).

FIG. 4.

Specific binding of recombinant B. anthracis SinR to the promoters of sinR-regulated genes calY and sipW. EMSAs were performed using 0.1 nM probe DNA and increasing concentrations of purified rSinR, 0 nM, 0.4 nM, 2 nM, 10 nM, and 50 nM. The Npr599 gene promoter probe was used as a negative control.

FIG. 5.

Effects of B. anthracis sinR and sinI on Camelysin, TasA, and InhA1 levels. The parent strain 7702 and sinR, sinI, and sinIR mutants (UT315, UT365, and UT371, respectively) were cultured in NBY, and samples were taken during the exponential phase (OD₆₀₀, ≈1.6; 4 h), transition phase (OD₆₀₀, ≈3.1; 6 h), and stationary phase (OD₆₀₀, ≈3.1; 8 h) of growth. Cell pellets were used to assess camelysin and TasA, and culture supernatant was used to assess InhA1. Western blot analyses were performed with camelysin-, TasA-, and InhA1-specific antibodies.

FIG. 6.

Relationship between camelysin and InhA1 levels. Western blot experiments employed cell-associated protein (for camelysin) and cell supernatant protein (for InhA1). (A) Endogenous camelysin and InhA1 levels in the parent strain. Strain 7702 was cultured in NBY, and samples were taken every 2 h, beginning at the transition phase of growth. A corresponding growth curve for 7702 is shown. (B) Camelysin and InhA1 levels in early-stationary-phase samples from the parent strain and the inhA1 and calY mutants (UT345 and UT356, respectively). (C) Camelysin and InhA1 levels produced by 7702 and a B. anthracis calY mutant carrying an IPTG-inducible calY gene (pUTE980). The abundances of calY and inhA1 relative to that of the parent strain 7702 containing the empty vector (EV) were determined using densitometry. Data are presented as fold changes.