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Infect Immun. Jul 2003; 71(7): 3914–3919.
PMCID: PMC161987

Detection of a luxS-Signaling Molecule in Bacillus anthracis

Abstract

Quorum-sensing regulation of density-dependent genes has been described for numerous bacterial species. The partially annotated genome sequence of Bacillus anthracis contains an open reading frame (BA5047) predicted to encode an ortholog of luxS, required for synthesis of the quorum-sensing signaling molecule autoinducer-2 (AI-2). To determine whether B. anthracis produces AI-2, the Vibrio harveyi luminescence bioassay was used. Cell-free conditioned media from vaccine (Sterne) strain 34F2 induced luminescence in V. harveyi reporter strain BB170, indicating its production of AI-2. Cloned BA5047, expressed in Escherichia coli DH5α cells, restored AI-2 activity to these cells. To evaluate whether BA5047 is essential for AI-2 synthesis, it was deleted through allelic exchange with marker rescue; the resulting mutant had no functional luxS activity and had reduced growth in vitro. In the wild-type strain, AI-2 activity was greatest during the exponential phase of growth. In total, these data indicate that BA5047 is a functional luxS ortholog in B. anthracis necessary for growth-phase-specific AI-2 expression. Thus, B. anthracis may utilize extracellular signaling molecules to regulate density-dependent gene expression.

Quorum sensing is the regulation of bacterial gene expression in response to changes in cell density (13). Bacteria that utilize quorum-sensing signaling pathways synthesize signaling molecules (autoinducers [AI]) which have been found in nature as N-homoserine lactones (8) or small peptides (13). AI levels are directly proportional to the size of the bacterial population (7), and at threshold levels detectable by bacterial cell receptors, AI binding alters bacterial gene expression (13). Quorum-sensing-based regulation of gene expression is critical for the pathogenesis of clinically important bacterial infections, such as those due to Pseudomonas aeruginosa in patients with cystic fibrosis (4) or to Vibrio cholerae (14).

Quorum sensing has been well characterized in Vibrio harveyi, a bioluminescent bacterium that freely lives in the ocean floor sediment or on the exterior of fish (17). The luminescence genes are expressed only when the V. harveyi populations are at high cell density under the control of the lux quorum-sensing system (13). The luxCDABE operon is regulated by two-component systems that are stimulated by the AI ligands, AI-1 (acyl-homoserine lactone [AHL]) and AI-2 (13). Synthesis of AI-1 requires luxLM. AI-1 diffuses freely through the cell wall into the extracellular milieu, and when sufficient quantities are recognized by its sensor histidine kinase, luxN, a hybrid two-component system-signaling cascade is initiated (13). The lux cascade also is regulated by another AI molecule, AI-2, which is predicted to be a furanosyl borate diester and is synthesized by the product of luxS (2). The luxS product converts S-ribosylhomocysteine to 4,5-dihydroxyl-2,3-pentanedione, catalyzing AI-2 formation (2). V. harveyi strain BB170, in which luxN is mutated, is unable to detect AI-1 molecules and may be used to detect AI-2 or AI-2-like molecules in its milieu (1).

Bacillus anthracis, a gram-positive, nonmotile, spore-forming bacterium, is the etiological agent of anthrax (15). Spores from B. anthracis are extremely resistant to a wide range of adverse environmental conditions, such as heat, UV and ionizing radiation, and chemical agents (15). With the emergence of B. anthracis spores as a weapon of terrorism (11), it is essential to develop new vaccines to prevent and new therapies to control B. anthracis infections. In the present report we demonstrate that B. anthracis possesses a luxS ortholog and synthesizes a functional AI-2 molecule and that a luxS mutant has slowed growth compared to that of a wild-type strain. These observations suggest modalities for both prevention and treatment of anthrax.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

B. anthracis vaccine strain 34F2 (Colorado Serum Company, Denver, Colo.), a derivative of the Sterne strain (21) (Table (Table1),1), was routinely grown in brain heart infusion broth (BHI) at 37°C. E. coli strain DH5α was routinely grown in Luria-Bertani broth at 37°C. Ampicillin (50 μg/ml) was added for cultivation of DH5α strains harboring recombinant plasmids. V. harveyi strain BB170, kindly provided by Bonnie Bassler (Princeton University, Princeton, N.J.), was routinely grown in Auto-Inducer Bioassay medium (AB) (22) at 30°C.

TABLE 1.
Plasmids and strains used in this study

Generation of cell-free culture medium and V. harveyi bioluminescence assays.

B. anthracis and E. coli strains were grown overnight with aeration at 37°C. Cell-free conditioned culture medium (CFM) was prepared by centrifuging cultures at 8,000 × g and passing the medium through a 0.2-μm-pore-size Acrodisc syringe filter (Gelman Laboratory). CFM preparations were stored at −20°C until studied. CFM from V. harveyi strain BB170 was prepared in the same manner, except that cultures were grown at 30°C. V. harveyi bioluminescence assays were performed essentially as described by Surette and Bassler (22). Briefly, V. harveyi strain BB170 was grown at 30°C with aeration for 16 h, cultures were diluted 1:10,000 in fresh AB broth, and then 10% CFM from the bacterial cells to be tested was added. Aliquots of 1.0 ml were taken 2 and 4 h after CFM was added, and bioluminescence was measured and expressed as relative light units (RLU) by using a luminometer.

Construction of B. anthracis ΔluxS strain.

To construct a luxS mutant, 1.18-kb and 989-bp fragments flanking BA5047 were amplified by using oligonucleotides BAluxSKOF1 (5′-GACTCAGTAACAGAACGTCGG-3′), BAluxSKOR1 (5′-CGCAATCTCTTACATAAGGTG-3′), BAluxSKOF2 (5′-CACATGTGGTCAAGCGAAG-3′), and BAluxSKOR2 (5′-GCCACATCATATCCAGTATTCG-3′). The PCR-amplified products were purified by using a Qiagen PCR purification kit and subsequently were digested with HindIII. Digest fragments were cloned into pGEM-T Easy and were screened by PCR with primers BAluxSKOF1 and BAluxSKOR2; the plasmid with the correct insert is designated pMJ301. pMJ301 was digested with HindIII and aphA, conferring kanamycin resistance, and was introduced into pMJ301 to create pMJ301K (9). pMJ301K was digested with EcoRI, releasing the insert region, which was cloned into a pUC19 derivative (pUS19) with spectinomycin resistance to create pMJ301KS. Since methylation inhibits transformation into B. anthracis, pMJ301KS was cloned into dam-defective E. coli strain SCS110. Purified pMJ301KS from SCS110 was electroporated into B. anthracis strain 34F2, and colonies were selected for Kanr and Sper. Transformants were picked on medium containing 50 μg of kanamycin/ml and 100 μg of spectinomycin/ml and then were subcultured daily in the absence of antibiotics at 37° with aeration for 15 days. Individual colonies were subsequently screened to identify clones that were both Kanr and Spes. Clones with the correct antibiotic phenotype were confirmed by PCR to have allelic exchange of aphA in the luxS locus by using oligonucleotides SterneF (5′-GCAAATTGAAAACGACTCAG-3′) and SterneR (5′-GTATGCTTATAAACATTCCGTCG-3′), with HindIII digestion of the PCR products.

Construction and screening for pMJ501.

Chromosomal DNA of B. anthracis strain 34F2 was purified by using the Wizard Genomic DNA Purification kit (Promega, Madison, Wis.) and was used as template for PCR amplification of open reading frame (ORF) BA5047. The oligonucleotides used were designated BAluxSF1 (5′-ATGCCATCAGTAGAAAGCTTTG-3′) and BAluxSR2 (5′-CCAAATACTTTCTCAAGTTCATC-3′). In the PCR, DNA was denatured for 1 min at 94°C, with annealing for 1 min at 51°C and extension for 1 min at 72°C. The amplified product was cloned into pGEM-T Easy, yielding pMJ501, which then was transformed into E. coli strain DH5α with selection for ampicillin resistance. The insert from pMJ501 was subjected to sequence analysis by using vector primers T7F and SP6R to ensure that no nucleotide errors had been introduced in the cloning process. E. coli DH5α cells also were transformed with pGEM-T Easy alone for use as a control.

Genomic analysis.

LuxS protein sequences were retrieved from the National Center for Biotechnology Information database, and alignments were created by using ClustalW (23). Phylograms based on amino acid alignments were generated by using Paup 4.0b8 (Sinauer Associates, Sunderland, Mass.) with generation of 1,000 replicate trees by using a full heuristic search (5).

Growth phase regulation of AI-2 synthesis in B. anthracis.

Overnight cultures of B. anthracis strain 34F2 were diluted to an optical density at 600 nm (OD600) of 0.03 in 50 ml of BHI and were grown at 37°C with aeration. Every 60 min, ODs of the cultures were measured by reading 1-ml aliquots with a Beckman DU7400 spectrophotometer. At sequential intervals, quantitative cultures were performed to determine bacterial CFU. From these same aliquots CFMs were prepared for use in the bioluminescence AI-2 reporter assay. All assays were repeated in triplicate. In separate experiments B. anthracis strains 34F2 and 34F2ΔluxS were grown overnight at 37°C with aeration. Cultures were used to inoculate fresh media to an OD600 of 0.03 and then were grown at 37°C with aeration. OD was measured over a 24-h period, since this may be more accurate than cell count due to chaining in B. anthracis cultures (16).

RESULTS

Identification and organization of the B. anthracis luxS locus.

The unfinished genomic sequence of the Ames strain of B. anthracis has been made publicly available by The Institute for Genomic Research (www.TIGR.org). By using the nucleotide sequence of the 471-bp Bacillus subtilis luxS gene as a template, the partially annotated B. anthracis genome was subjected to BLASTN search, which revealed a 474-bp ORF, BA5047, with 72% similarity to luxS (also known as ytjB) from B. subtilis. To further characterize the putative B. anthracis luxS locus, flanking nucleotide sequences were submitted for BLASTN analysis. Sequence analysis of the region upstream of the B. anthracis luxS ortholog revealed a high level of conservation in which B. subtilis genes ytjA and ytiB had homologs with nucleotide similarities of 70 and 67%, respectively. However, the region downstream of the B. anthracis luxS ortholog showed substantial variation compared to B. subtilis. Only one proximate downstream B. subtilis gene, ytkD, had an ortholog in B. anthracis. Immediately downstream of BA5047 are two ORFs (BA5045 and BA5046) of 201 and 231 bp, respectively, with no significant homologies in GenBank. The orientations of the flanking ORFs indicate that BA5047 is in a monocistronic operon (Fig. (Fig.1)1) (18).

FIG. 1.
Schematic of B. anthracis chromosomal organization in the region of the putative luxS homolog. The B. subtilis nucleotide sequence was used to identify a putative luxS homolog in the B. anthracis genome. BLASTN analysis revealed an ORF (BA5047) in the ...

Characterization of the B. anthracis luxS ortholog.

Further data suggesting that BA5047 may be a functional luxS ortholog are provided by an alignment of the translated sequence with 17 other LuxS protein sequences. Although size variations exist in the luxS products, conserved regions essential for function across prokaryotic genera have been defined (13). Alignment of protein sequences from 17 known luxS orthologs with BA5047 reveal a number of conserved amino acids, including those hypothesized to be essential for LuxS enzymatic activity (10). These data provide evidence that B. anthracis ORF BA5047 encodes a LuxS protein with function. Phylogenetic analysis was done to further characterize the B. anthracis luxS ortholog (Fig. (Fig.2).2). As expected, the phylogram revealed that the B. anthracis luxS translated product is most closely related to the luxS products of B. subtilis (10) and B. halodurans; the high bootstrap values indicate that the analysis is robust. Interestingly, the Helicobacter pylori luxS product (6) was found to be more closely related to Staphylococcus aureus (24) than to Campylobacter jejuni (3), suggesting horizontal gene transfer.

FIG. 2.
Phylogenetic analysis of translated products of luxS orthologs from 17 bacterial species and B. anthracis ORF BA5047. Sequences were aligned by using the GCG Pileup program and were subjected to phylogenetic analysis by using PAUP 4.0b4a. Bootstrap values ...

Synthesis of a functional AI-2 molecule by B. anthracis cells.

Utilizing the V. harveyi AI-2 reporter assay, liquid cultures of B. anthracis vaccine (Sterne) strain 34F2 were examined to determine whether B. anthracis cells synthesize an AI-2 or AI-2-like molecule. The AI-2 assay utilizes a deficiency in the AI-1 sensor in V. harveyi strain BB170 (22). Without the luxN AI-1-encoded sensor, strain BB170 can only exhibit bioluminescence in response to AI-2 or an AI-2-like molecule. Growing a culture of strain BB170 overnight and then diluting it 1:10,000 (to yield low cell density) reduces the level of endogenous AI-2 below the threshold required for luminescence. In this experimental system the addition of exogenous AI-2 from bacteria possessing luxS function can restore the bioluminescence phenotype of the BB170 cells (22). As a negative control the V. harveyi reporter strain BB170 was incubated with sterile CFM alone; as a positive control CFM from a high-density culture of strain BB170 also was used (Fig. (Fig.3).3). Addition of sterile CFM to cells of BB170 served as the standard for baseline in luminescence, whereas as expected, addition of CFM from the high-density BB170 culture induced greater than 100-fold increases in luminescence. In multiple experiments, CFM from B. anthracis strain 34F2 had activity similar to that of the positive control, with substantial increases in luminescence compared to that of the negative control (Fig. (Fig.3).3). The results of these experiments indicate that B. anthracis synthesizes AI-2 or an AI-2-like molecule that is involved in the lux quorum-sensing system.

FIG. 3.
Induction of bioluminescence in V. harveyi reporter strain by CFM from B. anthracis cells. V. harveyi strain BB170 is deficient in the AI-1 sensor encoded by luxN, and thus upregulates only the expression of the lux operon (measured as RLU) when AI-2 ...

Evidence that BA5047 encodes a functional luxS.

To determine whether BA5047 is the B. anthracis ORF responsible for synthesis of AI-2 or an AI-2-like molecule, we took advantage of the inability of E. coli strain DH5α to synthesize a functional AI-2 molecule (6). The B. anthracis luxS ortholog (BA5047) was amplified by PCR and was cloned into the E. coli shuttle vector pGEM-T Easy to create pMJ501. Only the ORF was cloned into pGEM-T Easy, into a site downstream of the vector's isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter. CFM from high-density cultures of DH5α containing vector pMJ501 were induced with IPTG and then were screened for the synthesis of AI-2, as measured in the V. harveyi bioluminescence assay (Fig. (Fig.4).4). As negative controls, the reporter strain BB170 was incubated with sterile CFM alone or with CFM from high-density IPTG-induced cell cultures of E. coli strain DH5α without vector or containing pGEM-T Easy with no insert. As positive controls, CFM was used from high-density cultures of strain BB170 and B. anthracis strain 34F2. As previously shown, when sterile CFM alone was used as the baseline for bioluminescence, CFM from high-density cultures of strain BB170 and B. anthracis 34F2 induced substantial bioluminescence (Fig. (Fig.4).4). As expected, no bioluminescence was induced by CFM from cultures of DH5α or from DH5α containing pGEM-T Easy without insert. In contrast, CFM from DH5α containing pMJ501 induced a high level of bioluminescence, greater than that induced by CFM from the positive controls. Compared to the control E. coli CFMs, there was nearly a 1,000-fold mean increase in induction of bioluminescence by pMJ501 (Fig. (Fig.44).

FIG. 4.
Induction of bioluminescence in the V. harveyi reporter strain by cloned BA5047 in E. coli. V. harveyi strain BB170 upregulates only the expression of bioluminescence when AI-2 or AI-2-like molecules are present in its milieu, as described in the legend ...

B. anthracis 34F2ΔluxS has a defect in AI-2 activity.

To analyze the effect of AI-2 signaling in B. anthracis, we created a ΔluxS mutant in strain 34F2 by replacement of the luxS homolog with a kanamycin resistance cassette (see Materials and Methods). After electroporation of the wild-type strain with the mutated locus on pMJ301KS, kanamycin-resistant transformants were serially passed for 15 days in vitro to select for a double-crossover event. Screening of one such Kanr Spes transformant by PCR showed the expected products (Fig. (Fig.5),5), indicating the proper construction. Additionally, in lane 3 of Fig. Fig.5,5, the ~0.4-kb band corresponds to a fragment of luxS amplified from genomic DNA from the wild-type strain. To determine the effect of the mutation on AI-2 synthesis, we utilized the V. harveyi bioassay as described above (Fig. (Fig.3).3). Compared to the baseline level of AI-2 activity in reporter strain BB170, CFM from a high-density culture of 34F2ΔluxS had no additional AI-2 activity. The data collected provided evidence that luxS is necessary for AI-2 synthesis in B. anthracis strain 34F2 (Fig. (Fig.33).

FIG. 5.
PCR confirmation of the creation of B. anthracis 34F2 ΔluxS. Primers flanking the regions of recombination were used to confirm the construction of 34F2ΔluxS. The PCR products were from chromosomal DNA from wild-type strain 34F2 (lane ...

Growth defect in B. anthracis 34F2ΔluxS.

When cultured in liquid medium, B. anthracis 34F2ΔluxS exhibits noticeable growth defects compared to wild-type B. anthracis 34F2 (Fig. (Fig.6A).6A). As determined by cell density, 34F2ΔluxS has a brief delay (approximately 30 to 60 min) in the transition between lag and early exponential phase compared to those for wild-type 34F2. Subsequently, exponential growths for the wild-type and mutant strains are parallel, but the mutant enters into stationary phase at a much lower cell density. Thus, under the conditions tested AI-2 function appears necessary for full B. anthracis growth in vitro.

FIG. 6.
Growth rate and AI-2 production of B. anthracis strains 34F2 and 34F2ΔluxS. (A) B. anthracis strains 34F2 and 34F2ΔluxS were grown overnight in BHI medium and were inoculated with fresh BHI medium at an adjusted OD600 of 0.03. Cells of ...

Growth phase dependence of AI-2 synthesis in B. anthracis.

To determine whether B. anthracis synthesis of AI-2 was growth phase dependent, CFMs were collected from the 34F2 cells at various time points in the growth cycle and were used in the V. harveyi bioluminescence assay. The CFMs were diluted in sterile medium to reflect equal numbers of cells and were incubated with strain BB170 (Fig. (Fig.6B).6B). Sterile CFM alone and CFM from 34F2ΔluxS were used as negative controls, and CFM from a high-density culture of V. harveyi strain BB170 was used as a positive control. Analysis of CFM collected from B. anthracis showed that AI-2 is maximally synthesized during the mid-exponential phase of growth and diminishes during stationary phase. CFM collected from a 6-h culture of the wild-type strain did not enhance the growth of 34F2 (data not shown).

DISCUSSION

In this study we confirmed that an ORF (BA5047) in the partially annotated B. anthracis genome possesses extensive homology to the luxS ortholog (ytjB) in B. subtilis (10). Although the B. anthracis locus for BA5047 is not highly conserved compared to that of B. subtilis, the orientations of the ORFs indicate that BA5047 is in a monocistronic operon, which facilitates examination of its function and regulation. In other organisms, luxS appears essential for the synthesis of a quorum-sensing molecule (AI-2) (12), first identified in the marine bacterium V. harveyi (1). Both multiple protein alignments and phylogenetic analyses (Fig. (Fig.2)2) of the B. anthracis luxS ortholog (BA5047) revealed strong evolutionary relationships with those of B. subtilis and B. halodurans, indicating strong conservation of luxS within the genus Bacillus. Phylogenetic analysis of luxS orthologs reveals two major groupings, generally clustering gram-positive and gram-negative species separately (Fig. (Fig.2);2); one exception is H. pylori, providing evidence consistent with horizontal transfer of luxS across genera (Fig. (Fig.2).2). Taken together, the phylogenetic studies and the protein alignments indicating the presence of conserved amino acids confirm that BA5047 encodes a luxS ortholog.

That CFM from strain 34F2 was able to stimulate luminescence in V. harveyi strain BB170 (Fig. (Fig.3)3) indicates that B. anthracis produces AI-2 or an AI-2-like molecule, likely similar in structure to AI-2 from V. harveyi (2). With this evidence we next focused on BA5047, the luxS ortholog. Expression of B. anthracis BA5047 in E. coli strain DH5α demonstrates its central role in synthesis of AI-2 or an AI-2-like molecule (Fig. (Fig.4)4) and suggests the capability of B. anthracis to conduct density-dependent gene expression. Isogenic deletion of luxS (Fig. (Fig.5)5) resulted in an inability of the B. anthracis mutant (34F2ΔluxS) to produce AI-2 or an AI-2-like molecule that could be detected in the V. harveyi bioassay (Fig. (Fig.3).3). Similarly, compared to the wild-type strain the mutant showed delay in the transition from lag to exponential growth phase and entered stationary phase early (Fig. (Fig.6A).6A). In total, the ΔluxS culture grew more slowly and produced fewer cells compared to the wild type.

B. anthracis synthesis of AI-2 or an AI-2-like molecule mediated by luxS thus plays an important role in the regulation of growth. As such, targets of the hypothesized density-dependent gene expression must include genes regulating vegetative growth and cell cycle. If B. anthracis regulates gene expression by means of an AI molecule, as do other pathogens (14), cells might have the ability to suppress virulence gene expression until the total population reaches a threshold density. Suppression of virulence gene expression by quorum sensing could allow B. anthracis to evade immune detection until its population is at a density sufficiently high to overwhelm the host's innate and adaptive defenses. That AI-2 synthesis is maximal during exponential phase growth is consistent with this hypothesis.

This hypothesis suggests that a possible means of treating anthrax could be via inhibitors of AI-2 to downregulate density-dependent gene expression. Recent data has shown that a synthetic furanone, (5Z)-4-bromo-5-(bromethylene)-3-butyl-2(5H)-furanone, has the ability to inhibit AI-2-mediated quorum sensing in E. coli and V. harveyi (19) as well as swarming and biofilm formation by B. subtilis (20). Examination of this or similar molecules could permit ascertainment of the role of AI-2-mediated mechanisms in virulence gene expression of B. anthracis.

Acknowledgments

We thank Bonnie Bassler for providing V. harveyi reagents and Michael Garabedian for instrumentation.

This work was supported in part by RO1 GM 63270 from the National Institutes of Health, by the Medical Research Service of The Department of Veterans Affairs, by the David and Lucile Packard Foundation, and by the Ellison Medical Foundation.

Notes

Editor: W. A. Petri, Jr.

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