An overlap between the control of programmed cell death in Bacillus anthracis and sporulation

Abstract

The Staphylococcus aureus cid and lrg operons have been shown to control cell death and lysis in a manner thought to be analogous to programmed cell death (apoptosis) in eukaryotic organisms. Although orthologous operons are present in a wide variety of bacterial species, members of the Bacillus cereus group are unique in that they have a total of four cid-/lrg-like operons. Two of these operons are similar to the S. aureus cid and lrg operons, while the other two (designated clhAB(1) and clhAB(2)) are unique to this group. In the present study, the functions and regulation of these loci were examined. Interestingly, the Bacillus anthracis lrgAB mutant displayed decreased stationary-phase survival, whereas the clhAB(2) mutant exhibited increased stationary-phase survival compared to the parental and complementation strains. However, neither mutation had a dramatic effect on murein hydrolase activity or autolysis. Furthermore, a quantitative analysis of the sporulation efficiency revealed that both mutants formed fewer spores than did the parental strain. Similar to S. aureus, B. anthracis lrgAB transcription was shown to be induced by gramicidin and CCCP, agents known to dissipate the proton motive force, in a lytSR-dependent manner. Northern blot analyses also demonstrated a positive role for lytSR in the clhAB(2) transcription. Taken together, the results of the present study demonstrate that B. anthracis lrgAB and clhAB(2) play important roles in the control of cell death and lysis and reveal a previously unrecognized role of this system in sporulation.

FIG. 1.

Schematic diagram of cid and lrg orthologues of B. anthracis. The cid and lrg operons are located adjacent to cidR and lytSR loci, respectively, as in S. aureus. Two additional loci with similarities to the S. aureus cid/lrg operons are also present in the B. anthracis genome. These genes have been designated clhA₁ (BAS3599), clhB₁ (BAS3598), clhA₂ (BAS4960), and clhB₂ (BAS4959). The gntR (BAS3600) and secG (BAS4958) genes encode a potential transcriptional regulator and a subunit of the SecG preprotein translocase, respectively. Open reading frames whose putative products do not match known proteins are labeled “orf.” Arrows above the genes represent the direction and sizes of transcripts identified by Northern blot analyses presented here and in previous studies (1).

FIG. 2.

Northern blot analysis of clhAB₂ transcription. B. anthracis Sterne, its lytSR mutant derivative (lytSR), and the lytSR complementation strain (comp) were grown in the presence (+Glu) or absence (−Glu) of 35 mM glucose, and RNA was collected at 2, 6, and 12 h. Portions (5 μg) of samples were separated in a 1.0% formaldehyde-agarose gel, transferred to a nylon membrane, and hybridized to DIG-labeled probes derived from the clhB₂ gene. An ethidium bromide-stained gel of the RNA used in these experiments is also shown as a loading control.

FIG. 3.

Northern blot analysis of lrgAB transcription. B. anthracis Sterne, its lytSR mutant derivative (lytSR), and the lytSR complementation strain (comp) were grown in the presence (+Glu) or absence (−Glu) of 35 mM glucose, and RNA was collected at 2, 6 and 12 h. Portions (5 μg) of samples were separated in a 1.0% formaldehyde-agarose gel, transferred to a nylon membrane, and hybridized to DIG-labeled probes derived from the lrgB gene. An ethidium bromide-stained gel of the RNA used in these experiments is also shown.

FIG. 4.

Δψ-mediated control of lrgAB and chlAB₂ transcription. B. anthracis Sterne, its lytSR mutant derivative (lytSR), and the lytSR complementation strain (comp) were grown without glucose for 6 h and treated with gramicidin or CCCP for 15 min prior to RNA isolation. Portions (5 μg) of samples were separated in a 1.0% formaldehyde-agarose gel, transferred to a nylon membrane, and hybridized to DIG-labeled probes derived from the lrgB gene (a) or the clhB₂ gene (b). An ethidium bromide-stained gel of the RNA used in these experiments is also shown.

FIG. 5.

Stationary-phase survival assays. B. anthracis Sterne (squares), its lrgAB mutant derivative (a and b, triangles), the lrgAB complementation strain (a and b, circles), its clhAB₂ mutant derivative (c and d, diamonds), and the clhAB₂ complementation strain (c and d, circles) were grown in the presence (open symbols) or absence (solid symbols) of 35 mM glucose, and samples were removed and assayed to determine the optical density (OD₆₀₀ [a and c]) and cell viability (log CFU/ml [b and d]). The data presented are representative of three independent experiments. Additional replicates are presented in Fig. S2 in the supplemental material.

FIG. 6.

Microscopic examination of cell viability. Fluorescence microscopy using Live/Dead BacLight staining of the B. anthracis Sterne, its lrgAB mutant, its clhAB₂ mutant derivative, and the clhAB₂ complementation strain after incubation in NZY broth for 48 h. Red staining is indicative of dead or damaged cells, while green staining indicates live cells.

FIG. 7.

Impact of lrgAB and clhAB₂ mutations on sporulation. B. anthracis Sterne, lrgAB mutant, clhAB₂ mutant, clhAB₂ complementation strain KB6050 (comp), and spo0A mutant were grown in Schaeffer's medium. After 48 h of incubation at 37°C, dilutions were spread on BHI agar before and after heat treatment at 65°C for 30 min. The sporulation efficiency was calculated as the ratio of the number of spores to the number of viable cells for each strain.