Dormant spores of Bacillus anthracis germinate during host infection and their vegetative growth and dissemination precipitate anthrax disease. Upon host death, bacilli engage a developmental programme to generate infectious spores within carcasses. Hallmark of sporulation in Bacillus spp. is the formation of an asymmetric division septum between mother cell and forespore compartments. We show here that sortase C (SrtC) cleaves the LPNTA sorting signal of BasH and BasI, thereby targeting both polypeptides to the cell wall of sporulating bacilli. Sortase substrates are initially produced in different cell compartments and at different developmental stages but penultimately decorate the envelope of the maturing spore. srtC mutants appear to display no defect during the initial stages of infection and precipitate lethal anthrax disease in guinea pigs at a similar rate as wild-type B. anthracis strain Ames. Unlike wild-type bacilli, srtC mutants do not readily form spores in guinea pig tissue or sheep blood unless their vegetative forms are exposed to air.

Introduction

Bacillus anthracis, the causative agent of anthrax, is a spore-forming, Gram-positive microbe that requires oxygen for vegetative growth and spore formation (Roth et al., 1955; Koch, 1876). Upon entry into host tissues, spores, the infectious form of B. anthracis, germinate within phagocytes and the resulting vegetative forms then replicate in the cytosol of immune cells (Koch, 1876; Ruthel et al., 2004; Hu et al., 2006; Ribot et al., 2006). B. anthracis dissemination and replication in all organ tissues of the infected host generates up to 10¹⁰ vegetative bacilli per gram of blood or tissue (Koch, 1876; Ivins et al., 1994). Vegetative bacilli secrete lethal factor (LF), protective antigen (PA) and oedema factor (OF) (Smith et al., 1955; Leppla, 1982; 2000; Vodkin and Leppla, 1983; Robertson and Leppla, 1986; Klimpel et al., 1994; Duesbery et al., 1998). Assembly of LF and PA into lethal toxin and of OF and PA into oedema toxin (OT) are critical events in the pathogenesis of anthrax disease, enabling toxin-mediated killing of infected hosts (Milne et al., 1994; 1995; Collier and Young, 2003).

At the time of death, anthrax-infected animals do not harbour spores (Koch, 1876). B. anthracis spore formation in carcasses of anthrax-infected small animals, such as rabbits, guinea pigs or mice, occurs within 24–48 h (Toschkoff and Veljanov, 1970). Spore formation in large animals, cows or sheep, however, requires cadaver opening and exposure of infected tissues to air (Koch, 1876). These observations are consistent with the general hypothesis, initially derived from bacterial growth studies in laboratory media, whereby B. anthracis sporulation occurs only in the presence of oxygen (Roth et al., 1955).

The cell wall envelope of Gram-positive bacteria represents a unique cellular structure, which contains proteins that require specific sorting signals for their anchoring (Marraffini et al., 2006). Sortases cleave motif sequences of sorting signals and form amide bonds between the C-terminal carboxyl groups of proteins and amino groups of peptide cross-bridges within cell wall peptidoglycan (Schneewind et al., 1995; Ton-That et al., 1999). The genome of B. anthracis encodes three sortases (Read et al., 2003; Gaspar et al., 2005). Sortase A cleaves LPXTG motif sorting signals of seven surface proteins destined for the cell wall of vegetative bacilli: BasA, BasB, BasC, BasD, BasE, BasF and BasG (B. anthracissurface protein). Physiological functions are thus far known for BasC, a collagen adhesin that promotes bacterial binding to connective tissues (Xu et al., 2004), and BasD, the surface protein receptor of γ-bacteriophage (Davison et al., 2005). B. anthracis sortase B recognizes the NPKTG motif of BasK (IsdC, iron-regulated surface determinant C), a haem-binding protein involved in uptake of iron during infection (Gaspar et al., 2005; Maresso et al., 2006). The third sortase-like gene, sortase C (srtC) (BA5069), is located in the basI-srtC-sctR-sctS operon, where basI (BA5070) encodes for a surface protein with an LPNTA-motif sorting signal and sctR-sctS encodes for a two-component regulatory system (Fig. 1A). A second gene specifying a polypeptide with LPNTA-motif sorting signal, basH (BA0397), is positioned elsewhere in the B. anthracis genome (Gaspar et al., 2005) (Fig. 1A). Although bioinformatic analysis provides a guide for the discovery of physiological gene function, the assignment of sortase/substrate relationships does require experimental proof. Further, the contribution of sortases and surface proteins to the pathogenesis of anthrax and the replication cycle of B. anthracis cannot be gleaned from bioinformatics analysis alone. This report examines the molecular and physiological properties of sortase C and its substrates.

Details are in the caption following the image — **Figure 1**
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Biochemical characterization of *B. anthracis* sortase C.
A. The *basI-srtC-sctR-sctS* operon contains *basI*, a sortase substrate with a LPNTA-motif sorting signal (B. anthracissurface protein I), sortase C (*srtC*), *sctR* response regulator (sortase Ctwo-component system regulator) and *sctS* (sensory kinase). *basH* encodes a second sortase substrate with LPNTA sorting signal.
B. SrtC_ΔN was purified from *E. coli* (Coomassie-stained SDS-PAGE, inset) and incubated with peptide substrate (a-GEKLPNTASNN-d) in the presence or absence of thiol reagent MTSET or reducing agent DTT. Peptides were conjugated to the fluorophore 2-aminobenzoyl (a) and the quencher 2,4-dinitrophenyl (d) groups at the N- and C-terminus respectively. 2-aminobenzoic acid fluorescence increases as a result of peptide cleavage due to loss of intramolecular quenching. Peptide cleavage was measured as arbitrary fluorescence units.
C. SrtC_ΔN was incubated with different peptides harbouring pentapeptide motifs of C-terminal cell wall sorting signals of *B. anthracis* surface proteins (Gaspar *et al*., 2005). SrtC_ΔN cleaved only LPNTA, but not LPATG (SrtA substrate), LGATG nor NPKTG (SrtB substrate) peptides.
D. rpHPLC separation and mass spectrometry of SrtC_ΔN substrate and reaction products reveals that sortase C cleaves the LPNTA motif between the threonine and the alanine residues.
E. MALDI-TOF mass spectrometry of the compound in the 42 min fraction (D). A species with m/z 877.46 was detected that could be assigned the compound structure a-GEKLPNT-OH (calculated m/z 877.98). Asterisks indicate signals present in matrix control spectra.
F. MALDI-TOF mass spectrometry of the compound in the 40 min fraction (D). The main cleavage product with m/z 656.25 was detected and assigned the compound structure NH₂-ASNN-d with a calculated m/z of 656.59.

Results

Sortase C (SrtC) cleaves LPNTA motif peptides

Due to their genetic linkage (Fig. 1A), we hypothesized that sortase C may specifically recognize and cleave the BasI LPNTA motif. Recombinant SrtC_ΔN, with a replacement of the N-terminal signal peptide of sortase C for six histidyl, was purified from Escherichia coli lysate by affinity chromatography on Ni-NTA (Fig. 1B, inset) and its activity was tested towards substrate peptides encompassing sorting signal motif sequences of different types of B. anthracis surface proteins – LPNTA, LPATG, LGATG and NPKTG. A fluorescence-based cleavage assay was used to measure sortase activity. Fluorescence of 2-aminobenzyl-GEKLPNTASNN-dinitrophenyl (a-GEKLPNTASNN-d) is quenched due to the close proximity of the fluorophore (amino-benzyl) and the quencher (dinitrophenyl). Incubation of a-GEKLPNTASNN-d with SrtC_ΔN increased sample fluorescence more than 30-fold above background, indicating that in vitro peptide cleavage did occur (Fig. 1B). SrtC_ΔN cleaved only LPNTA, but not LPATG, LGATG and NPKTG peptides (Fig. 1C). In contrast, SrtA_ΔN and SrtB_ΔN, which recognize LPKTG and NPKTG, respectively, did not cleave LPNTA motif peptide (Fig. 1B) (Gaspar et al., 2005; Maresso et al., 2006). Incubation of SrtC_ΔN with methylmethane-thiosulphonate (MTSET) abolished substrate cleavage (Fig. 1B), suggesting MTSET may form a disulphide with the active site cysteine (Cys¹⁸¹) of sortase C, a property observed for all other sortases examined thus far (Ton-That and Schneewind, 1999; Ton-That et al., 1999; Mazmanian et al., 2002; Gaspar et al., 2005; Maresso et al., 2006).

Reaction products of SrtC_ΔN or mock-treated peptide substrate were separated by reversed phase HPLC and elution monitored by absorbance (Fig. 1D). Mock-treated peptide eluted at 49 min (39% acetonitrile). MALDI-TOF mass spectrometry of the eluted compound confirmed substrate integrity. Incubation of substrate with SrtC_ΔN generated two product peaks eluting at 40 (36% acetonitrile) and 42 min (32% acetonitrile). MALDI-TOF mass spectrometry of the compound in the 40 min fraction (Fig. 1F) revealed the main cleavage product with m/z 656.25 and assigned the compound structure NH₂-ASNN-d (calculated m/z of 656.59). MALDI-TOF mass spectrometry of the compound in the 42 min fraction (Fig. 1E) revealed a species with m/z 877.46 and assigned compound structure a-GEKLPNT-OH (calculated m/z 877.98). Together these data indicate that SrtC_ΔN cleaves peptides encompassing the LPNTA motif between threonine (T) and alanine (A). This reaction is inhibited by MTSET, consistent with the hypothesis that the thiol of Cys¹⁸¹ functions as the active site SrtC_ΔN (Fig. 1B). These results suggest further that basH and basI, whose precursor products harbour LPNTA-motif sorting signals, likely encode in vivo substrates of sortase C.

srtC is expressed during sporulation

A hallmark of sporulation in the soil bacterium Bacillus subtilis is the formation of an asymmetrically positioned (polar) septum that divides the developing cell into dissimilarly sized progeny called the forespore (the smaller cell) and the mother cell (Losick and Stragier, 1992). Both compartments further differentiate by activating different sigma (σ) factors (Losick and Pero, 1981), each of which directs transcription of a specific set of genes (Losick and Stragier, 1992). Activation of σ^F in the forespore leads to the expression of forespore-specific genes as well as activation of σ^E in the mother cell (Karow et al., 1995; Londono-Vallejo and Stragier, 1995). Differential gene expression in the two cell types brings about engulfment of the forespore as the mother cell membrane migrates around the forespore (Rubio and Pogliano, 2004). Following engulfment, the forespore matures into a fully formed spore (Chada et al., 2003), which is released after mother cell lysis (Nicholson et al., 2000).

To examine sporulation of B. anthracis, an overnight culture of B. anthracis strain Sterne in brain–heart infusion (BHI) broth was diluted 1:10 into modG sporulation medium (Kim and Goepfert, 1974) and incubated at 30°C. Optical density (OD₆₀₀) of culture aliquots was used as a measure for vegetative growth values in hourly intervals. T₀ marks the time when exponential growth reaches plateau and sporulation commences (Fig. 2A). FM4-64 membrane dye was used to monitor sporulation by fluorescence microscopy (Fig. 2B). Polar septation of B. anthracis strain Sterne begins at T₊₁ and forespore engulfment neared completion at T₊₃. Bright endospores were observed at T₊₅. Henceforth, optical density measurements of B. anthracis cultures and fluorescence microscopy of FM4-64-stained samples were used to examine gene expression during spore development.

Specific antibodies [rabbit antibodies raised against purified SrtC (αSrtC) and BasI (αBasI)] and gfp transcriptional fusions were used to probe expression of srtC::gfp and its substrates genes, basH::gfp and basI::gfp, in B. anthracis Sterne under sporulating conditions. SrtC was detected by immunoblotting in bacillus extracts at T₋₁, T₀ and T₊₁ (Fig. 3A), but only when basI-srtC-sctR-sctS expression was increased in sporulating cells harbouring a multicopy plasmid with the sctR response regulator (pLM218, Fig. 3B). SrtC was not detected in B. anthracis with vector control plasmid (data not shown). Immunoblot analysis indicated that SrtC abundance peaked at T₀ and declined at T₊₁ (Fig. 3A). Disappearance of SDS-soluble SrtC appears to be at least in part due to SrtC sequestration within developing spores (vide infra). Fluorescent signal generated from a chromosomal srtC::gfp transcriptional fusion was first detected in sporulating cells at T₋₁, 2 h prior to asymmetric septum formation (T₊₁) (Fig. 3B). As a test for the regulatory function of SctR, the sctR gene was placed under control of the IPTG-inducible P_spac promoter and expression induced by the addition of IPTG during exponential growth (Fig. 3C). GFP fluorescence was observed in vegetative srtC::gfp bacilli during exponential growth, indicating that the SctR response regulator is sufficient to induce expression of basI-srtC-sctR-sctS in the absence of other sporulation-specific factors (Fig. 3C).

Sortase C substrate BasH is expressed in the forespore

Nucleotide sequence upstream of basH matches the σ^F consensus (Wang et al., 2006), i.e. the promoter recognition sites of σ^F RNA polymerase for genes that are only transcribed in the forespore (Schmidt et al., 1990; Fig. 4A). To visualize basH expression, bacteria were transformed with a plasmid containing gfp under control of the basH promoter (Fig. 4B). Fluorescence microscopy of sporulating bacilli revealed forespore-restricted expression of P_basH–gfp (4, 5). Previous work using DNA microarray analysis also revealed basH transcription during sporulation (Liu et al., 2004). Fluorescence of a translational hybrid, BasH–GFP (Fig. 5B), was detected at forespore boundaries, consistent with signal peptide-mediated targeting of BasH–GFP to the forespore plasma membrane (Fig. 5C and D). To determine whether σ^F RNA polymerase is sufficient to drive basH expression, σ^F (spoIIAC) expression was induced via the IPTG-inducible P_spac promoter (pLM223, Fig. 4B) during vegetative growth of bacilli. GFP fluorescence was indeed observed during exponential growth in the presence but not in the absence of IPTG, indicating that σ^F RNA polymerase directs basH expression (Fig. 4D) and that its product, BasH, must be targeted to the cell wall compartment of B. anthracis forespores (Fig. 5).

BasH is anchored to the forespore cell wall envelope by sortase C

To examine whether BasH is indeed targeted to forespore cell wall, codons for the FLAG epitope were inserted into the basH open reading frame, upstream of nucleotide sequences that specify the LPNTA sorting signal. basH_FLAG was cloned on a plasmid and expressed from its σ^F-dependent promoter in sporulating bacilli (pLM230, Fig. 4E). Culture aliquots were treated with PlyL N-acetylmuramoyl-l-alanine amidase (Low et al., 2005) and lysates of the cell wall compartment analysed by immunoblotting with anti-FLAG monoclonal antibody. BasH_FLAG was detected at T₊₁, when the first polar septa appeared (Fig. 4F). Abundance of BasH_FLAG peaked at T₊₂, declined at T₊₃ and, upon further incubation (T₊₄ and T₊₅), no immunoreactive species could be detected (Fig. 4F). Similar to SrtC, BasH polypeptide appears to be sequestered upon spore maturation, presumably preventing PlyL-mediated solubilization of spore peptidoglycan with anchored BasH. In B. subtilis, cortex peptidoglycan biosynthesis has not yet commenced during the T₊₁–T₊₃ interval (Meador-Parton and Popham, 2000). Assuming that this may also be true for B. anthracis, BasH may be anchored to primordial peptidoglycan surrounding the newly formed forespore. BasH targeting to forespore cell wall occurred in wild-type B. anthracis, but not in an isogenic srtC mutant (Fig. 4G). This defect was complemented in sporulating srtC mutant bacilli harbouring plasmid-encoded wild-type srtC (pLM238, Fig. 4G).

BasI is anchored to the cell wall envelope by sortase C

Expression and anchoring of BasI were also analysed in sporulating bacteria using optical density measurements and fluorescence microscopy of FM4-64-stained B. anthracis cultures. When measured with GFP fluorescence generated from a chromosomal basI::gfp transcriptional fusion in sporulating bacilli, basI expression commenced at T₋₁ (Fig. 6B). PlyL digestion of the cell wall followed by immunoblotting revealed that BasI is anchored to peptidoglycan of sporulating bacilli in a srtC-dependent manner (Fig. 6C). The defect in anchoring of BasI was restored by plasmid encoded srtC, whose expression was activated by the sctR response regulator also encoded on the same plasmid (pLM233, Fig. 6A and C). As a fractionation control, ribosomal subunit protein L6 was not found in the cell wall compartment (Fig. 6C).

SrtC–mCherry is inherited by B. anthracis forespores

srtC is expressed by sporulating cells prior to septum formation between mother cell and forespore, i.e. before the cellular compartment that represents the final destination of BasH has been formed. Forespore inheritance of SpoIIIJ, a regulatory factor, has been described in B. subtilis (Serrano et al., 2003). To test whether SrtC is also inherited by the forespore compartment, plasmid-encoded translational srtC–mCherry fusion was expressed via the basI-srtC promoter. As expected, SrtC–mCherry was detected in sporulating bacilli that had not yet formed an asymmetric septum (T₋₁, Fig. 7). Upon septum formation (T₊₁) SrtC–mCherry was detected by fluorescence microscopy in both forespore and mother cell compartments. Upon further incubation, SrtC–mCherry fluorescence was detected in mature spores (T₊₂₀, Fig. 7). Thus, even though basH is expressed in a cell that does not synthesize SrtC, mother cell membranes ensure inheritance of sortase C into the forespore compartment, thereby enabling BasH anchoring to the forespore cell wall envelope.

srtC contributions during the pathogenesis of anthrax in guinea pigs

basI-srtC-sctR-sctS operon and basH homologues were found only in genomes of members of the Bacillus cereus group (Jensen et al., 2003) (B. cereus, B. anthracis and Bacillus thuringiensis), but not in the genome of other Bacillus species. Isogenic variants of the non-virulent vaccine strain, B. anthracis Sterne (Sterne, 1937), carrying a deletion of the srtC gene, displayed no defect in germination, growth, spore formation or virulence (data not shown). This prompted us to derive srtC mutants from the fully virulent isolate B. anthracis Ames (Welkos et al., 1989). Similar to Sterne variants, srtC mutants of B. anthracis Ames displayed no defect in spore formation and germination in laboratory media (data not shown) and no defect in the ability to cause lethal anthrax disease in a guinea pig model of subcutaneous infection (Ivins et al., 1994; Fellows et al., 2001; Fig. 8A). At the time of death, organ tissues of animals infected with B. anthracis Ames or its isogenic srtC mutant were teeming with vegetative bacilli in spleen and blood (Table 1). Within 3 weeks, tissues of animals that had been infected with wild-type bacilli generated infectious spores (∼20% of vegetative bacilli), whereas tissues infected with the srtC mutant strain did not (Table 1). To investigate whether deletion of srtC renders spores heat-sensitive or even abolishes spore formation, tissue samples were stained for spores and viewed by microscopy. Abundant spores were detected in heart blood and in spleens from animal carcasses that had been infected with B. anthracis Ames, but not in animals that succumbed to infection by srtC mutants (Fig. 8B).

Table 1. Spore formation of B. anthracis Ames and srtC variants in animal tissues.

Strain	Organ^a	Vegetative bacilli^b ^c	Colony-forming units (day 21)^c	Heat-resistant spores (day 21)^c	Sporulation efficiency^d
Wild type	Heart blood	1.05 × 10⁸	2.60 × 10⁸	5.10 × 10⁷	19.6
Wild type	Heart blood	3.25 × 10⁷	1.90 × 10⁷	3.80 × 10⁶	20.0
Wild type	Heart blood	7.75 × 10⁸	1.40 × 10⁷	2.50 × 10⁶	17.9
Wild type	Spleen	1.05 × 10⁹	2.00 × 10⁷	7.10 × 10⁶	35.5
Wild type	Spleen	1.92 × 10⁹	1.80 × 10⁷	4.10 × 10⁶	22.8
Wild type	Spleen	6.00 × 10⁹	3.50 × 10⁷	9.00 × 10⁶	25.7
srtC	Heart blood	2.57 × 10⁷	3.60 × 10⁶	< 100	< 0.001
srtC	Heart blood	4.00 × 10⁷	7.60 × 10⁵	< 100	< 0.001
srtC	Heart blood	4.00 × 10⁶	3.10 × 10⁵	< 100	< 0.001
srtC	Spleen	7.75 × 10⁸	2.70 × 10⁶	< 100	< 0.001
srtC	Spleen	1.00 × 10⁸	3.00 × 10⁷	< 100	< 0.001
srtC	Spleen	1.20 × 10⁹	3.50 × 10⁶	< 100	< 0.001

a. Organs were removed from guinea pigs (infected with B. anthracis Ames or isogenic srtC mutant spores) at time of death (4 days following infection), homogenized and incubated in sterile tubes at room temperature.
b. Enumerated as colony-forming units per millilitre of tissue harvested at time of death. No spores were detected at this time and no statistically significant difference in the number of vegetative bacilli between wild type and srtC mutant were detected.
c. Numbers represent the average of two measurements of tissues samples from the same animals. < 100 indicates below the limit of detection.
d. Per cent amount of colony-forming units resistant to heat treatment (65°C for 30 min).

Delayed spore formation of srtC mutants in sheep blood

To monitor spore formation in animal blood, B. anthracis cultures were inoculated into sheep blood and grown to a density of 10⁷ vegetative bacilli per millilitre of tissue. Within 24 h in sheep blood, B. anthracis Ames generated heat-resistant, viable spores, a process that was essentially completed within 2–3 days (Fig. 9A). srtC mutants did not generate heat-resistant spores during the first 12 days; however, spore formation in sheep blood rose eventually to the same level as that observed for B. anthracis Ames (Fig. 9A). Microscopic analysis of bacilli stained with FM4-64 revealed that srtC mutants failed to form polar septa during the first 10 days of incubation in sheep blood at room temperature (Fig. 9B). Nevertheless, although spore formation was significantly delayed in sheep blood, srtC mutants eventually completed the developmental programme.

Earlier work revealed B. anthracis spore formation in carcasses of anthrax-infected small animals, such as rabbits, guinea pigs and mice (Toschkoff and Veljanov, 1970). Spore formation in large animals, cows or sheep, however, requires opening of cadavers and exposure of infected tissues to air (Koch, 1876). These and other observations support a general hypothesis, whereby B. anthracis sporulation occurs only in the presence of oxygen (Roth et al., 1955). We wondered whether the sporulation defect of srtC mutants can be rescued by exposure of bacilli to air and measured vegetative growth and spore formation in sheep blood cultures with rotation. Exposure to air indeed restored spore formation of srtC mutants to a level similar to that observed for wild-type B. anthracis Ames (data not shown). Moreover, microscopic analysis revealed synchronous formation of polar septa and endospores in wild type and srtC mutants (Fig. 10).

Discussion

Dramsi et al. (2005) used bioinformatic tools and assigned 14 sortases as class D with three subclusters as a reflection of host phylogeny (bacilli, clostridia and actinomycetales). Members of the Bacillus (B. anthracis, B. cereus and B. thurigensis), Streptomyces (S. coelicolor, S. avermitilis) and Clostridium spp. (C. difficile, C. perfringens, C. tetani) all engage in a developmental programme that leads to the formation of spores (Dramsi et al., 2005). Only Corynebacterium diphtheriae, a member of the actinomycetales and the causative agent of diphtheria, encodes a class D sortase yet this species does not form spores.

Previous work examined the role of class D sortases in S. coelicolor, a Gram-positive, filamentous soil bacterium that is capable of producing many natural antibiotics (Hodgson, 2000). The life cycle of S. coelicolor commences with the formation of a feeding (submerged) mycelium, which differentiates into aerial hyphae that septate and develop into spore chains. The hyphal surface becomes hydrophobic, a property that promotes outgrowth into the air and facilitates the dispersion of spores, eventually giving rise to new mycelia. Six surface proteins of S. coelicolor, collectively called chaplins, are involved in aerial hyphae formation (Claessen et al., 2003; Elliot et al., 2003). All chaplins are synthesized during mycelium formation as precursors with an N-terminal signal sequence and a short hydrophobic domain (chaplin domain) of about 40 residues. Chaplins D–H are small polypeptides, whereas chaplins A–C are larger and carry a C-terminal sorting signal (LAXTG motif). Deletion of all six chaplin genes (chpA–H) hinders the formation of aerial hyphae, a defect that can be rescued by addition of purified chaplin proteins (Elliot et al., 2003). As mixtures of ChpD–H isolated from cell walls of aerial hyphae are highly surface active (Claessen et al., 2003), it seems plausible that these proteins lower aqueous surface tension for the emergence of aerial hyphae at the air–water interface. Although this has not yet been demonstrated, large chaplins ChpA–C are thought to be anchored by sortase to the bacterial peptidoglycan, serving as scaffold for assembly of small chaplins. Furthermore, chaplins may provide a hydrophobic surface for deposition of RdlA and RdlB, a highly insoluble, outer protein layer that facilitates spore dispersion (Claessen et al., 2004).

In this report, we have used purified sortase C to analyse its substrate requirements. SrtC_ΔN, lacking the N-terminal signal peptide and membrane anchor, cleaved peptides bearing the LPNTA motif, but not peptides with LPXTG, NPKTG or LGATG sequences. LPXTG and NPKTG motif sorting signals are the substrates of sortase A and sortase B respectively (Gaspar et al., 2005; Maresso et al., 2006). A sorting signal-like sequence with LGATG motif has been identified at the predicted C-terminal end of BasJ; however, a sortase enzyme responsible for cell wall anchoring of this internalin A homologue has thus far not been revealed (Gaspar et al., 2005). Two substrates, BasH and BasI, are anchored by sortase C to the cell wall of sporulating B. anthracis. basI, along with sortase C, is expressed by the basI-srtC-sctR-sctS operon. Both srtC::gfp and basI::gfp translational products were first detected at T₋₁, 2 h prior to the formation of the asymmetric septum that separates the mother cell from the developing forespore. The abundance of immunoreactive BasI and SrtC in B. anthracis extracts peaked at T₀ and rapidly declined at T₊₁ (Fig. 3A, data not shown). BasI and SrtC species were no longer detectable at T₊₂. A similar, although delayed, expression pattern was observed for BasH_FLAG; here the peak occurred at T₊₂, declined at T₊₃ and BasH_FLAG was no longer detectable at T₊₄ (Fig. 4F). Nevertheless, fluorescence microscopy allowed detection of SrtC–mCherry in forespores and endospores even at T₊₂₀, suggesting that SrtC, and perhaps even BasH and BasI, may be sequestered within developing spores in a manner that resists treatment with PlyL enzyme and hot SDS.

sctR and sctS, which encode a two-component regulatory system, appear to autoregulate the basI-srtC-sctR-sctS operon, as IPTG-induced synthesis of SctR from another promoter is sufficient to induce expression of the sortase C operon. Expression of basH, which encodes another sortase C substrate, is driven by σ^F RNA polymerase and the translation product of basH::gfp is located only in the forespore. As cell wall anchoring of BasH_FLAG absolutely requires srtC, it follows that sortase C must act in the intermembranous space that encloses forespore peptidoglycan.

What could be the role of protein targeting to the cell wall of developing B. anthracis spores? Initial pathogenesis experiments with srtC mutants derived from either B. anthracis strain Sterne in A/J mice (data not shown) or B. anthracis strain Ames in guinea pigs revealed no defect in spore-mediated virulence and development of lethal anthrax disease. Surprisingly, vegetative bacilli derived from srtC mutants failed to form spores in guinea pig carcass tissues (spleen, liver or heart's blood) that had been excised post-mortem and incubated for 21 days, whereas wild-type bacilli sporulated with remarkable efficiency. Microscopic analysis of guinea pig carcass tissue revealed the absence of spores with the Schaeffer–Fulton stain and suggested that spore formation must be hindered in mutants lacking srtC. This phenotype was also observed when srtC mutants were incubated in defibrinated sheep blood, although that spore formation eventually commenced after 12 days of incubation. It should be noted that spore formation of wild-type B. anthracis in sheep blood is essentially complete within 2–3 days. Fluorescence microscopy of FM4-64-stained bacilli revealed that srtC mutants do not form asymmetric division septa and hence cannot develop spores. This phenotype of arrested sporulation was completely rescued when B. anthracis srtC mutants were rotated in sheep blood to increase their exposure to air and oxygen.

We wonder how srtC and its substrate genes (basH and basI), whose products are targeted to the cell wall envelope of sporulating bacilli, can enable formation of infectious B. anthracis spores under oxygen-limiting conditions as has been observed in carcasses of small animals. BasI and BasH are small polypeptides, seemingly unstructured when analysed with protein fold predictions and one of them harbours GXGS sequence repeat. In future experiments, we will need to consider how sortase C deposition of BasH and BasI may increase exposure to molecular oxygen, for example, by altering the physical properties of developing forespores, as has been reported for sortase-dependent deposition of chaplins by S. coelicolor. Other work will need to consider the two-component regulator sctR/sctS, whose physiological function may be to sense of environmental conditions in the host that permit spore formation.

Experimental procedures

Bacterial strains and plasmids

Bacillus anthracis strains Sterne 34F2, Ames or its variants were grown in BHI or LB broth at 30°C. Sporulation was induced by diluting overnight BHI cultures 1:10 into modG medium (Kim and Goepfert, 1974) and further incubation at 30°C. FM4-64 membrane dye (Invitrogen) (Pogliano et al., 1999) was added to monitor sporulation (1:1000 dilution). Growth media were supplemented with kanamycin (20 μg ml⁻¹) or chloramphenicol (10 μg ml⁻¹) to provide for plasmid selection. Isopropyl-β-d-thiogalactopyranoside (IPTG) was added at 1 mM where indicated. B. anthracis chromosomal DNA was extracted with the Wizard Genomic DNA Purification Kit (Promega).

Plasmids

Table 2 lists primers used for plasmid construction. PCR with primers P23/P24 was used to append BamHI and XmaI restriction sites to srtC and the product was inserted into pQE-30 (Qiagen) to generate pLM116 (encoding SrtC_ΔN). Insertion of P18/P19 PCR product flanked by NdeI/BamHI sites into pET15b (Novagene) generated pLM114, which provided for expression His-tagged BasI and affinity chromatography as well as rabbit antibody production. Plasmid pLM4 was used for allelic replacement and constructed as follows. pTS1 (McDevitt et al., 1994) sequence containing the cat gene was excised using HpaI and StuI and replaced by the SmaI fragment of pUT618 containing the aphA3 gene (gift of Dr T. Koehler) to generate pLM3. The bla gene was excised from pLM3 using AdhI and EcoNI, protruding ends blunted with Klenow polymerase and ligated, thereby generating pLM4. For IPTG-inducible expression, spac promoter and lacI repressor sequences were amplified from pMutin-HA (Kaltwasser et al., 2002) using primers P146/P149 and inserted into pLM4 using EcoRI and XmaI restriction sites to generate pLM5. Insertion of P85/P86 (gfp mut2 coding sequence) (Cormack et al., 1996) and P107/P120 (basH promoter) PCR products into pOS1 via EcoRI, BamHI and PstI restriction sites produced pLM200. Insertion of P141/P142 (spoIIAC coding sequence) PCR product into pLM5 via KpnI sites generated pLM223. Insertion of P4/P88 (basI promoter sequence) and P95/P80 (sctR coding sequence) PCR products into pLM4 (EcoRI and BamHI sites) generated pLM218. Insertion of P132/P126 (sctR coding sequence) PCR product into pLM5 (KpnI sites) generated pLM226. Insertion of P12/P131 (srtC 1 kb 5′ flanking sequence) and P132/P13 (srtC 1 kb 3′ flanking sequence) and P112/P111 (gfp coding sequence) PCR products into pLM4 (EcoRI, KpnI and XmaI sites) generated pLM212; this plasmid was used for replacement of srtC coding sequence with gfp in strain LAM012 (srtC::gfp). Insertion of P12/P58 (srtC 1 kb 3′ coding sequence) and P14/P13 (srtC 1 kb 3′ flanking sequence) PCR products into pLM4 (EcoRI, KpnI and XmaI sites) generated pLM201 for in-frame deletion of srtC (strain LAM005). Insertion of the EcoRI fragment of pLM218 (basI promoter–sctR fusion), P107/P108 PCR product (basH promoter and 5′ coding sequence) and PCR product P109/P110 (basH sorting signal and terminator) into pOS1 (EcoRI, KpnI and XmaI sites) generated pLM230. Replacement of the pLM230 EcoRI fragment with the P12/P13 PCR product (on pLM233 template) generated pLM238. Insertion of P73/P52 PCR product (basI 1 kb 5′ flanking sequence) and P9/P13 product (basI 1.5 kb 3′ flanking sequence containing srtC and sctR) into pLM4 (EcoRI, KpnI and XmaI sites) generated pLM233. Insertion of P112/P111 product (gfp) into pLM233 (KpnI sites) generated pLM234, a plasmid that was used for replacement of basI with gfp in strain LAM010 (basI::gfp). Insertion of P12/P154 product (basI promoter and coding sequences as well as srtC coding sequences), P159/P158 product (mCherry orf from pRSET-B-mCherry; Shaner et al., 2004) and P132/P13 product (sctR) into pLM4 (EcoRI, XbaI, KpnI and XmaI sites) generated pLM243 for SrtC–mCherry expression. PCR reactions were performed with Pfu DNA polymerase (Stratagene). Ligation products were transformed into E. coli K1077(dam^–dcm^–) and purified (non-methylated) plasmid DNA was transformed into B. anthracis following a previously developed protocol (Kim et al., 2004).

Table 2. Primers used in this study.

Primer	Restriction site added	Sequence^a
P4	EcoRI	aaGAATTCcaatgaaatggagtaagtggag
P9	KpnI	aaGGTACCcaatgatggcactaagcgcg
P12	EcoRI	aaGAATTCaggtttgaagaagagacccc
P13	XmaI	aaCCCGGGgatctaatgtcgtaatccacg
P14	KpnI	aaGGTACCacactgacgacttgttaccc
P18	BamHI	aaGGATCCttaagttgcagcattttttggttg
P19	NdeI	aaCATATGattgaagaagagaaaaagag
P23	BamHI	aaaGGATCCgcgcaagaattaacgaatgagg
P24	XmaI	aaaCCCGGGtcattttgaataactaccagttaac
P52	KpnI	aaGGTACCcattttatttctctccttttatac
P58	KpnI	aaGGTACCctttatttcctcattcgttaattc
P73	EcoRI	aaaGAATTCaaataaagtcgattgttacatcg
P80	PstI	aaaCTGCAGttcaagagcatcaatgcttatc
P85	BamHI	aaaGGATCCagtaaaggagaagaacttttcac
P86	PstI	aaaCTGCAGttatttgtatagttcatccatgcc
P88	BamHI	aaaGGATCCcattttatttctctccttttatac
P95	BamHI	aaaGGATCCaattgtagtttcgaaaagcggg
P107	EcoRI	aaaGAATTCccatatttaatttgtgtaccacc
P108	KpnI	aaaGGTACCcttatcgtcgtcatccttgtaatcttgttttttcgcattatcttttac^b
P109	KpnI	aaaGGTACCatgcatcaccatcaccatcacggcgataagttaccgaatactgc
P110	BamHI	aaaGGATCCttttgaacaagaacatttagcag
P111	KpnI	aaaGGTACCatgagtaaaggagaagaacttttcac
P112	KpnI	aaaGGTACCtttgtatagttcatccatgccatg
P120	BamHI	aaaGGATCCtttcgtcattgatttcctcctg
P131	KpnI	aaaGGTACCcatatttcatccctccatgcc
P132	KpnI	aaaGGTACCtgaaattgtagtttcgaaaagcg
P141	KpnI	aaaGGTACCaaggaggatagcctatggacatag
P142	KpnI	aaaGGTACCttgcgtttgatctttatagtagcg
P146	XmaI	aaaCCCGGGaggttttcaccgtcatcaccg
P149	EcoRI	ttatggGAATTCtacacagccc
P154	XbaI	aaaTCTAGAttttgaataactaccagttaacttcg
P158	KpnI	aaaGGTACCagcaagggcgaggaggataac
P159	XbaI	cgTCTAGAgtgagcaagggcgaggaggataacatg

a. Capital letters indicate the sequence of the restriction site inserted.
b. Underlined nucleotides encode for the FLAG epitope (Hopp et al., 1988).

Bacillus anthracis mutants

Bacillus anthracis variants were generated with pLM4, containing a thermosensitive origin of replication. Plasmids with 1 kb DNA sequence flanking each side of the mutation were transformed into B. anthracis and transformants grown at 30°C (permissive temperature) in LB broth (20 μg ml⁻¹ kanamycin). Cultures were diluted 1:100 and incubated at 43°C overnight (restrictive temperature). After four growth cycles at 43°C, cultures were diluted 1:100 into LB broth without antibiotics and grown overnight at 30°C. To ensure loss of pLM4-based plasmid, cultures were diluted four times into fresh media and propagated at 30°C. Bacteria were plated on LB agar and colonies examined for kanamycin resistance. DNA from kanamycin-sensitive colonies was analysed by PCR for the presence or absence of mutant alleles. Nucleic acid sequences of wild-type and mutant alleles were verified by DNA sequencing.

SrtC_ΔN cleavage of LPNTA peptide

Expression of SrtC_ΔN in E. coli XL-1 Blue (pLM116) was induced with IPTG. Six-histidyl-tagged enzyme was purified from cleared lysate by affinity chromatography on Ni-NTA agarose. Activity was assayed in buffer A (50 mM Tris-HCl pH 7.5, 150 mM NaCl) after 16 h incubation at 37°C (50 μM SrtC_ΔN and 10 μM peptide substrate). Modified peptide substrates (BiopeptideCo., LLC) were conjugated to 2-aminobenzoyl fluorophore (a) and 2,4-dinitrophenyl quencher (d) at the N- and C-terminus respectively. Reactions were quenched by boiling samples for 5 min and peptide cleavage monitored by quantification of 2-aminobenzoyl fluorescence at 320 nm excitation and 420 nm emission. Mean and standard deviation of three independent measurements are reported. SrtC_ΔN was treated with 5 mM MTSET ([2-(trimethylammonium)ethyl]methanethiosulphonate) (Smith et al., 1975) and 10 mM dithiothreitol (DTT). For chromatography, sortase reactions were quenched by centrifugation at 7500 g through Centricon-10 membrane (Millipore) to separate enzyme from substrate and product. Filtrate was separated by rpHPLC (2 × 250 mm, C18 Hypersil, Keystone Scientific) and elution (1–60% linear gradient in acetonitrile) monitored at 215 nm absorbance. Vacuum-dried fractions were analysed by MALDI-TOF and tandem mass spectrometry (ABI 4700, operated in reflection mode) to determine peptide structures.

Immunoblotting

Bacillus anthracis cultures were grown in modG sporulation media and aliquots removed at 1 h intervals until spores were detectable. Bacilli were sedimented by centrifugation at 10 000 g, washed and suspended in 300 μl of PlyL buffer (0.5 M sucrose, 1 mM DTT, 1 mM PMSF, 1 mM EDTA, 5 mM sodium phosphate, pH 6.0) with 250 μg ml⁻¹ PlyL amidase (Low et al., 2005). After digestion of cell wall for 16 h at 37°C, protoplasts were sedimented by centrifugation at 10 000 g. Aliquots of supernatant (cell wall lysate) were mixed with sample buffer, separated on SDS-PAGE and electrotransferred to PVDF membrane. Proteins were detected with specific serum (αBasI, αSrtC or αL6 – raised against purified proteins in rabbits) or anti-FLAG mouse monoclonal antibody (Stratagene), followed by species-specific secondary antibody conjugate to horseradish peroxidase and chemiluminescent detection.

Microscopy

Bacillus anthracis culture aliquots were viewed by DIC or fluorescence microscopy under Olympus Provis axial microscope (1000-fold magnification) equipped with CCD camera and digital images of FM4-64, GFP or mCherry fluorescence captured.

Sporulation

Overnight cultures of bacilli in BHI were diluted 1:10 into modG sporulation medium and incubated at 30°C for 48 h. Culture aliquots were heated for 30 min at 65°C to kill vegetative bacilli and 10-fold serial dilutions of heat-and non-heat-treated cultures were spread on LB agar followed by incubation and enumeration of total colony-forming units (cfu) (no heat treatment) and viable spores (heat treatment). Sporulation efficiency was calculated as the ratio (viable spores)/(total cfu).

Animal experiments

Bacillus anthracis spores (wild type and srtC) were washed three times with sterile distilled water and examined for the presence of vegetative bacilli by microscopy. Contaminating bacilli, typically < 1% of spore preparations, were heat-killed by incubating the spore preparation at 65°C for 30 min prior to additional washes. Colony-forming units were enumerated after serial dilution and plating on LB agar. To investigate the virulence of B. anthracis Ames wild-type and srtC spores, six guinea pigs each were infected subcutaneously with 30 or 300 spores for each of the two strains (wild-type and srtC). Animals were monitored for anthrax disease over 7 days following infection and LD₅₀ values were calculated (Reed and Muench, 1938).

Sporulation in host tissues

Shortly following anthrax death, guinea pig organs were removed from three animals infected with either wild-type or srtC mutant bacilli. Tissue aliquots were homogenized (under aseptic conditions) and total cfu as well as spore concentrations enumerated. Following incubation of homogenized tissues for 3 weeks, total cfu as well as spores were enumerated [homogenized tissues of all animals tested generated B. anthracis colonies (ground glass appearance) and no contaminations on LB agar]. For sporulation in sheep blood, 300 µl of culture (wild type or srtC at OD₆₀₀ 0.7, ∼10⁷ cfu ml⁻¹) was mixed with an equal volume of defibrinated sheep blood in a 1.5 ml tube and samples were incubated at room temperature for 21 days. At timed intervals, total cfu and viable spores were enumerated. To determine spore formation in rotating blood, the same experiment was carried out in 15 ml tubes with rapid agitation. Homogenized tissues and blood samples were fixed with 10% buffered formalin prior to microscopic analysis.

Acknowledgements

We thank Dominique Missiakas, Bill Blaylock, Justin Kern and Anthony Maresso for experimental help or critical reading of this manuscript. We are indebted to the Great Lakes RCE Small Animal Core for help with B. anthracis guinea pig infections and to Jonathan Budzik for PlyL purification. This work was supported by Grants AI38897 and AI52474 from the National Institute of Allergy and Infectious Diseases (Infectious Diseases Branch). O.S. acknowledges membership within and support from the Region V ‘Great Lakes’ Regional Center of Excellence in Biodefense and Emerging Infectious Diseases Consortium (GLRCE, National Institute of Allergy and Infectious Diseases Award 1-U54-AI-057153).

References

Chada, V.G., Sanstad, E.A., Wang, R., and Driks, A. (2003) Morphogenesis of bacillus spore surfaces. J Bacteriol 185: 6255–6261.
10.1128/JB.185.21.6255-6261.2003
CAS PubMed Web of Science® Google Scholar
Claessen, D., Rink, R., De Jong, W., Siebring, J., De Vreugd, P., Boersma, F.G., et al. (2003) A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils. Genes Dev 17: 1714–1726.
10.1101/gad.264303
CAS PubMed Web of Science® Google Scholar
Claessen, D., Stokroos, I., Deelstra, H.J., Penninga, N.A., Bormann, C., Salas, J.A., et al. (2004) The formation of the rodlet layer of streptomycetes is the result of the interplay between rodlins and chaplins. Mol Microbiol 53: 433–443.
10.1111/j.1365-2958.2004.04143.x
CAS PubMed Web of Science® Google Scholar
Collier, R.J., and Young, J.A. (2003) Anthrax toxin. Annu Rev Cell Dev Biol 19: 45–70.
10.1146/annurev.cellbio.19.111301.140655
CAS PubMed Web of Science® Google Scholar
Cormack, B.P., Valdivia, R.H., and Falkow, S. (1996) FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33–38.
10.1016/0378-1119(95)00685-0
CAS PubMed Web of Science® Google Scholar
Davison, S., Couture-Tosi, E., Candela, T., Mock, M., and Fouet, A. (2005) Identification of the Bacillus anthracis {gamma} phage receptor. J Bacteriol 187: 6742–6749.
10.1128/JB.187.19.6742-6749.2005
CAS PubMed Web of Science® Google Scholar
Dramsi, S., Trieu-Cuot, P., and Bierne, H. (2005) Sorting sortases: a nomenclature proposal for the various sortases of Gram-positive bacteria. Res Microbiol 156: 289–297.
10.1016/j.resmic.2004.10.011
CAS PubMed Web of Science® Google Scholar
Duesbery, N.S., Webb, C.P., Leppla, S.H., Gordon, V.M., Klimpel, K.R., Copeland, T.D., et al. (1998) Proteolytic inactivation of Map-kinase-kinase by anthrax lethal factor. Science 280: 734–737.
10.1126/science.280.5364.734
CAS PubMed Web of Science® Google Scholar
Elliot, M.A., Karoonuthaisiri, N., Huang, J., Bibb, M.J., Cohen, S.N., Kao, C.M., and Buttner, M.J. (2003) The chaplins: a family of hydrophobic cell-surface proteins involved in aerial mycelium formation in Streptomyces coelicolor. Genes Dev 17: 1727–1740.
10.1101/gad.264403
CAS PubMed Web of Science® Google Scholar
Fellows, P.F., Linscott, M.K., Ivins, B.E., Pitt, M.L., Rossi, C.A., Gibbs, P.H., and Friedlander, A.M. (2001) Efficacy of a human anthrax vaccine in guinea pigs, rabbits, and rhesus macaques against challenge by Bacillus anthracis isolates of diverse geographical origin. Vaccine 19: 3241–3247.
10.1016/S0264-410X(01)00021-4
CAS PubMed Web of Science® Google Scholar
Gaspar, A.H., Marraffini, L.A., Glass, E.M., DeBord, K.L., Ton-That, H., and Schneewind, O. (2005) Bacillus anthracis sortase A (SrtA) anchors LPXTG motif-containing surface proteins to the cell wall envelope. J Bacteriol 187: 4646–4655.
10.1128/JB.187.13.4646-4655.2005
CAS PubMed Web of Science® Google Scholar
Hodgson, D.A. (2000) Primary metabolism and its control in streptomycetes: a most unusual group of bacteria. Adv Microb Physiol 42: 47–238.
10.1016/S0065-2911(00)42003-5
CAS PubMed Web of Science® Google Scholar
Hopp, T.P., Prickett, K.S., Price, V.L., Libby, R.T., March, C.J., Cerretti, P., et al. (1988) A short polypeptide marker sequence useful for recombinant protein identification and purification. Biotechnology 6: 1204–1210.
10.1038/nbt1088-1204
CAS Web of Science® Google Scholar
Hu, H., Sa, Q., Koehler, T.M., Aronson, A.I., and Zhou, D. (2006) Inactivation of Bacillus anthracis spores in murine primary macrophages. Cell Microbiol 8: 1634–1642.
10.1111/j.1462-5822.2006.00738.x
CAS PubMed Web of Science® Google Scholar
Ivins, B.E., Fellows, P.F., and Nelson, G.O. (1994) Efficacy of a standard human anthrax vaccine against Bacillus anthracis spore challenge in guinea-pigs. Vaccine 12: 872–874.
10.1016/0264-410X(94)90027-2
PubMed Web of Science® Google Scholar
Jensen, G.B., Hensen, B.M., Eilenberg, J., and Mahillon, J. (2003) The hidden lifestyles of Bacillus cereus and relatives. Environ Microbiol 5: 631–640.
10.1046/j.1462-2920.2003.00461.x
CAS PubMed Web of Science® Google Scholar
Kaltwasser, M., Wiegert, T., and Schumann, W. (2002) Construction and application of epitope- and green fluorescent protein-tagging integration vectors for Bacillus subtilis. Appl Environ Microbiol 68: 2624–2628.
10.1128/AEM.68.5.2624-2628.2002
CAS PubMed Web of Science® Google Scholar
Karow, M.L., Glaser, P., and Piggot, P.J. (1995) Identification of a gene, spoIIR, that links the activation of sigma E to the transcriptional activity of sigma F during sporulation in Bacillus subtilis. Proc Natl Acad Sci USA 92: 2012–2016.
10.1073/pnas.92.6.2012
CAS PubMed Web of Science® Google Scholar
Kim, H.S., Sherman, D., Johnson, F., and Aronson, A.I. (2004) Characterization of a major Bacillus anthracis spore coat protein and its role in spore inactivation. J Bacteriol 186: 2413–2417.
10.1128/JB.186.8.2413-2417.2004
CAS PubMed Web of Science® Google Scholar
Kim, H.U., and Goepfert, J.M. (1974) A sporulation medium for Bacillus anthracis. J Appl Bacteriol 37: 265–267.
10.1111/j.1365-2672.1974.tb00438.x
CAS PubMed Web of Science® Google Scholar
Klimpel, K.R., Arora, N., and Leppla, S.H. (1994) Anthrax toxin lethal factor contains a zinc metalloprotease consensus sequence which is required for lethal toxin activity. Mol Microbiol 13: 1093–1100.
10.1111/j.1365-2958.1994.tb00500.x
CAS PubMed Web of Science® Google Scholar
Koch, R. (1876) Die Ätiologie der Milzbrand-Krankheit, begründet auf die Entwicklungsgeschichte des Bacillus anthracis. Beiträge Zur Biologie der Pflanzen 2: 277–310.
Google Scholar
Leppla, S.H. (1982) Anthrax toxin edema factor: a bacterial adenylate cyclase that increases cyclin AMP concentrations in eukaryotic cells. Proc Natl Acad Sci USA 79: 3162–3166.
10.1073/pnas.79.10.3162
CAS PubMed Web of Science® Google Scholar
Leppla, S.H. (2000) Anthrax toxin. In Bacterial Protein Toxins. K. Aktories, and I. Just (eds). Berlin: Springer, pp. 445–472.
10.1007/978-3-662-05971-5_19
Google Scholar
Liu, H., Bergman, N.H., Thomason, B., Shallom, S., Hazen, A., Crossno, J., et al. (2004) Formation and composition of the Bacillus anthracis endospore. J Bacteriol 186: 164–178.
10.1128/JB.186.1.164-178.2004
CAS PubMed Web of Science® Google Scholar
Londono-Vallejo, J.A., and Stragier, P. (1995) Cell–cell signaling pathway activating a developmental transcription factor in Bacillus subtilis. Genes Dev 9: 503–508.
10.1101/gad.9.4.503
CAS PubMed Web of Science® Google Scholar
Losick, R., and Pero, J. (1981) Cascades of sigma factors. Cell 25: 582–584.
10.1016/0092-8674(81)90164-1
CAS PubMed Web of Science® Google Scholar
Losick, R., and Stragier, P. (1992) Criss-cross regulation of cell-type specific gene expression during development in B. subtilis. Nature 355: 601–604.
10.1038/355601a0
CAS PubMed Web of Science® Google Scholar
Low, L.Y., Yang, C., Perego, M., Osterman, A., and Liddington, R.C. (2005) Structure and lytic activity of a Bacillus anthracis prophage endolysin. J Biol Chem 280: 35433–35439.
10.1074/jbc.M502723200
CAS PubMed Web of Science® Google Scholar
McDevitt, D., Francois, P., Vaudaux, P., and Foster, T.J. (1994) Molecular characterization of the clumping factor (fibrinogen receptor) of Staphylococcus aureus. Mol Microbiol 11: 237–248.
10.1111/j.1365-2958.1994.tb00304.x
CAS PubMed Web of Science® Google Scholar
Maresso, A.W., Chapa, T.J., and Schneewind, O. (2006) Surface protein IsdC and sortase B are required for heme-iron scavenging of Bacillus anthracis. J Bacteriol (in press).
Google Scholar
Marraffini, L.A., DeDent, A.C., and Schneewind, O. (2006) Sortases and the art of anchoring proteins to the envelopes of gram-positive bacteria. Microbiol Mol Biol Rev 70: 192–221.
10.1128/MMBR.70.1.192-221.2006
CAS PubMed Web of Science® Google Scholar
Mazmanian, S.K., Ton-That, H., Su, K., and Schneewind, O. (2002) An iron-regulated sortase enzyme anchors a class of surface protein during Staphylococcus aureus pathogenesis. Proc Natl Acad Sci USA 99: 2293–2298.
10.1073/pnas.032523999
CAS PubMed Web of Science® Google Scholar
Meador-Parton, J., and Popham, D.L. (2000) Structural analysis of Bacillus subtilis spore peptidoglycan during sporulation. J Bacteriol 182: 4491–4499.
10.1128/JB.182.16.4491-4499.2000
CAS PubMed Web of Science® Google Scholar
Milne, J., Furlong, D., Hanna, P.C., Wall, J.S., and Collier, R.J. (1994) Anthrax protective antigen forms oligomers during intoxication of mammalian cells. J Biol Chem 269: 20607–20612.
10.1016/S0021-9258(17)32036-7
CAS PubMed Web of Science® Google Scholar
Milne, J.C., Blanke, S.R., Hanna, P.C., and Collier, R.J. (1995) Protective antigen-binding domain of anthrax lethal factor mediates translocation of a heterologous protein fused to its amino- or carboxy-terminus. Mol Microbiol 15: 661–666.
10.1111/j.1365-2958.1995.tb02375.x
CAS PubMed Web of Science® Google Scholar
Nicholson, W.L., Munakata, N., Horneck, G., Melosh, H.J., and Setlow, P. (2000) Resistance of Bacillus endospores to extreme terrestrial and extraterrestrial environments. Microbiol Mol Biol Rev 64: 548–572.
10.1128/MMBR.64.3.548-572.2000
CAS PubMed Web of Science® Google Scholar
Pogliano, J., Osborne, N., Sharp, M.D., Abanes-De Mello, A., Perez, A., Sun, Y.L., and Pogliano, K. (1999) A vital stain for studying membrane dynamics in bacteria: a novel mechanism controlling septation during Bacillus subtilis sporulation. Mol Microbiol 31: 1149–1159.
10.1046/j.1365-2958.1999.01255.x
CAS PubMed Web of Science® Google Scholar
Read, T.D., Peterson, S.N., Tourasse, N., Baille, L.W., Paulsen, I.T., Nelson, K.E., et al. (2003) The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 423: 81–86.
10.1038/nature01586
CAS PubMed Web of Science® Google Scholar
Reed, L.J., and Muench, H. (1938) A simple method of estimating fifty per cent endpoints. Am J Hyg 27: 493–497.
Google Scholar
Ribot, W.J., Panchal, R.G., Brittingham, K.C., Ruthel, G., Kenny, T.A., Lane, D., et al. (2006) Anthrax lethal toxin impairs innate immune functions of alveolar macrophages and facilitates Bacillus anthracis survival. Infect Immun 74: 5029–5034.
10.1128/IAI.00275-06
CAS PubMed Web of Science® Google Scholar
Robertson, D.L., and Leppla, S.H. (1986) Molecular cloning and expression in Escherichia coli of the lethal factor gene of Bacillus anthracis. Gene 44: 71–78.
10.1016/0378-1119(86)90044-2
CAS PubMed Web of Science® Google Scholar
Roth, N.G., Livery, D.H., and Hodge, H.M. (1955) Influence of oxygen uptake and age of culture on sporulation of Bacillus anthracis and Bacillus globigii. J Bacteriol 69: 455–459.
CAS PubMed Google Scholar
Rubio, A., and Pogliano, K. (2004) Septal localization of forespore membrane proteins during engulfment in Bacillus subtilis. EMBO J 23: 1636–1646.
10.1038/sj.emboj.7600171
CAS PubMed Web of Science® Google Scholar
Ruthel, G., Ribot, W.J., Bavari, S., and Hoover, T. (2004) Time-lapse confocal imaging of development of Bacillus anthracis in macrophages. J Infect Dis 189: 1313–1316.
10.1086/382656
PubMed Web of Science® Google Scholar
Schaeffer, A.B., and Fulton, M. (1933) A simplified method of staining endospores. Science 77: 194.
10.1126/science.77.1990.194
CAS PubMed Google Scholar
Schmidt, R., Margolis, P., Duncan, L., Coppolecchia, R., Moran, C.P.J., and Losick, R. (1990) Control of developmental transcription factor sigma F by sporulation regulatory proteins SpoIIAA and SpoIIAB in Bacillus subtilis. Proc Nat Acad Sci USA 87: 9221–9225.
10.1073/pnas.87.23.9221
CAS PubMed Web of Science® Google Scholar
Schneewind, O., Fowler, A., and Faull, K.F. (1995) Structure of the cell wall anchor of surface proteins in Staphylococcus aureus. Science 268: 103–106.
10.1126/science.7701329
CAS PubMed Web of Science® Google Scholar
Serrano, M., Corte, L., Opdyke, J., Moran, C.P.J., and Henriques, A.O. (2003) Expression of spoIIIJ in the prespore is sufficient for activation of sigma G and for sporulation in Bacillus subtilis. J Bacteriol 185: 3905–3917.
CAS Google Scholar
Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., and Tsien, R.Y. (2004) Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat Biotechnol 22: 1567–1572.
10.1038/nbt1037
CAS PubMed Web of Science® Google Scholar
Smith, D.J., Maggio, E.T., and Kenyon, G.L. (1975) Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes. Biochemistry 14: 764–771.
Google Scholar
Smith, H., Keppie, J., and Stanley, J.L. (1955) The chemical basis of the virulence of Bacillus anthracis. V. The specific toxin produced by Bacillus anthracis in vivo. Brit J Exp Pathol 36: 460–472.
CAS PubMed Web of Science® Google Scholar
Sterne, M. (1937) Avirulent anthrax vaccine. Onderstepoort J Vet Sci Anim Ind 21: 41–43.
Google Scholar
Ton-That, H., and Schneewind, O. (1999) Anchor structure of staphylococcal surface proteins. IV. Inhibitors of the cell wall sorting reaction. J Biol Chem 274: 24316–24320.
10.1074/jbc.274.34.24316
CAS PubMed Web of Science® Google Scholar
Ton-That, H., Liu, G., Mazmanian, S.K., Faull, K.F., and Schneewind, O. (1999) Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif. Proc Natl Acad Sci USA 96: 12424–12429.
10.1073/pnas.96.22.12424
CAS PubMed Web of Science® Google Scholar
Toschkoff, A., and Veljanov, D. (1970) Sporulation and virulence of Bacillus anthracis in opened and unopened animal carcasses. Arch Exp Veterinarmed 24: 1153–1160.
CAS PubMed Google Scholar
Vodkin, M.H., and Leppla, S.H. (1983) Cloning of the protective antigen gene of Bacillus anthracis. Cell 34: 693–697.
10.1016/0092-8674(83)90402-6
CAS PubMed Web of Science® Google Scholar
Wang, S.T., Setlow, B., Conlon, E.M., Lyon, J.L., Imamura, D., Sato, T., et al. (2006) The forespore line of gene expression in Bacillus subtilis. J Mol Biol 358: 16–37.
10.1016/j.jmb.2006.01.059
CAS PubMed Web of Science® Google Scholar
Welkos, S.L., Trotter, R.W., Becker, D.M., and Nelson, G.O. (1989) Resistance to the Sterne strain of B. anthracis: phagocytic cell responses of resistant and susceptible mice. Microb Pathog 7: 15–35.
10.1016/0882-4010(89)90108-3
PubMed Web of Science® Google Scholar
Xu, Y., Liang, X., Chen, Y., Koehler, T.M., and Hook, M. (2004) Identification and biochemical characterization of two novel collagen binding MSCRAMMS of Bacillus anthracis. J Biol Chem 279: 51760–51768.
10.1074/jbc.M406417200
CAS PubMed Web of Science® Google Scholar

Citing Literature

Volume62, Issue5

December 2006

Pages 1402-1417

Targeting proteins to the cell wall of sporulating Bacillus anthracis

Summary

Introduction

Results