DNA isolation and manipulation

Preparation of plasmid DNA from E. coli, transformation of E. coli, and recombinant DNA techniques were carried out by standard procedures (Sambrook & Russell, 2001). E. coli XL2-blue and SCS110 competent cells were purchased from Stratagene and E. coli TOP10 competent cells from Invitrogen. Recombinant plasmid construction was carried out in E. coli XL2-Blue or TOP10. Plasmid DNA from B. anthracis/B. cereus was isolated according to the QIAGEN Plasmid Protocol: Purification of Plasmid DNA from B. subtilis (QIAGEN Inc., Valencia, CA). Chromosomal DNA from B. anthracis/B. cereus was isolated with the Wizard Genomic Purification Kit (Promega, Madison, WI) in accordance to the protocol for isolation of genomic DNA from Gram-positive bacteria (Promega). B. anthracis/B. cereus was electroporated with unmethylated plasmid DNA isolated from E. coli SCS110. Electroporation-competent cells of B. cereus were prepared using the same method as previously described for B. anthracis (Park & Leppla, 2000).

Restriction enzymes, T4 ligase, T4 DNA polymerase and alkaline phosphatase were purchased from MBI Fermentas or New England Biolabs. Taq polymerase kits were purchased from TaKaRa Shuzo or Invitrogen/Life Technologies. Ready-To-Go PCR Beads (Amersham Biosciences) were used for DNA rearrangement analysis. For routine PCR analysis a single colony was suspended in 200 μl of TE buffer, pH 8.0, heated to 95°C for 60 sec and then cooled to room temperature. Cellular debris was removed by centrifugation at 15,000g for 10 min. One microliter of the lysate contained sufficient template to support a PCR reaction with the PCR beads. The GeneRuler DNA ladder mix (MBI Fermentas) or the 1 kb plus ladder (Invitrogen) was used for determination of DNA fragment length. All constructs were verified by DNA sequencing and/or restriction enzyme digestion. All plasmids used in this study and their relevant characteristics are listed in Table 1. Oligonucleotide primer sequences are listed in Table 2.

TABLE 1

Plasmids and strains used in this study

Plasmid or strain	Relevant characteristic(s)	Source or reference
Plasmid
pAE5	Encodes B. cereus 569 plcR under control of B. anthracis protective antigen gene (pagA) promoter; Apr in E. coli; Kmr in B. anthracis	(Pomerantsev et al., 2003)
pAE51	pAE5 with stop codon in plcR truncating six C-terminal amino acids	This work
pUTE29-plcR-papR	B. cereus 569 plcR-papR DNA fragment inserted into pUTE29; Apr in E. coli; Tcr in B. anthracis	(Pomerantsev et al., 2004)
pFP12	pUTE29-plcR fusion to papR	(Pomerantsev et al., 2004)
pUTE29-papR	B. cereus 569 papR DNA fragment inserted into pUTE29	(Pomerantsev et al., 2004)
p102	pUTE29-nprB-plcR-papR	This work
p182	pUTE29-nprB-plcR fusion to papR	This work
pUTE29-nprB	P182 without BstZ17I-PstI fragment encoding fused PlcR-PapR	This work
pJRS312	pUC18 carrying an Ω element with spectinomycin resistance marker (Ω-sp)	J.R. Scott
p102Sp	p102 with Ω-sp cassette flanked by two directly repeated loxP and inserted into BglII-site of nprB	This work
p182Sp	p182 with Ω-sp cassette flanked by two directly repeated loxP and inserted into BglII-site of nprB	This work
pHY304	Contains strongly temperature-sensitive replicon for both E. coli and gram-positive bacteria; Emr in both E. coli and B. anthracis	(Pritzlaff et al., 2001)
pHU304	Large PvuII-fragment of pUC18 combined with large Ecl136II-fragment of pHY304	This work
pHU102	PvuII fragment of p102 containing nprB- plcR-papR gene cluster inserted into BstZ17I-site of pHU304	This work
pHU102Sp	PvuII fragment of p102Sp containing nprB(loxP-Ω-sp-loxP) - plcR-papR gene cluster inserted into BstZ17I-site of pHU304	This work
pCrePA	pHY304 with Cre recombinase gene under control of pagA promoter	(Pomerantsev et al., 2006)
pDG148-Stu	Gram-Positive-E. coli shuttle vector featuring ligation-independent cloning and inducible expression; Apr in E. coli; Kmr in B. anthracis	(Joseph et al., 2001)
pB148	pDG148-Stu carrying B. cereus 569 nprB gene under control of Pspac promoter	This work
pB148Lac-	pB148 without lacI repressor gene	This work
Strain
B. cereus
569	Wild strain, produces extracellular PC-PLC	(Reddy et al., 1987)
541	569; intact plcR lacks a promoter; Emr; does not produce extracellular PC-PLC	(Pomerantsev et al., 2003)
537	569; nprB knockout, Spr; nprB contains Ω-sp cassette flanked by two directly repeated loxP; demonstrates very low PC-PLC activity	This work
536	569; nprB knockout containing one loxP site; demonstrates very low PC-PLC activity	This work
535	569; papR replaced by loxP site; demonstrates very low PC-PLC activity	This work
B. anthracis
SdT	Sterne 34F2 cured of pXO1; therefore pXO1- pXO2-	(Ivins et al., 1986)
SdT2	SdT electroporated with pAE5; Kmr; produces intracellular PlcR; does not produce a halo when grown on LB agar with lecithin	(Pomerantsev et al., 2003)
SdT251	SdT electroporated with pAE51; Kmr; produces intracellular PlcR with truncated six C-terminal amino acids; does not produce a halo when grown on LB agar with lecithin	This work
SdT12	SdT electroporated with pFP12; Tcr; produces intracellular PlcR-PapR fused protein; demonstrates does not produce a halo when grown on LB agar with lecithin	(Pomerantsev et al., 2004)
Ames 33	pXO1- pXO2- Ames derivative strain	(Pomerantsev et al., 2003)
BH450	Ames 33 ba_0599 protease knockout, spo0A knockout	(Pomerantsev et al., 2006)

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DNA cloning and sequencing

The nprB region from B. cereus 569 chromosomal DNA was obtained as a 2.21-kb DNA fragment by PCR using primers Npr-f and Npr-r (Table 2). The PCR fragment restricted with BstZ17I and BamHI was isolated from SeaKem agarose gel (Rockland, ME) and cloned into the corresponding sites of the pUTE29-plcR-papR or pFP12 plasmid (Pomerantsev et al., 2004) using E. coli XL2-Blue. The resulting p102 (pUTE29-nprB-plcR-papR) or p182 (pFP12-nprB-plcR-papR) plasmids were electroporated into B. cereus with selection for tetracycline resistance. The nprB gene was also amplified with primers StuBf and StuBr and introduced into the pDG148-Stu vector under control of the P_spac promoter (Joseph et al., 2001). The resulting pB148 plasmid was electroporated into B. anthracis/B. cereus with selection for kanamycin resistance. The LacI repressor gene in pB148 was eliminated by cleavage with SplI and BamHI, filling of overhangs with T4 DNA polymerase and ligation of the resulting blunt ends with T4 ligase.

The PlcR-expressing plasmid pAE5 containing the B. cereus 569 plcR gene was generated in our previous study by directional cloning of a PCR fragment amplified with primers designated R1 and R2 (Pomerantsev et al., 2003). Here we repeated this cloning using an alternative reverse primer, R3 (Table 2), which deleted a base so as to introduce a terminal codon TGA in place of a methionine codon. The resulting plasmid pAE51 that produces a truncated PlcR protein was introduced into B. anthracis SdT strain to produce B. anthracis SdT251 strain.

Nucleotide sequencing of the cloned fragments and DNA-fragments used for the analysis of mutations was performed by the dideoxy chain-termination technique with a Taq dye primer cycle sequencing kit (Food and Drug Administration Core Facility, National Institutes of Health, Bethesda, MD). The nucleotide sequences encoding the B. cereus 569 nprB gene was previously submitted to Genbank under accession number AY195601 (Pomerantsev et al., 2003). The NCBI BLAST and FASTA programs (http://www.ncbi.nlm.nih.gov/) were used for homology searches in the GenBank and SWISS Protein databases.

Strain construction

Strains, plasmids, and their relevant characteristics are listed in Table 1. All B. cereus strains used here were derived from strain 569. The B. cereus nprB mutant strains 536 and 537 were constructed using Cre/LoxP technology. Briefly, the Ω-spectinomycin (Ω-sp) cassette of the pΩL plasmid (Pomerantsev et al., 2006) was amplified with LoxSB/LoxEB primers to produce a BglII-loxP-Ω-sp-loxP-BglII DNA fragment. The fragment was inserted into the unique BglII-restriction site located in the nprB gene in the p102 plasmid. The PvuII-fragment of the resulting p102Sp plasmid containing the nprBsp-plcR-papR – gene cluster was cloned into the BstZ17I-site of the pHU304 plasmid. pHU304 is a recombinant plasmid made by combining the large PvuII-fragment from pUC18 and the Ecl136II-fragment from pHY304 (Pritzlaff et al., 2001). The resulting pHU102Sp plasmid was electroporated into B. cereus 569 with selection for erythromycin resistance. Transformants were selected at 30°C on BHI agar containing erythromycin. Erythromycin-resistant colonies were transferred to agar containing spectinomycin, incubated at 37°C to limit replication from the pHY304 origin, and again transferred to fresh agar containing spectinomycin. After the third passage at 37°C, colonies were selected and screened for spectinomycin-resistance and erythromycin-sensitivity. Isolates in which a double-crossover recombination event had occurred were confirmed by PCR and phenotype on blood or lecithin agar. Primer Sp5, located inside the Ω-sp cassette, was used as an internal primer; Bc-ext, located downstream of the Npr-r primer, was used as an external primer. The resulting nprB mutant was designated B. cereus 537.

For elimination of spectinomycin resistance, plasmid pCrePA (Pomerantsev et al., 2006) was introduced by electroporation followed by selection for erythromycin resistance at 30°C. Erythromycin-resistant colonies were transferred to antibiotic-free agar and incubated at 37°C to eliminate pCrePA. Colonies were then patched to three replicate agar plates containing erythromycin, spectinomycin, or no antibiotic. The presence of a single loxP site within the nprB gene of the resulting fully antibiotic-sensitive B. cereus mutant strain 536 was confirmed by PCR and/or sequence analysis. Primer pair BseqF/BseqR was used for the final verification.

The B. cereus papR mutant strain 535 was constructed by replacing the papR coding sequence with a loxP site using a Cre/LoxP technology similar to that described above. Primer pair PapRF/PapRR was used for the final verification.

For the trans-complementation experiments, the pB148 and pB148Lac^- plasmids expressing nprB were used for the transformation of B. cereus 536 (nprB^-). Kanamycin-resistant clones that reestablished hemolytic and PC-PLC activities were selected on blood or lecithin-containing agar. The in situ nprB gene replacement was accomplished using plasmid pHU102, which was made by combining the PvuII-fragment of the p102 plasmid containing the nprB-plcR-papR gene cluster and the BstZ17I-fragment of pHU304. The pHU102 plasmid was electroporated into B. cereus 536 (nprB^-) with selection for erythromycin resistance. Transformants were selected at 30°C on lecithin agar containing erythromycin. Colonies that were erythromycin-resistant and formed halos were transferred to lecithin agar without antibiotic and incubated at 37°C to limit replication from the pHY304 origin and encourage the insertion of the pHU102 plasmid into the chromosomal DNA of B. cereus 536 as a single-crossover event. Halo forming colonies were again transferred to fresh lecithin agar without antibiotic and incubated at 37°C. The entire process was repeated until erythromycin sensitive halo-forming isolates in which a double-crossover recombination event had occurred were obtained. The restoration of the parental nprB gene sequence in the fully antibiotic-sensitive strain was confirmed by PCR and sequence analysis.

In further trans-complementation experiments, the pUTE29-papR (Pomerantsev et al., 2004) plasmid was used for the transformation of B. cereus 535(papR^-). Tetracycline-resistant clones that reestablished PC-PLC activity were selected on lecithin-containing agar.

Identification of extracellular B. cereus 569 NprB and its orthologue in B. anthracis

The extracellular proteomes of PlcR-positive B. cereus 569 and B. anthracis SdT12, as well as for the PlcR-negative variants, were determined during the exponential phase (5 h), stationary phase (10 h), and pre-spore growth phase (24 h). Bacteria were grown in LB broth at 37°C. Aliquots of growing cultures were centrifuged for 15 min at 8500 × g. The supernatants were filtered through 0.22-μm (pore size) Millex-GS membrane (Millipore Corp. Bedford, MA) and the proteins were concentrated using Centriprep YM-10 units (Amicon, Inc., Beverly, MA). Samples containing concentrated proteins were focused on pH 3 to 10 IPG strips and separated on 4-12% Bis-Tris NuPAGE gels. The gels were stained with Coomassie Blue. All visible spots were extracted and proteins were identified by mass spectrometry using MALDI-TOF MS (FDA Core Facility). The National Center for Biotechnology Information (NCBI) BLAST and FASTA programs (http://www.ncbi.nlm.nih.gov/) were used for MS peptide homology searches in the GenBank and SWISS Protein databases. Genomic DNA sequences extending 1000 bp upstream of the identified proteins start codons were examined for possible PlcR-binding sites.

NprB purification

Plasmid pB148Lac^- was transformed into the sporulation-negative, protease-deficient B. anthracis BH450 strain (formerly designated MSLL33) (Pomerantsev et al., 2006) with selection for kanamycin resistance. The strain was grown in FA medium (Singh et al., 1989) with kanamycin (50 μg mL^-1) at 37°C until the beginning of the stationary phase. The supernatant was filter sterilized using a Pellicon cassette system (Millipore Corp., Bedford, MA) with Durapore 0.22-mm membranes. Ammonium sulfate was added to obtain 40% saturation and the sample was loaded onto a Fast Flow Phenyl–Sepharose column (Amersham Pharmacia, Piscataway, NJ) pre-equilibrated with buffer containing 1.5 M ammonium sulfate, 10 mM Tris and 0.5 mM EDTA (pH 7.5). The column was washed with the same buffer and NprB was eluted with an ammonium sulfate gradient from 1.5 to 0 M. Fractions were analyzed on casein agar and NprB-containing fractions were collected and dialyzed with Slide-A-Lyzer Dialysis Cassettes (Pierce, Rockford, IL) overnight against 10 mM Tris (pH 7.5). Finally, the NprB was concentrated using Centriprep YM-10 units (Amicon, Inc., Beverly, MA) and stored at −80°C until used. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of NprB. The protein was heated in 1x sample buffer (6x sample buffer contains 0.35 M Tris-HCl, pH 6.8, 10% sodium dodecyl sulfate [SDS], 36% glycerol,0.6 M DTT, and 0.01% bromphenol blue) at 95°C for 5 min and separated on SDS-polyacrylamide (10-20%) gel using the PhastSystem (Amersham Pharmacia, Piscataway, NJ). The N-terminal sequence of NprB was determined by the Edman degradation method, using a gas-phase sequencer at the Research Technologies Branch, NIAID/NIH Twinbrook I Facility, Rockville, MD.

Determination of NprB/PapR cleavage specificity

The PapR propeptide containing 27 C-terminal amino acids was synthesized at the Peptide Synthesis and Analysis Unit of Research Technologies Branch, NIAID/NIH Twinbrook I Facility, Rockville, MD. PapR was incubated with the purified NprB protease (100:1 by mass) in ammonium carbonate buffer, pH 7.5, for 1, 12, or 24 h at 37°C. The hydrolysis products were separated with a C18 reverse phase column and a 5-60% B/40 min gradient (solvent A: 0.1% trifluoroacetic acid/H₂O; solvent B: 0.1% trifluoroacetic acid/20% H₂O/80% acetonitrile) using a Shimadzu VP series HPLC system (Peptide Synthesis and Analysis Unit). Peptides were identified by mass spectrometry (MS) using a Voyager RP BioSpectrometry Workstation (Peptide Synthesis and Analysis Unit).

PlcR/PapR supplementation test

Bacillus strains were analyzed for phosphatidylcholine hydrolysis activity in two ways. Plates containing LB agar supplemented with 0.04% L-α-phosphatidylcholine (lecithin, Sigma-Aldrich) and different concentrations of the corresponding peptide (titrated from 200 to 0.2 μM) were prepared for visual analysis of the halo. C-terminal peptides of 5, 7, 8, and 27 amino acids of B. cereus 569 PapR were synthesized at the Peptide Synthesis and Analysis Unit of Research Technologies Branch, NIAID/NIH Twinbrook I Facility, Rockville, MD. Kanamycin and tetracycline were added to media when appropriate to give final concentrations of 50 and 5 μg mL^-1, respectively. Suspensions of the B. anthracis strains derived from SdT or B. cereus strains were prepared with an A₆₀₀ close to 0.25, and 3 μl of each suspension was inoculated on plates which were then incubated overnight at 37°C. For quantitative determination of the enzymatic activities, synthetic peptides (5 μM) were added to a culture of the B. cereus strains 535 and 536 grown in LB broth at 37°C until stationary phase (OD₆₀₀ 1.5 ± 0.2). Aliquots of growing cultures were centrifuged for 15 min at 8500 × g 1 h after peptide addition. The supernatants were filtered through 0.22-μm (pore size) Millex-GS membrane and PC-PLC assays were performed. In the same way, B. anthracis SdT2 cells were grown in LB broth supplemented with kanamycin (50 μg mL^-1) at 37°C until stationary phase (OD₆₀₀ 1.3 ± 0.2). The culture was then divided and each synthetic peptide was added to individual fractions to final concentrations of 1, 10 and 100 μM. PC-PLC assays were performed 1 h after peptide addition.

Inactivation of B. cereus 569 nprB and papR genes

The nprB gene of B. cereus 569 was disrupted as described in the Materials and Methods section according to the scheme presented in Fig. 2A and verified by PCR in Fig. 2B. The presence of a loxP sequence created a frameshift that terminated translation of NprB in the resulting mutant B. cereus strain 536. The B. cereus nprB mutant strain 536 was examined for phenotypic changes (Fig. 2 B,C,D). Hemolytic activity was completely lost (Fig. 2B, lower panel, lane 2). The Enz Chek Protease Assay Kit and Amplex Red Phosphatidilcholine-Specific Phospholipase C Assay Kit provided convenient ways to assess PlcR/PapR dependent casein protease and PC-PLC activities, respectively. The nprB mutant showed greatly decreased activities both for casein and phosphatidylcholine digestions as compared to its B. cereus 569 parent (Fig. 2C and D, respectively).

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Figure 2

Inactivation of B. cereus 569 nprB gene. (A) Diagram of the genetic organization of the B. cereus 569 mutants, B. cereus 537 and B. cereus 536. Construction of the mutants is described in Materials and Methods. Arrows indicate the position and orientation of the loxP sites. Half-head arrows mark the position and orientation of the primers used for PCR analysis. (B) (Upper panel) The PCR fragments amplified with BseqF/BseqR primers from the chromosomal DNA of B. cereus 569 (lane 1), B. cereus 536 (lane 2), B. cereus 536 with plasmid pB148 (lane 3), B. cereus 536 with plasmid pB148Lac^- (lane 4), B. cereus 536 with plasmid pFP12 (lane 5), B. cereus 536 with plasmid pHU102 integrated into chromosomal DNA as a single crossover event (lane 6), B. cereus 569 NprB-revertant obtained as a result of a double crossover event between pHU102 and B. cereus 536 chromosomal DNA (lane 7). Locations of the primers correspond to those shown in panel A. The upper fragment (398 bp) presents nprB-loxP allele; the lower fragment (302 bp) presents nprB allele. (Lower panel) Ability of B. cereus 569 to hydrolyze fresh blood is appreciably depressed in B. cereus 536. Transformation with pB148, pB148Lac^-, pFP12 or pHU102 restored ability of B. cereus 536 to hydrolyze fresh blood. The locations of the samples correspond to those of the above PCR. (C and D) Casein proteolytic (C) and PC-PLC (D) activities of B. cereus 569, B. cereus 536, and B. cereus 536 (pB148Lac^-) extracellular proteins. The strains were grown at 37°C in LB broth for 10 h with supernatant samples taken every hour. Equal amounts of extracellular protein (2-10 μg for PC- PLC and 10-50 μg for casein proteases analysis) were assayed either by the Red Amplex (PC- PLC) or green-fluorescent BODIPY FL (protease) reagents. Maximum root-mean-square deviations did not exceed 10% for the protease (C) and PC-PLC (D) determinations.

The papR gene of B. cereus 569 was replaced by a loxP site as described in the Materials and Methods according to the scheme presented in Fig. 3A. The resulting B. cereus papR mutant strain 535 showed greatly decreased activity for phosphatidylcholine digestion as compared to its B. cereus 569 parent (Fig. 3B).

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Figure 3

Inactivation of B. cereus 569 papR gene. (A) Diagram of the genetic organization of the B. cereus 569 papR mutant, B. cereus 535. Arrows indicate the position and orientation of plcR, papR and loxP. Half-head arrows mark the position and orientation of the primers used for PCR analysis. (B) (Upper panel) The PCR fragments amplified with PapRF/PapRR primers from the chromosomal DNA of B. cereus 569, B. cereus 535, and B. cereus 535 with plasmid pUTE29-papR. Locations of the primers correspond to those shown in panel A. The upper fragment (624 bp) presents the plcR-papR allele; the lower fragment (550 bp) presents the plcR-loxP allele. (Lower panel) Ability of B. cereus 569 to hydrolyze lecithin is appreciably depressed in B. cereus 535. Transformation with pUTE29-papR restored ability of B. cereus 535 to hydrolyze lecithin. The locations of the samples correspond to those of the above PCR. The strains were grown at 37°C in LB broth for 8 h. Equal amounts of extracellular protein were assayed by Red Amplex (PC-PLC). (C) Complementation of both mutant B. cereus 535 and 536 with plasmid producing NprB (pB148Lac-) restores PC-PLC activity only in B. cereus 536 while supplementation of growth medium with 10 μM of 27-aa C-terminal PapR peptide restores phosphatidylcholine hydrolyzing activity only in B. cereus 535. Bacteria were grown at 37°C in LB broth for 8 h with or without the PapR peptide. Equal amounts of extracellular protein were assayed by Red Amplex (PC-PLC).

We reported previously that the hemolytic and lecithinase activities of a B. cereus 569 plcR mutant were markedly reduced or eliminated as compared to the wild-type parent strain (Pomerantsev et al., 2003). Here we obtained very similar data for the nprB and papR mutants B. cereus 536 and 535, respectively. To confirm that B. cereus 536 phenotypic changes were due to nprB disruption, we complemented the mutation with a native B. cereus 569 nprB gene carried on plasmids pB148 or pB148Lac^- and also restored the gene in the chromosome (see below). The plasmid transformants partially regained the ability to express the hemolytic activity characteristic of the parent B. cereus 569 strain (Fig. 2B, lanes 3,4). Casein protease and PC-PLC activity were also restored in the B. cereus (pB148Lac^-) strain (Fig. 2C and D). In an alternative approach to correct the phenotypic consequences of nprB disruption, the nprB mutant strain was transformed with plasmid pFP12, which contains a translational fusion of the B. cereus PlcR and PapR proteins. The resulting transformant also partially regained the hemolytic activity of the parent strain (Fig. 2B, lane 5). These data are consistent with the previous demonstration that the PlcR-PapR fusion protein activates transcription of PlcR-dependent genes in B. anthracis. In this experiment we found that the fused PlcR-PapR protein can also activate transcription of PlcR-dependent genes in the B. cereus nprB mutant, bypassing the need for extracellular maturation of the PapR protein that appears to depend on the action of NprB. Finally, the hemolytic activity of B. cereus 536 was completely restored by transformation with pHU102 so as to reinsert an intact nprB gene into the original chromosomal location (Fig. 2B, lane 7).

To confirm that the B. cereus 535 phenotypic changes were due to papR replacement with loxP, we complemented the mutation with a native B. cereus 569 papR gene carried on plasmid pUTE29-papR. PC-PLC activity was restored in B. cereus 535(pUTE29-papR) (Fig. 3B). In contrast, no digestion of phosphatidylcholine was found for the B. cereus 535(pB148Lac^-) strain, in which the plasmid expresses nprB, while B. cereus 536(pB148Lac^-) clearly digested phosphatidylcholine in the same conditions (Fig. 3C). On the other hand, supplementation of LB broth with the 27-aa C-terminal PapR peptide induced PC-PLC activity in B. cereus 535 but not in B. cereus 536 (Fig. 3C). This experiment indicates that PapR cannot be processed in the nprB mutant to produce active peptide.

B. cereus and B. anthracis protease identification

The data shown in Fig. 2C demonstrated that the casein proteolytic activity of B. cereus 569 found at early phases of growth results mostly from NprB. This finding indicates that the nprB gene is required in B. cereus 569 for expression of normal casein proteolytic activity level at the early phase because the B. cereus 569 nprB mutant had drastically decreased activity in the same time interval. Later the activities of both strains became similar. We also found that the hemolytic B. anthracis SdT12 strain expressing the fused PlcR/PapR protein demonstrated a similar induction of proteolytic activity at early phases of growth, while the parent B. anthracis SdT did not. Similarly to the B. cereus 569 nprB mutant, the B. anthracis SdT strain revealed casein proteolytic activity later, but at a reduced level compared with the B. anthracis SdT12 strain (Fig. 4). Because the B. anthracis Sterne strain does not have nprB in the vicinity of plcR (Fig. 1A), we then asked what protease is induced by PlcR in B. anthracis SdT12 at the early phase of growth.

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Figure 4

Growth curves (A) and extracellular protein casein proteolytic activity (B) of B. anthracis SdT and B. anthracis SdT12 strains. The growth conditions and activity determinations were performed as was done for B. cereus strains (see description to Fig. 2C).

To identify the proteases produced by the B. cereus and B. anthracis strains being examined here, we used two-dimensional (2D) gel electrophoresis (Fig. 5). In the culture supernatant (secretome) of B. cereus 569 grown for 5 h (corresponding to the peak in Fig. 2C), we identified six proteins, of which only one was a protease - NprB. Expression of four proteins (hemolysin BL, non-hemolytic enterotoxin, NprB, and PC-PLC) was apparently regulated by PlcR because a PlcR-binding site is found upstream of each corresponding gene (data not shown). No PlcR-binding site was found upstream of the genes responsible for the synthesis of either the chitin binding protein or flagellins. Both the chitin binding protein and the flagellin spots were found at the same position on the 2D-gel of B. cereus 536 (Fig. 5, upper right gel). However, spots corresponding to the proteins with PlcR-dependent regulation disappeared, to make way for three proteins with PlcR-independent regulation: N-acetylmuramoyl-L-alanine amidase (BC_5234), hypothetical membrane spanning protein (BC_1726), and cytochrome aa3 quinol oxidasepolypeptide (BC_0698). It is interesting that a protease (BAS2543) of the same M4 neutral protease class as NprB was one of the three proteins found in the 5 h secretome of B. anthracis SdT12 (Fig. 5). Expression of all three proteins containing a PlcR-binding site upstream of the corresponding genes (neutral protease M4, murein endopeptidase and enterotoxin A) was obviously regulated by PlcR because the corresponding secretomes of B. anthracis SdT strain contained only one protein, the S-layer SAP protein (BAS0841), which lacks PlcR regulation. Neither NprB from B. cereus 569 nor neutral protease M4 from B. anthracis SdT12 were found in the secretomes after 10 and 24 h of growth (data not shown).

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Figure 5

Secretome analyses. Two-dimensional electrophoretic separation of proteins secreted by B. cereus 569 and 536 as well as B. anthracis SdT12 and SdT grown in LB broth for 5 h. M_r, molecular mass (in kDa); NL, nonlinear. The numbers mark protein spots which were identified by MALDI-TOF-MS in accordance with the B. cereus 14579 (accession number NC_004722) and B. anthracis Sterne (accession number NC_005945) databases. B. cereus 569: 1 - chitin binding protein (BC_2798); 2, 3 - hemolysin BL lytic and binding components (BC_3104 and BC_3103, respectively); 4, 6, 7 - three isoforms of non-hemolytic enterotoxin corresponding to lytic component L2 (enterotoxin A, BC_1809) and lytic component L1 (enterotoxin B, BC_1810); 5 - neutral protease B (BC_5351); 8 - flagellins A, B, C (BC_1657-1659); 9 -phosphatidylcholine-specific phospholipase C (BC_0670). B. cereus 536: 1- chitin binding protein (BC_2798); 2 - flagellins A, B, C (BC_1657-1659); 3 - cytochrome aa3 quinol oxidasepolypeptide (BC_0698); 4 - hypothetical membrane spanning protein (BC_1726); 5 - N-acetylmuramoyl-L-alanine amidase (BC_5234); B. anthracis SdT12: 1 - murein endopeptidase (BAS1852); 2 - neutral protease M4 (BAS2543); 3,4 - enterotoxin A (BAS1749) ; B. anthracis SdT: 1, 2 - S-layer protein, Sap (BAS0841).

NprB characteristics

NprB of B. cereus 569 (peptidase M04.012 in accordance with the MEROPS peptidase database, http://merops.sanger.ac.uk) is a 583-aa zinc metalloprotease of the M4 family. The M4 family is composed of secreted eubacterial endopeptidases that contain an HEXXH motif (where X is any amino acid), which has been shown in crystallographic studies to form part of the metal-binding site (Gomis-Ruth, 2003). The B. cereus 569 NprB contains a typical 26-aa signal peptide (http://www.cbs.dtu.dk/services/SignalP/) and a Zn²⁺-binding site with the sequence HEFGH. The most similar orthologue of B. cereus 569 NprB is that of B. cereus ATCC 14579 (identities = 574/583 [98%], positives = 578/583 [99%]) (Ivanova et al., 2003). The B. anthracis orthologue BAS2543 (567 aa) is less similar (identities = 32%, positives = 50%) and has a slightly different 29-aa signal peptide and Zn²⁺-binding site, HEFTH (GenBank and SWISS protein Database). Although the B. cereus NprB resembles this and other close relatives over its entire length, it is interesting to note that an extended sequence at the Zn⁺²-binding site, HEFGHAV, exactly matches that of an otherwise more distantly related protease, the anthrax toxin lethal factor (Bragg & Robertson, 1989).

In the course of this work, we also noted that overexpression of NprB is not tolerated in either B. cereus or B. anthracis when PlcR and PapR are highly expressed from a plasmid. Thus, the average efficiency of B. cereus 569 and B. cereus 536 transformation with pUTE29-plcR-papR, pFP12 or pUTE29-nprB was similar for all indicated plasmids (100 ± 20 for B. cereus 569 and 600 ± 100 cfu/μg for B. cereus 536) whereas in the case of p102 and p182 the efficiency was only 6 ± 2 cfu/μg for both host strains. Both p102 (pUTE29-nprB-plcR-papR) and p182 (pUTE29-nprB-plcR fusion to papR) plasmids encode nprB in the same gene cluster with plcR and papR. The nprB gene is overexpressed both in p102 and p182 transformants due to high copy number of nprB along with plcR and papR. In the case of B. anthracis Ames 33, the average efficiency of transformation with pUTE29-plcR-papR, pFP12 and pUTE29-nprB was 900 ± 200 cfu/μg. The average efficiency of transformation was only 150 ± 30 cfu/μg with the p102 plasmid and no transformants were obtained with p182. Thus, the introduction of a transcriptionally active nrpB gene was not tolerated when its expression was controlled by a fused PlcR-PapR protein (p182, constitutive synthesis). In the case of the p102 plasmid the synthesis of NprB was apparently diminished due to PapR regulation and therefore it was better tolerated in B. anthracis.

The calculated molecular masses of NprB are 64.4 and 61.6 kDa for the protein with and without the signal peptide, respectively. However, analysis of the 2D-gel of the B. cereus 569 secretome (Fig. 5), along with data from Gohar et al. on the B. cereus 14579 secretome (Gohar et al., 2005), demonstrated that NprB migrates as if it were approximately 37 kDa. To determine the true size of the mature form of the NprB we produced the protease in the sporulation negative B. anthracis BH450 strain which has reduced proteolytic activity. B. anthracis BH450 transformed with pB148 or its parent plasmid pDG148-Stu were spotted on casein agar plates containing 50 μg mL^-1 kanamycin. Large zones of casein hydrolysis were observed only for the pB148 transformant (Fig. 6A). This production of NprB occurred even without induction of the P_spac promoter. Addition of IPTG even up to 2 mM did not greatly enhance expression of the nprB gene from the P_spac promoter (Fig. 6B, upper and lower panels). To better understand the behavior of this plasmid we eliminated the lacI repressor gene from pB148, producing pB148Lac^-. Expression remained high (data not shown). These data suggest that expression of nprB in these plasmids may depend on either the P_spac promoter operating even in the presence of the LacI repressor, or else on the bleomycin resistance gene promoter that is located immediately upstream of the P_spac promoter. Regardless of the mechanism, the resulting plasmid, pB148Lac-, provided expression levels that were sufficient for isolation and purification of NprB from B. anthracis BH450. The purified protein was transferred to a PVDF membrane and subjected to N-terminal sequence determination. The sequence of the first 11 residues at the N-terminus was determined to be VTSPRLSPASE, a sequence corresponding to that encoded beginning at nucleotide 703 of Genbank accession number AY195601. This shows that NprB is synthesized as a preproenzyme with a 234-amino acid-long prepro sequence (Fig. 6C). The mature enzyme is therefore composed of 349 amino acids and has a calculated mass of 38.2 kDa, corresponding well to that determined by SDS-polyacrylamide gel electrophoresis. The enzyme is resistant to 10 mM phenylmethylsulphonyl fluoride and can be completely inhibited by the metal chelator EDTA (10 mM) (data not shown).

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Figure 6

Characterization of B. cereus 569 NprB. (A) 2.5, 5 and 7.5 μl of overnight cultures of B. anthracis BH450 transformed with either pDG148-Stu (upper row) or pB148 (lower row) were spotted on casein agar plates containing 50 μg mL^-1 kanamycin. Plates were incubated overnight at 37°C. (B) B. anthracis BH450 (pB148) was cultured in FA broth in the presence of kanamycin (50 μg mL^-1) either in the absence or presence of 2 mM IPTG. Supernatants collected at T₂ (2 h after the end of exponential growth) were concentrated and applied to SDS-polyacrylamide (10 to 20%) gel using the PhastGel System (Amersham Pharmacia). The gel was stained with Coomassie blue. M designates molecular mass markers. The arrowhead indicates the 38.2-kDa neutral protease B. Results are presented in the upper panel. Protease activities of the corresponding supernatants were determined by spotting them on casein agar (lower panel). The zones of casein proteolysis were observed after 2 h incubation at 37°C. (C) Graphic presentation of the three functional segments of the B. cereus 569 NprB preproenzyme. The protein includes the signal peptide (SP), propeptide region (Pro) and mature protease.

NprB/PapR cleavage

Because PapR is cleaved outside the cells, we wished to characterize its secretion and the initial processing by the signal peptidase. For this purpose the amino acid sequence of the theoretical 48-aa PapR polypeptide was submitted to the SignalP V1.1 Prediction Server, Center for Biological Sequence Analysis, Department of Biotechnology, Technical University of Denmark (http://www.cbs.dtu.dk/services/SignalP/). The SignalP-Neutral Networks prediction for Gram-positive bacteria yielded nearly equal probability scores for 21- and 26-aa signal peptides. However, the SignalP-Hidden Markov Models prediction that was also developed for Gram-positive bacteria demonstrated a much higher score for the 21-aa signal peptide. Therefore, we synthesized a 27-aa C-terminal peptide as a target for cleavage by the purified, recombinant NprB protease. In general, the NprB protease cleaved the synthetic PapR peptide very slowly. We found that only 30-40% of the peptide was digested after 6 h and approximately 70-80% after 24 h of incubation at 37°C. The efficiency of the synthetic PapR cleavage was estimated as a percent ratio of HPLC peak areas for cleaved/uncleaved PapR (data not shown). It was not possible to discriminate between primary and secondary cleavage sites since all cleavage peptides could be detected after 6 h of digestion. Analysis of the cleavage products by reverse phase HPLC and mass spectrometry showed that NprB processed the synthetic 27-aa PapR propeptide to produce a set of C-terminal peptides 11, 8, 7, and 5 amino acids in length (Table 3). The following cleavage sites of the synthetic pro-PapR were obtained: E16-↓-V17, L19-↓-A20, A20-↓-S21 and D22-↓-M23.

We thank M. Garfield and J. Lukszo from the NIAID core facility for peptide synthesis and protein sequencing as well as Dr. Nga Nguyen from the FDA core facility for assistance with mass spectrometry analysis. We also acknowledge T. Read for the disclosure of the B. cereus 569 chromosomal sequences and K. Beliakov for assistance with the figure formatting. This research was supported by the Intramural Research Program of the NIH, National Institute of Allergy and Infectious Diseases.