CURRENT MICROBIOLOGY Vol. 38 (1999), pp. 233–238

An International Journal

R Springer-Verlag New York Inc. 1999

A Nonhemolytic Phospholipase C from Burkholderia cepacia Christine L. Weingart, Anne Morris Hooke Department of Microbiology, Miami University, Oxford, OH 45056, USA Received: 13 October 1998 / Accepted: 6 November 1998

Abstract. Burkholderia cepacia is an opportunistic pathogen that causes serious pulmonary infections in cystic fibrosis patients. Although several potential virulence factors—a protease, lipase, and two phospholipases C (one hemolytic and one nonhemolytic)—have been identified, only two, the protease and the lipase, have been described in detail. The goal of this study was to purify and characterize a nonhemolytic phospholipase C secreted by B. cepacia strain Pc224c. The enzyme was concentrated from culture supernatants and purified by polyacrylamide gel electrophoresis. The 54-kDa protein was stable in the presence of sodium dodecyl sulfate (up to 10%) and at 4°, 22°, and 37°C; it was, however, inactivated at 100°C. The enzyme bound to glass, chromatography matrices, and polyvinylidene difluoride and cellulose membranes, suggesting that it is hydrophobic. In a genetic approach, primers based on conserved sequences of a B. cepacia Pc69 hemolytic phospholipase C and both the Pseudomonas aeruginosa hemolytic and nonhemolytic proteins were designed to identify the Pc224c nonhemolytic phospholipase C gene. One polymerase chain reaction product was identified; it was sequenced and the sequence compared with sequences in the BLAST database. The best match was the Pseudomonas aeruginosa hemolytic phospholipase C. Ten additional B. cepacia strains were screened for the gene by Southern hybridization; five had the 4-kb band, suggesting that these strains have a similar form of the PLC gene. Nine of the ten strains reacted with the probe, suggesting that similar sequences were present, but in another form.

Burkholderia cepacia was first identified as a plant pathogen causing soft rot in onions [3]. It was originally classified as a pseudomonad but, on the basis of rRNA sequence analysis, it was transferred to a new genus, Burkholderia, in 1992 [18]. Within the last 20 years, this plant pathogen has emerged as a serious opportunistic pathogen in nosocomial infections, chronic granulomatous disease, and particularly in cystic fibrosis (CF) patients [4]. Pseudomonas aeruginosa, like B. cepacia, is a serious opportunistic pathogen in CF patients. Pseudomonas aeruginosa produces a battery of virulence factors: exoenzyme S, exotoxin A, elastase, alkaline protease, and two phospholipases (one hemolytic, the other nonhemolytic) [12,14]. Clinical isolates of B. cepacia produce extracellular protease, lipase, and a hemolytic phospholipase C [8, 9, 11, 17], although their role in virulence has not been defined. Burkholderia cepacia Pc224c, a clinical

Correspondence to: A.M. Hooke

isolate from the sputum of a CF patient, expresses a heat-labile nonhemolytic phospholipase C (PLC) activity [8]. We have purified and partially characterized this nonhemolytic PLC and cloned a portion of the gene. Materials and Methods Bacterial strains. Burkholderia cepacia strains [8] and Pseudomonas aeruginosa PAO-1 were maintained on Pseudomonas isolation agar (PIA, Difco, Detroit, MI) at 37°C. Escherichia coli DH5a (pUC18) and E. coli HB101 (pDK5) (a gift from Michael L. Vasil, University of Colorado Health Sciences Center) were maintained on trypticase soy agar (TSA, Difco) with ampicillin (50 µg/ml) and carbenicillin (100 µg/ml), respectively. All strains were stored in trypticase soy broth (TSB) with 20% glycerol at 280°C. Purification of PLC. Burkholderia cepacia Pc224c was grown in MOPS-minimal salts-tryptose medium (3 mM KCl, 12 mM [NH4 ] 2SO4, 3.2 mM MgSO4, 0.02 mM FeSO4, 3 mM NaCl, 0.1% tryptose in 50 mM 3-[N-morpholino]propanesulfonic acid [MOPS]) with 0.8% glycerol (MMSTG) [8] at 37°C in a New Brunswick Controlled Environment incubator shaker for 16–17 h at 200 rpm. The supernatant was collected by centrifugation in a Sorvall RC-5B Refrigerated Superspeed Centrifuge GSA rotor (Du Pont Instruments, Norwalk, CT) at 18,000 g for 20 min at 4°C and sterilized by filtration. Cell-free supernatant was

234 concentrated in an Amicon CH2PRS ultrafiltration system (Amicon, Danvers, MA) at 20 psi at room temperature (RT). The supernatant was further concentrated in an Amicon stirred-cell system (Model 8010) under nitrogen pressure (20 psi) at room temperature (RT). The Spectra/Por polyvinylidene difluoride (PVDF) hydrophobic filter, which retains molecules greater than 20 kDa, was washed with 2 ml Tris buffer, 0.1% SDS, in a polypropylene centrifuge tube (Fisher, Pittsburgh, PA) to elute the bound PLC. Samples were assayed for PLC activity on p-nitrophenylphosphorylcholine (NPPC, Sigma, St. Louis, MO) [2, 6]. Total protein was measured with a microtiter version of the Pierce bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL). Specific activity was calculated as absorbance units at 410 nm per mg total protein. Gel electrophoresis and protein blotting. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) in the Laemmli discontinuous electrophoresis buffer system [7]. The separating gel consisted of 10% (wt/vol) acrylamide:N,N8methylene-bis-acrylamide solution (Bio-Rad, Richmond, CA) in 0.375 M Tris-HCl (pH 8.8) and a stacking gel of 4% (wt/vol) acrylamide: N,N8-methylene-bis-acrylamide solution, 0.125 M Tris-HCl (pH 6.8). Samples were diluted in 23 treatment buffer (0.125 M Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, and 0.01% bromphenol blue), and electrophoresis was run at 40 mA constant current. The PLC band was located after electrophoresis by flooding the gel with NPPC reagent at RT; the yellow reaction product was visible within seconds. Gels were stained in Coomassie blue (0.25% Coomassie brilliant blue-R250 [Bio-Rad], 40% methanol, and 7% acetic acid) and destained in 50% methanol/10% acetic acid and then 5% methanol/10% acetic acid. Two-dimensional gel electrophoresis. The first dimension, isoelectric focusing, was performed with a Bio-Rad tube cell system (Model 175) [13]. The tube gel (1.5 mm ID, 6.0 OD, 150 mm length) consisted of 8 M urea, 26% acrylamide: N,N8-methylene-bis-acrylamide (30:1.8, BioRad), 3% NP-40, 2% ampholytes (3–10, 5–7, and 7–9, Bio-Rad), 0.5% (wt/vol) APS, and 0.05% (vol/vol) TEMED and polymerized at RT for 6 h. The sample was treated with 9.5 M urea and two vol urea sample buffer [9.5 M urea, 2% ampholytes (3–10), 5% b-mercaptoethanol and 8% NP-40] and added to the tubes. The upper buffer chamber was filled with 20 mM NaOH; the bottom chamber was filled with 10 mM H3PO4. The proteins were separated at constant voltage (400 V) at 4°C; after 21 h, the voltage was adjusted to 700 V for 1 h. Tube gels were removed, and three were placed on a 4% stacking gel, adjacent to the mid-range molecular weight standards (35 µl, Promega, Madison, WI). The 12% SDS-PAG (14 cm 3 13 cm 3 0.15 cm) was run at 100 V for 2 h at RT and then adjusted to 400 V until the dye was 1 cm from the bottom of the gel. The gel was stained with the Bio-Rad silver stain kit according to the directions of the manufacturer. Amino-terminus sequence and analysis. The proteins from the PVDF wash were separated on a 10% SDS-PAG and electroblotted onto Immobilon-P PVDF (Millipore, Bedford, MA) with a Hoefer TE42 Transphor system. Proteins were transferred at constant current (100 mA) for 16 h in 7 mM N-ethylmorpholine (Sigma) electroblotting buffer. The membrane was stained for 2 min in Coomassie blue (0.1% Coomassie brilliant blue-R250, 40% methanol, and 10% acetic acid), destained in 45% methanol, 7% acetic acid for 15 min, and dried at RT. The PLC band was excised with an ethanol-cleaned razor blade and sequenced by The Medical College of Wisconsin, Protein/Nucleic Acid Shared Facility (Milwaukee, WI). Isolation and manipulation of DNA. Burkholderia cepacia chromosomal DNA was isolated by the method described by Strom et al. [16]. Plasmid DNA was isolated by the Qiagen large-scale plasmid isolation kit (Qiagen Inc., Chatsworth, CA). Purified DNA was digested with

CURRENT MICROBIOLOGY Vol. 38 (1999) restriction enzymes purchased from New England Biolabs (Beverly, MA). The DNA was separated in a 1% agarose gel with Tris-acetate buffer and visualized with ethidium bromide [15]. Primer design and polymerase chain reaction. Forward (58GGAATTCAACCGCGCATTCGACCATTACTT-38) and reverse (58GGAATTCCCACCCGCCCTTCGACCAGGG-38) primers for polymerase chain reaction (PCR) were designed based on the conserved regions of B. cepacia Pc69 hemolytic PLC (M. Vasil, personal communication) and the P. aeruginosa phospholipase amino acid sequences. The sequences were aligned with Sequence Application 2.05. For cloning purposes, EcoRI sites were added to the 58 and 38 ends of the primers. The polymerase chain reaction was composed of Promega PCR buffer B (13), 300 nM MgCl2, 500 nM dNTPs (Boehringer-Mannheim, Indianapolis, IN), forward primer (2 µg), reverse primer (2 µg), genomic or plasmid DNA (1 µg), and Taq polymerase (2.5 U). The reaction mixture (100 µl) was incubated in a Perkin-Elmer Cetus DNA thermal cycler (Norwalk, CT) for 30 cycles under these conditions: denature, 94°C, 1 min; anneal, 30°C, 3 min; extend, 72°C, 3 min. Cloning and sequencing the amplification product. The Pc224c amplification product was purified with the Gene Clean Bio101 (LaJolla, CA) kit and ligated into EcoRI-digested pUC18. The ligation reaction consisted of EcoRI-digested insert DNA (3 µg), vector DNA (250 ng), T4 DNA ligase buffer (13, New England Biolabs), and T4 DNA ligase (10 U, New England Biolabs). The positive control for transformation was EcoRI-digested vector DNA (250 ng), 13 ligase buffer, ligase (10 U), and water to 20 µl. The reactions were incubated at RT overnight. Escherichia coli DH5a cells were rendered competent for transformation by growing them in 100 ml Luria-Bertani (LB) broth (1% tryptone, 0.5% sodium chloride, and 0.5% yeast extract) at 37°C, 200 rpm. At late-log phase (A600 nm 5 0.65), the cultures were chilled in an ice bath for 10 min with frequent stirring. The cells were harvested by centrifugation at 4000 g for 5 min at 4°C. The pellet was suspended in 1/10th vol 0.1 M CaCl2 and placed on ice for 1 h. The cells were collected by centrifugation at 5000 g for 5 min at 4°C. The pellet was suspended in 1/20th vol 0.1 M CaCl2, 10% glycerol, and stored at 280°C. For transformation, the competent E. coli cells were thawed on ice. The positive control, 0.5 µl pUC18, was added to 100 µl competent cells. The ligation mixture (10 µl) was added to 100 µl of competent cells. The samples were gently mixed and transferred to ice for 30 min. The cells were shocked at 42°C for 2 min, and then 1 ml warmed LB broth was added to each tube. The cells were incubated at 37°C with constant shaking (100 rpm) for 1 h. The pUC18 clones were selected by the blue/white color assay [15]. The transformation mixture and the positive control were plated on LB agar containing ampicillin (50 µg/ml, Sigma) and spread with 5-bromo-4-chloro-3-indoyl-b-D-galactoside (800 µg in 40 µl dimethylformamide, Promega) and isopropyl-b-thiogalactopyranoside (800 µg in 4 µl ddH2O, Promega), and incubated at 37°C for 17 h. Successfully cloned inserts were indicated by white colonies. We sequenced a portion of the amplification product with the Promega fmolt DNA sequencing kit. The sequencing reactions proved challenging because Burkholderia DNA is 67% G1C rich [18], so we had the sequence confirmed on an automated Perkin-Elmer ABI 377 DNA sequencer by Robert Munson (Children’s Hospital Research Foundation, Ohio State University, Columbus, OH). Southern blot hybridization. Chromosomal DNAs were digested with HindIII. The fragments were separated by electrophoresis on a 1% agarose gel (9.9 cm 3 6.7 cm 3 0.35cm) at 80 V. The gel was washed in 0.25 N HCl, 0.5 M NaOH/1.5 M NaCl, and 1.5 M NaCl/0.5 M Tris-HCl (pH 7.4) for 20 min each. The DNA was transferred to positively

C.L. Weingart and A.M. Hooke: Nonhemolytic Phospholipase C from B. cepacia

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Table 1. Recovery of PLC activity by ultrafiltration of Pc224c culture supernatant

Sample a Supernatant Concentrate Retentate Filtrate Membrane e

Total PLC activity b (U)

Total protein c (mg)

Specific activity (U/mg)

Yield d (%)

403,300 6,980 137 332 419

629.0 19.6 3.8 14.7 0.1

641 356 36 23 4,190

100.00 2.00 0.03 0.08 0.10

a Four 2-L flasks containing 500 ml MMSTG each were inoculated with Pc224c and incubated at 37°C, 200 rpm for 16–17 h. Cell-free supernatant was concentrated 81-fold by ultrafiltration in an Amicon CH2PRS ultrafiltration system containing a cellulosic-type spiral filter (30,000 mwco) and an Amicon stirred-cell ultrafiltration system containing a PVDF membrane (20,000 mwco). b Absorbance units at 410 nm/ml 3 total sample volume. c Protein was measured by the Pierce bicinchoninic assay. d Yield was determined by dividing total activity of the sample by the total activity of the supernatant. e The PVDF membrane was washed with 2 ml Tris buffer, 0.1% SDS.

charged nylon (Boehringer-Mannheim) in 53 SSC (0.6 M NaCl, 0.07 M sodium citrate, pH 7) overnight at RT and immobilized by UV cross-linking (Bio-Rad GS Linker) at 150 mjoules. To prevent nonspecific binding, the membrane was soaked in prehybridization solution [25% formamide, 2% SDS, 63 SSC (pH 7), 13 Denhardt’s solution (2% Ficoll, 2% polyvinylpyrrolidone, and 2% bovine serum albumin)] for 3 h at 37°C. For the hybridization, sonicated-salmon sperm DNA (2 mg) was added to 10 µl radiolabeled Pc224c amplification product (Pharmacia Biotech Oligolabeling Kit), heated at 100°C for 10 min, and added to the membrane for overnight incubation at 37°C. The membrane was washed with 53 SSC, 0.1% SDS, and 23 SSC, 0.1% SDS at 50°C. The membrane was dried at RT, placed in an XC autoradiography cassette (Fisher, Pittsburgh, PA), and exposed to Fuji RX film at 280°C overnight.

Results Concentration of PLC from Pc224c culture supernatants. Initial efforts to concentrate the Pc224c PLC by ammonium sulfate precipitation resulted in an insoluble protein aggregate. Ultrafiltration proved more useful— the proteins did not aggregate, but the yield of PLC activity was very low (Table 1). Further investigation revealed that the PLC was binding to the PVDF membrane, and it could be eluted with SDS (Table 1). The proteins in the ultrafiltration samples were analyzed by separating them on a 10% SDS-PAG. Immediately after electrophoresis, the gel was flooded with NPPC reagent to detect active PLC. Only one area on the gel turned yellow, suggesting there was only one active PLC and it was stable in the presence of 0.1% SDS in the gel. The gel was subsequently stained to visualize all the proteins. A band that migrated around 54 kDa correlated with the yellow band detected with NPPC (Fig.

Fig. 1. Separation of Pc224c supernatant proteins concentrated by ultrafiltration. The samples were treated and electrophoresed in a 10% SDS-PAG as described in Materials and Methods. After electrophoresis, NPPC was applied to the gel to detect the PLC band (arrow), and the gel was stained with Coomassie blue. Lane 1, low-molecular-weight standards (Bio-Rad); lane 2, supernatant concentrated 14-fold (5.2 µg protein); lane 3, supernatant concentrated 67-fold (30 µg protein); lane 4, ultrafiltration filtrate (4.26 µg protein; and lane 5, eluate from PVDF filter washed with 0.1% SDS (1.28 µg protein).

1). Further investigation of the gel-purified PLC indicated that the band appeared homogeneous and the isoelectric point was approximately 7 (Fig. 2). Stability of PLC. Concentrated supernatant was incubated at 4°, 22°, and 37°C; samples were withdrawn at various times and assayed for PLC activity. The activity remained at 100% for at least 96 h at all three temperatures, but was completely lost after 5 min at 100°C (data not shown). Amino-terminus sequence and amino-acid composition. The gel-purified phospholipase C was sequenced at its amino-terminal end for comparison with other phospholipase C proteins. The sequence, NTGTIRDXLIYVXL, was aligned with those of other PLCs with the shareware program, SeqApp 1.9a169 (r1992 by D.G. Gilbert); there was 50%, 50%, 29%, 29%, and 29% identity with the B. cepacia PLC-H, P. aeruginosa PLC-H, P. aeruginosa PLC-N, M. tuberculosis PLC-A, and the M. tuberculosis PLC-B, respectively (Fig. 3). Amino acid analysis indicated that 32% of the 54-kDa protein was composed of hydrophobic amino acids (Table 2). Amplification and sequencing of the Pc224c chromosomal DNA. An 1100-bp product from B. cepacia Pc224c was amplified with the forward and reverse primers (Fig. 4, Lane 3). Pseudomonas aeruginosa DNA sequences were amplified with the same primers, and two sets of doublets at the 900- and 400-bp positions were

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CURRENT MICROBIOLOGY Vol. 38 (1999)

Fig. 2. Two-dimensional gel electrophoresis of concentrated (51-fold) Pc224c supernatant. The first dimension (1D), isoelectric focusing, separated the proteins in tube gels loaded with 7.5 µg protein at 400 V for 21 h. The tube gels were placed on a 12% SDS-PAG for the second dimension (2D), separated at 100 V for 2 h, and silver stained. Table 2. Amino acid composition of purified nonhemolytic phospholipase C a Amino acid

Fig. 3. Alignment of the B. cepacia Pc224c PLC amino terminus with other PLC protein sequences. The amino terminus was compared with the B. cepacia Pc69 hemolytic PLC (deduced from the nucleotide sequence determined by M. Vasil, and used with permission), P. aeruginosa hemolytic and nonhemolytic PLCs [14], and the M. tuberculosis PLCs [5]. The asterisks indicate the end of the predicted signal sequences for P. aeruginosa PLC-H and PLC-N. The termini for the signal sequences of the mycobacterial PLCs and B. cepacia PLC-H are three amino acids back, and not shown.

detected (Fig. 4, Lane 4). A 1000-bp product was amplified in the positive control B. cepacia Pc69 DNA, indicating that the primers bound to the PLC-H gene and amplified a portion of it (Fig. 4, Lane 2). The Pc224c 1100-bp product was subsequently digested with EcoRI and ligated into pUC18 (Fig. 4, Lane 5). Analysis of the digested clone indicated that the Pc224c DNA had an internal EcoRI site because the insert was reduced to 850 bp (Fig. 4, Lane 6). The amplification product was sequenced, and the results were entered into the BLAST database; the best match was P. aeruginosa hemolytic PLC. Identification of the PLC gene in other B. cepacia strains. Ten additional B. cepacia strains were screened by Southern hybridization for the presence of PLC-N sequence. Five of the ten strains shared one homologous band at approximately 4 kb, suggesting that these strains possess a PLC gene similar in form to the Pc224c PLC gene (Fig. 5). In addition, the probe did hybridize to higher molecular weight DNA in nine of the ten strains screened.

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine

Mole (%) 7.2 4.3 14.3 11.1 3.9 14.4 5.6 7.4 1.1 2.9 5.9 4.8 5.7 1.4 5.9 4.1

a Phospholipase C was prepared for amino acid analysis by treating the PLC band with 6 M HCl and 0.1% b-mercaptoethanol. The mixture was heated at 110°C under nitrogen pressure for 20 h. Amino acid analysis was performed by the Medical College of Wisconsin Protein/Nucleic Acid Shared Facility.

Discussion Our initial efforts to purify PLC involved ammonium sulfate precipitation. After removal of ammonium sulfate by dialysis, the proteins aggregated and were insoluble. This poor recovery of protein and the aggregation problems with ammonium sulfate precipitation (data not shown) forced us to try another method of concentration, ultrafiltration. We concentrated 2 L of culture supernatant and, although recovery was still poor (Table 1), there was no aggregation. We investigated the reasons for the low recovery by washing the glass container that held the cell-free supernatant and the filters used in ultrafiltration

C.L. Weingart and A.M. Hooke: Nonhemolytic Phospholipase C from B. cepacia

Fig. 4. Agarose gel electrophoresis of PCR amplification reactions and the cloned PCR product. Samples were separated electrophoretically at constant voltage (40 V). Lane 1, lambda DNA cut with HindIII (1 µg); lanes 2–4, pDK5 containing the B. cepacia Pc69 PLC-H gene, Pc224c, and P. aeruginosa PAO-1 DNA, respectively, amplified with the forward and reverse primers; lane 5, undigested pUC18 containing the Pc224c amplification product; lane 6, EcoRI-digested pUC18 and the Pc224c amplification product.

with the anionic detergent, SDS. The PLC bound not only to the glass container but also to the ultrafiltration membranes (data not shown). In fact, PLC bound very strongly to the PVDF filter; the specific activity of the SDS-eluate was increased almost sevenfold over that in the supernatant (Table 1). The compound PVDF promotes the binding of hydrophobic proteins, suggesting that PLC is hydrophobic, and we exploited this property to enhance its purification. Another interesting observation is that PLC-N was stable in the presence of SDS. Most proteins are denatured by SDS because it alters their conformation. In contrast, the Pc224c PLC-N remained active in up to 10% SDS (data not shown), and, even after electrophoresis on SDS-PAG (0.1% SDS), the PLC-N band could be detected by its activity on NPPC (Fig. 1). The molecular weight of the PLC, determined by SDS-PAGE, was approximately 54 kDa, much lower than that of the P. aeruginosa PLC-N (73,455 Da) and PLC-H (78,352 Da), and the B. cepacia PLC-H (72,000 Da) [1, 14]. Recently, two phospholipases, PLC-A and PLC-B, were described for M. tuberculosis, with molecular weights of 56,103 and 56,137 Da, respectively [5]. Thus, the molecular weight of the Pc224c PLC-N is reasonable when compared with the M. tuberculosis PLCs. We investigated the effect of other potential variables on the recovery of PLC during precipitation procedures. We studied the stability of PLC-N in culture supernatants

237

Fig. 5. Southern hybridization with the Pc224c PLC gene fragment and B. cepacia strains. Chromosomal DNA was isolated from 11 B. cepacia strains. The DNAs (250 ng) were digested with HindIII, separated by agarose gel electrophoresis, and the DNA was transferred to a positively charged nylon membrane. The membrane was treated with the radiolabeled Pc224c PLC gene fragment. The arrow denotes the 4-kb region. Lane 1, lambda DNA digested with HindIII; lane 2, Pc224c; lane 3, K33-1; lane 4, 22-20; lane 5, K43-3; lane 6, K19-2; lane 7, 90ee; lane 8, K30-6; lane 9, Pc445kk; lane 10, K56-2; lane 11, H1729-2; lane 12, K41-6.

at 4°C, 22°C, and 37°C. The PLC activity was stable at 4°, 22°, and 37°C, so the loss of PLC activity was not due to the effect of temperature (data not shown). Because there was no loss of PLC activity at 22° and 37°C for at least 96 h, it is unlikely that a protease also present in the culture supernatant was destroying the enzyme. The first 14 amino acids of the Pc224c PLC-N were sequenced and aligned with the deduced amino termini of other PLCs (Fig. 3). The Pc224c PLC-N amino terminus begins 5–8 amino acids after the end of the predicted signal sequences for the P. aeruginosa and M. tuberculosis PLCs. The pseudomonal and mycobacterial PLCs have predicted signal sequences of 38, 35, and 38 amino acids [5, 14]. The mature Pc224c PLC-N amino terminus begins shortly after those predicted signal peptides, suggesting the cleavage site for the Pc224c signal peptidase is different in this strain. Protein sequence alignments of the P. aeruginosa PLCs reveal 40% identity overall, and the first two-thirds of the proteins have 47% identity [14]. Using these sequences and a portion of the B. cepacia PLC-H amino acid sequences (M. Vasil, personal communication), we designed primers for the identification of the B. cepacia PLC-N gene. We amplified one PCR product at the very

238 low annealing temperature of 30°C (Fig. 4). The sequence results indicated the PCR product was a portion of a B. cepacia PLC gene; several of the nucleotide sequences were identical to those of other PLCs (data not shown). Nucleotide alignment indicated that the B. cepacia Pc224c PCR product shared 43% and 50% identity with the P. aeruginosa PLC-H and the P. aeruginosa PLC-N sequences, respectively. Because there was only one amplification product and not two, as seen in P. aeruginosa, we speculated that Pc224c possessed only one PLC gene (Fig. 4), or, if there were two genes, then the sequence between the two primers was the same length. Because PLC is a virulence factor for P. aeruginosa and all clinical isolates Berka et al. tested were PLC positive [2], we looked for the PLC-N gene in 10 B. cepacia strains, nine clinical isolates and one environmental strain (22-20). The HindIII-digested chromosomal DNAs from these strains were probed with the Pc224c PLC gene fragment, and five strains had one band that comigrated with the Pc224c 4-kb band (Fig. 4). This suggested that all these strains have a similar form of the PLC gene. Although strains 22-20, 90ee, and Pc445kk did not have the 4-kb HindIII restriction fragment, they were, however, positive for hybridization. The PLC gene probe reacted with the high-molecular-weight DNA, suggesting that the chromosomal DNAs were not completely digested (which is unlikely, given the conditions) or that these strains possessed a different form of the gene or the gene was located in a larger fragment. It is also possible that sequences similar to those in the PLC-N gene are also present in an unexpressed PLC-H gene [14, 17]. In summary, we have purified and partially characterized a nonhemolytic PLC expressed by B. cepacia Pc224c. The protein is approximately 54 kDa, stable at temperatures ranging from 4° to 37°C, and has an isoelectric point of 7. The amino terminus of the protein shares significant identity with the pseudomonal and mycobacterial PLCs. The nucleotide sequence of the Pc224c PLC gene fragment we cloned was most similar to the P. aeruginosa PLC nonhemolytic gene. We are currently isolating insertion mutants to investigate the role of the PLC-N in virulence. ACKNOWLEDGMENTS We thank Dr. Michael Vasil for the B. cepacia Pc69 PLC-H gene and its sequence. We also thank Dr. Robert Munson for sequencing the B. cepacia Pc224c PLC gene fragment. We thank Drs. Luis Actis and Gary

CURRENT MICROBIOLOGY Vol. 38 (1999) Janssen for their advice and suggestions. This project was partially funded with a Sigma Xi, the Scientific Research Society, grant-in-aid.

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