Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa is a ... also regulates the level of pyocyanin production by P. aeruginosa and that one or.
JouRNAL OF BACTERIOLOGY, Oct. 1982, p. 431-440 0021-9193/82/100431-10$02.00/0 Coypright C 1982, American Society for Microbiology
Vol. 152, No. 1
Cloning of a Phosphate-Regulated Hemolysin Gene (Phospholipase C) from Pseudomonas aeruginosa MICHAEL L. VASIL,* RANDY M. BERKA, GREGORY L. GRAY,t AND HIROSHI NAKAI Department of Microbiology and Immunology, University of Colorado Medical School, Denver, Colorado 80262 Received 31 March 1982/Accepted 15 June 1982
Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa is a phosphate (Pj-regulated extracellular protein which may be a significant virulence factor of this organism. The gene for this hemolytic enzyme was cloned on a 4.1megadalton (Mdal) fragment from a BamHI digest of P. aeruginosa PA01 genomic DNA and was inserted into the BamHI sites of the multicopy Escherichia coli(pBR322) and P. aeruginosa(pMW79) vectors. The E. coli and P. aeruginosa recombinant plasmids were designated pGV26 and pVB81, respectively. A restriction map of the 4.1-Mdal fragment from pGV26 was constructed, using double and single digestions with BamHI and EcoRI and several different restriction enzymes. Based on information from this map, a 2.4-Mdal BamHII BglII fragment containing the gene for phospholipase C was subcloned to pBR322. The hybrid plasmids pGV26 and pVB81 direct the synthesis of enzymatically active phospholipase C, which is also hemolytic. The plasmid-directed synthesis of phospholipase C in E. coli or P. aeruginosa is not repressible by Pi as is the chromosomally directed synthesis in P. aeruginosa. Data are presented which suggest that the synthesis of phospholipase C from pGV26 and pVB81 is directed from the tetracycline resistance gene promoter. The level of enzyme activity produced by E. coli(pGV26) is slightly higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions. In contrast, the levels produced by P. aeruginosa(pVB81) are at least 600-fold higher than the levels produced by P. aeruginosa(pMW79) under repressed conditions and approximately 20-fold higher than those produced by P. aeruginosa(pMW79) under derepressed conditions. The majority (85%) of the enzyme produced by E. coli(pGV26) remained cell associated, whereas >95% of the enzyme produced by P. aeruginosa(pVB81) was extracellular. Analysis of extracellular proteins from cultures of P. aeruginosa(pMW79) and P. aeruginosa(pVB81) by high-performance liquid chromotography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the phospholipase C gene was cloned intact, and it is likely that several additional genes were cloned on the 4.1-Mdal fragment of DNA. It was also found that some of these genes encode proteins which are the same molecular weight as some previously described Pi-repressible proteins of P. aeruginosa. The existence of a Pi regulon of P. aeruginosa is proposed. It is likely that one of these genes also regulates the level of pyocyanin production by P. aeruginosa and that one or more play a role in transport or binding of Pi. The availability of the hybrid plasmids described herein will be useful in further studies on the role of this hemolysin in the virulence of P. aeruginosa and in the study of the genetics and physiology of Pi-regulated proteins. There is increasing evidence that the hemolysins of several gram-negative bacteria, including Escherichia coli (7, 22, 33), Pseudomonas aeruginosa (2, 17, 19, 30), Vibrio parahemolyticus (23), and Aeromonas hydrophila (26, 35), contribute to the pathogenesis of certain kinds of
infections caused by these organisms. In E. coli, for example, hemolysin appears to play a role in permitting this organism to initiate or sustain extraintestinal infections, particularly those involving the urinary tract (7, 22). P. aeruginosa produces two hemolysins (19). One of the hemolysins is a heat-labile phospholipase C which
t Present address: Department of Molecular Biology, Gen- catalyzes the hydrolysis of phosphatidylcholine to phosphorylcholine and diacylglycerol. The entech, Inc., South San Francisco, CA 94080. 431
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VASIL ET AL.
other is a heat-stable glycolipid (15). Both hemolysins are produced during stationary phase in low-phosphate media but are repressed in highphosphate media (8, 9, 15). The proposed physiological role of these hemolysins in the organism is to act cooperatively with alkaline phosphatase in the liberation of Pi from phospholipids in the environment. In terms of pathogenesis, it has been proposed that the phospholipase C of P. aeruginosa is important in the initiation of infection, particularly in the lung, where there is ample substrate for this enzyme in the form of the phospholipids of surfactant (19). Studies on the substrate specificity of this hemolytic enzyme indicate that it is very active on the phospholipids found in eucaryotic cells but has very little, if any, activity on phospholipids found in procaryotic membranes (2a). The phospholipase C also appears to be a necrotic or cytolytic toxin, like the phospholipase C (alphatoxin) of Clostridium perfringens (19). In P. aeruginosa several studies have implicated the heat-labile hemolysin as a determinant of virulence in the pathogenesis of lung infections (17, 30). These studies are of particular interest in terms of patients with cystic fibrosis, in whom P. aeruginosa lung infections are associated with high mortality rates. Also, this laboratory has reported a significant association of increased heat-labile hemolysin production with isolates from urinary tract infection (2). The pathogenesis of P. aeruginosa infections is complex and appears to involve the production of several distinct toxins which have been implicated as virulence factors (36). Therefore, genetic methods including recombinant DNA technology, offer a rational approach for dissecting the complex mechanisms of the virulence of P. aeruginosa. We report here the molecular cloning and stable expression of the heat-labile hemolysin gene of P. aeruginosa in E. coli and in P. aeruginosa, a significant step in the study of the role of this toxin in disease. (In this paper hemolysin and phospholipase C will be used interchangeably.)
J. BACTERIOL.
TABLE 1. Bacterial strains and plasmidsa Stock no.
or Genotype phenotype
Bacterial strains E. coli HB101 leu thr pro lac gal Strr recA EndoI hsdR hsdM KL320 trp his ilv pro lac rspL H22 htx mutant of KL320 P. aeruginosa PAO1 Prototroph chl-3
PA02003 Plasmids pBR322
Source/reference
A. L. Taylor
R. K. Holmes (4) R. K. Holmes (4)
B. W. Holloway (8-10) argH3 str-39 cml- V. Krishnapillai 2 rec-2 FP(34)
Ap Tc
Bolivar and Backman (3) pMW79 Ap/Cb Sm (hybrid D. 0. Wood of RSF1010 and (34) pBR322) pGV26 Ap Hly+ This study pVB81 Ap Cb Sm Hly+ This study pRM82 Ap Hly+ This study a Genotype symbols are described by Bachmann and Low (1). Ap, Tc, Sm, and Cb refer to resistance to ampicillin, tetracycline, streptomycin, and carbenicilfin, respectively. Hly refers to the hemolytic or phospholipase C phenotype of the recombinant plasmid.
tration of Pi in dialvsate of Trypticase soy broth (10 mM) is sufficient to repress synthesis of phospholipase C by 20- to 100-fold (9). Phospholipase C activity was measured by p-nitrophenylphosphorylcholine hydrolysis as previously described (2, 8, 9). Blood agar for screening the genomic libraries consisted of basal medium previously described by Johnson and BoeseMarrazzo (15) and was supplemented with 5% defibrinated sheep erythrocytes. Isolation and manipulation of DNA. Genomic DNA of P. aeruginosa PA01 was prepared by growing the cells overnight at 37°C and then extracting the DNA as previously described (6). Plasmid DNA from E. coli and P. aeruginosa was isolated by a modification of the procedure of Hirt (13). Briefly, 300 ml of an 18- to 24-h culture of E. coli or P. aeruginosa grown at 37°C MATERIALS AND METHODS in L-broth was centrifuged at 5,000 rpm for 10 min, Chemicals. Propidium iodide and p-nitrophenyl- and the cells were suspended in 10 ml of 5 mM Trisphosphorylcholine were obtained from Sigma Chemi- hydrochloride (pH 8) in 25% sucrose. Lysozyme was cal Co., St. Louis, Mo. added to a final concentration of 1 mg/ml, and the cells Bacterial strains and plasmids. The bacterial strains were put on ice for 5 min. Then 5 ml of 0.2 M EDTA and plasmids used in these studies are given in Table 1. (pH 8.0) was added to each tube followed by 2 ml of Assay of phospholipase C and hemolysin production. 10% sodium dodecyl sulfate (SDS), and the tube Conditions for the production of phospholipase C have contents were then blended in a Vortex mixer. Next, been previously described and were used in this study 2.5 ml of 5 M NaCl was added to each tube and mixed except that in some cases, where noted, dialysate of gently by inversion of the tubes. This lysate was left on Trypticase soy broth (BBL Microbiology Systems, ice overnight and then centrifuged at 12,000 rpm for 1 Cockeysville, Md.) (10) was used instead of tryptose h. The supernatant was precipitated with an equal minimal media (2, 8, 9) because of the better growth volume of isopropyl alcohol for 2 h at -20°C, and it obtained, particularly with auxotrophic E. coli and P. was then centrifuged at 10,000 rpm for 20 min. The aeruginosa PA02003 Hly+ derivatives. The concen- pellet was redissolved in water at room temperature.
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Undissolved material was centrifuged from the lysate at 5,000 rpm for 5 min. The lysate was then subjected to propidium iodide-cesium chloride density gradients as previously described (34). Construction of P. aeruginosa PAO1 genomic libraries in E. coli HB101. P. aeruginosa PAO1 chromosomal DNA (200 ,ug/ml; 80 ,ul) was digested to completion with BamHI restriction endonuclease (Bethesda Research Laboratories, Gaithersburg, Md.), mixed with pBR322 (20 p.g/ml; 50 ,ul) cleaved with BamHI, and ligated in a total volume of 164 pd (T4 ligase [Bethesda Research Laboratories]; 1 U of DNA per ,ug, 12°C, 18 to 24 h). The ligated DNA was transformed into E. coli HB101 by standard methods (21). Ampicillin-resistant (Ap), tetracycline-sensitive (Tc') transformants were chosen for further study. The transformants were initially selected on L-agar containing 50 ,ug of ampicillin per ml. The frequency of transformants for this experiment was 4 x 105 Apr transformants per ,ug of input pBR322. The Apr transformants were scored for Tcs on L-agar plates containing 30 p.g of tetracycline per ml. It was necessary to score for Tc' transformants in this manner since the recently developed procedures for selection of Tc' did not work for E. coli HB101 (20). However, it was found that Tcs transformants were reasonably frequent (22%), making it relatively easy to choose large numbers of potential clones carrying P. aeruginosa genes. From a single ligation and transformation experiment, approximately 3,000 Apr Tc' clones were saved and, according to the formula of Clarke and Carbon (6), represent at least 95% of the genome of P. aeruginosa. A Sail library of P. aeruginosa PAO DNA was constructed in a similar manner. Cloning of the hemolysin gene in P. aeruginosa. The overall scheme for cloning the phospholipase C gene is shown in Fig. 1. The recombinant pBR322 plasmid carrying the gene for hemolysin production (pGV26) was digested with BamHI endonuclease and then ligated to BamHI-cleaved pMW79 (34), using the conditions described above. The ligated DNA was transformed to P. aeruginosa PA02003, using methods previously described (34). Transformants were selected on brain heart infusion agar (Difco Laboratories, Detroit, Mich.) containing 500 ,ug of carbenicillin per ml and scored for Tc' on brain heart infusion agar with 30 ,ug of tetracycline per ml and for Hly+ on blood agar containing 500 p.g of carbenicillin per ml. Restriction enzyme procedures and mapping strategy. All restriction enzymes were obtained from Bethesda Research Laboratories, and conditions for restriction enzyme reactions were as specified by the manufacturer. Restriction mapping was performed with the pGV26 recombinant plasmid. Restriction mapping follows. A series of restriction enchosen which would not digest the vector pBR322 or which had only one site in that vector. Also, enzymes were chosen which only cleaved the inserted DNA once or twice. Then, using double digestion with EcoRI or BamHI and the selected enzyme, cleavage sites were determined after agarose gel electrophoresis, using a HindIII digest of bacteriophage lambda as a molecular weight standard. In this manner, the restriction map was constructed. Restriction enzyme-digested samples were run in 0.8% agarose gels with Tris-acetate buffer (pH 7.5) for strategy
was as
zymes were
433
3 h at 100 V. DNA fragments were visualized by staining with ethidium bromide (1 ,ug/ml) and photographed in short-wavelength UV light. Nick hnslation of plasmids and Southern blot hybridization with PAO1 genomic DNA. Southern blot hybridization of the nick-translated 2P-labeled (27) pGV26 plasmid with BamHI-digested P. aeruginosa PAO1 DNA was performed as previously described
(29).
Preparation of extraceUlular proteins for high-performance liquid chromotography (HPLC) and SDSpolyacrylamide gel electrophoresis (PAGE). Strains to be examined for extracellular proteins were each grown in 500 ml of tryptose minimal medium (2) in 2liter flasks for 18 h at 32°C on a gyratory shaker (-100 rpm). Cultures were centrifuged at 5,000 rpm for 10 min to remove the cells. The proteins in the supernatants were precipitated with ammonium sulfate to 70%o saturation. The precipitated proteins were dissolved in 5 ml of 10 mM Tris-hydrochloride (pH 7.2) and dialyzed against the same buffer for 12 h at 4°C. The samples were adjusted to identical volumes for further study. HPLC of extracellular proteins. High-performance size exclusion chromatography of extracellular proteins was done with a Beckman model 334 liquid chromatograph equipped with a 60-cm Spherogel TSK-2000 column (Altex Scientific, Berkeley, Calif.). An isocratic buffer system composed of 50 mM Trishydrochloride buffer, pH 7.2, in 5% (vol/vol) glycerol was used. Absorbance at 280 nm was monitored with a Hitachi model 100-40 spectrophotometer equipped with a liquid flow cell. Retention times and peak assignments were made with an Altex model C-R1A recorder-integrator that was interfaced with the liquid chromatograph. The column was calibrated by chromatographing protein standards and plotting the log M, of each standard against its retention time. The protein standards included phosphorylase b (Mr = 94,000), bovine serum albumin (M, = 68,000), ovalbumin (Mr = 43,000), carbonic anhydrase (Mr = 30,000), soybean trypsin inhibitor (M, = 20,100), and a-lactalbumin (M, = 14,400). The retention times for the above proteins were 11.68, 16.05, 18.48, 20.52, 25.25, and 27.92 min, respectively. SDS-PAGE. SDS-PAGE was done as described previously (9). Protein bands were developed with ammoniacal silver nitrate as described by Oakley et al. (24).
RESULTS Isolation of an E. coli clone carrying the phospholipase C gene from P. aeruginosa. The BamHI and SalI genomic libraries were first screened for phospholipase C production. Each of the clones was inoculated into 0.1 ml of tryptose minimal medium in microtiter plates and incubated for 18 to 24 h at 32°C. Enzyme production was assayed by the hydrolysis of the artificial substrate p-nitrophenylphosphorylcholine, which yields a yellow color (2). There was no clone found to produce phospholipase C by this method. At this point it was considered that the intact gene was not cloned, that it was not expressed, and that the phospholipase C was not secreted in E. coli. E. coli produces few extra-
434
J. BACTERIOL.
VASIL ET AL. EcoR I
BamH I
~ Tc rApr pBR322 pBR322 2.9 M del
AA~
I
P. aeruginosa PAOI Chromosome BSmHl
Complete digestion BsmH 1\4
|T4 Ligase 4.1 Mdai insertion of PAOI DNA
N T4 Ligase EcoR I
SamHoRi I
BsmHl
asmHl1 T4 Ligse
pMW7O
sPic+MHSy2
9
.6 Mdc
6
BCmHH/Bglll EcoR I
pVB-S1
Cbr TcO mr 8ur Pic*(HIy+) 12.7 M dal
Smr
Cbr
aBmH I
EcoRli
FIG. 1. Construction of recombinant plasmids (pGV26 and pVB81) carrying a 4.1-Mdal BamHI-derived fragment (-) from P. aeruginosa genomic DNA and subcloning of a 2.4-Mdal BamHVBgllI fragment of the 4.1Mdal fragnent to pBR322. The 4.1- and 2.4-Mdal fragments both carry the gene for the synthesis of phospholipase C (Plc), which is a hemolytic (Hly+) enzyme.
cellular proteins, and transported proteins are usually exported only as far as the outer membrane (18). Thus, it was decided that the banks be rescreened after the E. coli cells were lysed either by artificial means (e.g., chloroform) or by natural death and lysis. The easiest way to do this is to inoculate the clones on blood agar and allow them to grow for several days. Fortunately, the host strain HB101 is not hemolytic on sheep blood agar even after several days. Thus, the banks were rescreened by inoculation of sheep blood agar made with a peptone basal medium which had been demonstrated to support phospholipase C (hemolysin) production
(Berka and Vasil, unpublished data). After several days, one slowly growing clone from the BamHI bank produced hemolysis. Subsequent restreaking of this clone on blood agar demonstrated that the isolated colonies were betahemolytic. This clone was further tested for phospholipase C production. Practically all of the enzyme activity, unlike in P. aeruginosa, was cell associated, confirming our suspicion that our failure to detect this clone previously was because the enzyme was not transported to the extracellular space. Easily detected levels of phospholipase C activity were found when stationary-phase (18- to 24-h) E. coli cultures were
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CLONING OF A HEMOLYSIN GENE FROM P. AERUGINOSA
lysed with lysozyme, EDTA, and SDS or with chloroform. The enzyme had all of the properties of the enzyme synthesized by P. aeruginosa (Table 2), and the enzyme activity from the E. coli clone was neutralized by antiserum to P. aeruginosa phospholipase C (Table 2). Characterization of the DNA carrying the hemolysin gene and transfer to a P. aeruginosa hostvector system. A 7.0-megadalton (Mdal) plasmid designated pGV26 was isolated from the HB101 Hly+ cells. A BamHI digest of pGV26 contained two fragments: the 2.9-Mdal band corresponding to the molecular weight of the pBR322 vector and a 4.1-Mdal fragment, presumably of P. aeruginosa origin (Fig. 2A). That the 4.1-Mdal fragment was in fact P. aeruginosa DNA was confirmed by Southern blot hybridization (29) of the nick-translated 32P-labeled (27) pGV26 plasmid with BamHI-digested P. aeruginosa PA01 genomic DNA and the lack of a detectable signal with E. coli DNA (Vasil, unpublished data). The hybridization results were positive with a fragment of PA01 genomic DNA which had a mobility identical to the 4.1-Mdal fragment from pGV26. HB101 was transformed to ampicillin resistance by pGV26, and 100% of the Apr transformants tested produced the hemolytic enzyme. The 4.1-Mdal fragment is of more than sufficient size to encode for the phospholipase C which has recently been purified in this laboratory and found to be a 78,000-dalton protein (2a).
TABLE 2. Comparison of the cloned phospholipase C (heat-labile hemolysin) in E. coli HB101(pGV-26) and the native enzyme from P. aeruginosa PA01" Characteristic PA01 HB101(pGV26) Heat-labile reactivity with + +
NPPCb
Resistance to 2% SDS + + Resistance to 1% 1+ + mercaptoethanol + Sensitivity to 2 M urea + + Sensitivity to 1 mM ZnCl2 + Neutralization of enzyme activ- + + ity by antiserum against P. aeruginosa phospholipase C aHB101(pGV26) cells (20 ml) were grown for 18 to 24 h, centrifuged, treated with 10 ml of STET buffer (8% sucrose, 5% Triton X-100, 50 mM EDTA, 50 mM Tris, pH 8.0), and incubated for 15 min at 370C. Lysozyme (1 ml; 10 mg/ml in water) was added, and the mixture was incubated for 15 min at 37°C. SDS (1 ml; 10%) was then added, and the mixture was incubated at 37°C for an additional 15 min. Enzyme from P. aeruginosa PAO1 was previously purified in this laboratory from culture supematants, and the above properties have been documented elsewhere (2a). b Enzyme activity was measured as described previously (2).
435
A 1
2 3 4 mw
B Mdal w-.---.-
mw I
2
1----6.4----
-6.4-~ 4.32. 9/"'
1.6 -
1.4
FIG. 2. Restriction endonuclease cleavage analysis of hybrid plasmids carrying the phospholipase C gene. (A) Restriction analysis of recombinant plasmids derived from E. coli: lanes-1, BamHI digestion of pBR322; 2, BamHI digestion of pGV26; 3, BamHI and BglII digestion of pGV26; 4, BamHI and BglII digestion of pRM82; MW, HindIII digestion of X. (B) Restriction analysis of recombinant plasmids derived from P. aeruginosa: lanes-MW, HindIII digestion of X; 1, BamHI digestion of pMW79; 2, BamHI and BglII digestion of pVB81. Arrows indicate BamHI and BglII fragments of pVB81.
A preliminary restriction map of the 4.1-Mdal cloned P. aeruginosa DNA is shown in Fig. 3. This map was constructed with the pGV26 plasmid from E. coli. While constructing a restriction map of the cloned P. aeruginosa DNA (Fig. 3), we found that there was a single BglII site at 2.4 Mdal in the cloned DNA which offered us an opportunity to reduce its size and somewhat localize the phospholipase C gene on the 4.1Mdal insert. To do this, the pGV26 plasmid was digested with BamHI and BglII and then the DNA was religated (Fig. 1). The religated DNA was then transformed to HB101, and Apr Hly+ clones which contained plasmids smaller than the pGV26 were chosen for further study. It was found that the phospholipase C activity was associated with a 5.3-Mdal plasmid designated pRM82 (Fig. 1 and 2A) and not with a plasmid carrying the 1.7-Mdal fragment from pGV26, indicating that the phospholipase C gene was located on the 2.4-Mdal end of the cloned fragment. When the 2.4-Mdal BamHI/BglII fragment was ligated to BamHI-digested pBR322, the BglII site was lost since digestion of pRM82 with BamHI and BglIl only yielded a linear 5.3Mdal molecule (Fig. 2A).
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VASIL ET AL.
J. BACTERIOL.
I
aI
I Ir-
pBR322
I
w
I\
/1
-0/0
\
1
Snai XheI
B a m H 1mal
/ II
0
0.1
Bgl PstI IIII
Kpni
EcoR1BamH 1
Pvull
II
x
I
I
1.5
1.9
3.6
2.4
BamHl
.1
pBR322
4.1 Mdal
\
Pvull Kpnl
p I I -1 0.7 0.8 0.9
1
FIG. 3. Restriction endonuclease cleavage map of the 4.1-Mdal fragment derived from PAO1 genomic DNA and which carries the phospholipase C gene. The cloned fragment was not cleaved by EcoRI, HindIII, XorII, XbaI, or ClaI.
With the recent availability of a P. aeruginosa host-vector system (34), it was possible to reclone the 4.1-Mdal DNA fragment carrying the phospholipase C from pGV26 and transfer the new recombinant plasmid (pVB81) to P. aeruginosa. This was do-ne as described in Fig. 1 and in Materials and Methods. The new hybrid plasmid, designated pVB81, directed the synthesis of substantially increased levels of phospholipase C in the rec-2 host strain PA02003. The pVB81 plasmid appears to carry the same size insert as the pGV26 plasmid (Fig. 1 and 2). However, since PA02003 is not a modificationless strain, it is somewhat difficult to digest the pVB81 plasmid isolated from P. aeruginosa with some restriction enzymes. Even when the plasmid is digested with as much as 5 U/Ipg for 24 h with BglII, it is only partially digested by this enzyme, but it is fully digested with BamHI (Fig. 2B). Similar results are seen when the pVB81 is digested with SalI. Upon digestion of the pVB81 plasmid with Sail, there are only two fragments seen by electrophoresis. The pMW79 vector only has one Sall restriction site in the Tcr gene, like pBR322. In contrast, digestion of the pGV26 plasmid isolated from E. coli HB101, a modificationless strain, yields at least seven fragments. Expression of the cloned phospholipase C gene in E. coli and P. aeruginosa. The availability of the phospholipase C gene on a multicopy plasmid in E. coli and P. aeruginosa offered an opportunity to compare the expression of this hemolytic enzyme in these two organisms. In P1rich cultures of E. coli, the phospholipase C was expressed at detectable levels, but the majority (-90%) of the enzyme activity remained cell associated (Table 3). This is consistent with the observation that HB1O1(pGV26) only becomes hemolytic on blood agar after 3 to 4 days of incubation. When the pGV26 plasmid was transformed to other E. coli strains, including a strain which has a chromosomal mutation leading to hyperproduction of plasmid-directed synthesis of E. coli enterotoxin (4; kindly supplied by R. K. Holmes), the levels of phospholipase C were not changed compared with HB101. There also were no differences in levels of enzyme
activity when HB1Ol(pGV26) was grown in P1deficient or Pi-rich media (data not shown), indicating that in E. coli synthesis of the phospholipase C is not regulated by Pi. Interestingly, when HB1O1(pGV26) was grown in the presence of autoclaved chlortetracycline, there was a reproducible 25% increase in the total phospholipase C activity (Table 3), suggesting that its synthesis in E. coli is directed from the pBR322 Tcr promoter. Inducibility of the pBR322 Tcr TABLE 3. Quantitation of the expression of the gene in E. coli and P. aeruginosa Phospholipase
phospholipase C
Strain
C activity' 0.5 h 18 h
E. coli HB101(pBR322) NDb Extracellular ND Cell associated E. coli HB101(pBR322)-CTc ND Extracellular ND Cell associated E. coli HB101(pGV26) ND Extracellular ND Cell associated E. coli HB101(pGV26)-CT ND Extracellular ND Cell associated P. aeruginosa PA02003(pMW79) 0.6 Extracellular Cell associated 0 P. aeruginosa PA02003(pMW79)-CT 0.5 Extracellular Cell associated 0 P. aeruginosa PA02003(pVB81) Extracellular 393.5 Cell associated 2.1 P. aeruginosa PA02003(pVB81)-CT Extracellular 442.8 Cell associated 0
0 0 0 0 17.8 121.0
25.8 151.0 77.6 ND 77.8 ND
ND ND ND ND
a Activity is expressed as the absorbance at 405 nm divided by the optical density of the culture at 590 nm (2). b ND, Not determined. c CT refers to growth in the presence of 20 ,ug of autoclaved chlortetracycline per ml as described previously (20).
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protein has been previously reported (P. W. Rueckert and F. Reusser, Abstr. Annu. Meet. Am. Soc. Microbiol. 1982, H40, p. 119). Similar results with the pVB81 plasmid in PA02003 were found, perhaps not surprisingly, since the pMW79 vector is a hybrid plasmid of pBR322 and RSF-1010 (34). Additional support of the idea that phospholipase synthesis is being directed from the Tcr promoter is that the synthesis of the enzyme from the chromosomal gene in PA02003(pMW79) is not increased in the presence of autoclaved chlortetracylcline (Table 3). As in E. coli, the plasmid-directed synthesis in P. aeruginosa PA02003(pVB81) is not P1 regulated; however, >95% of the enzyme activity from P. aeruginosa is extracellular in contrast to E. coli HB101(pGV26) enzyme. On low-Pi blood agar plates, after only 24 h, the hemolytic zone surrounding PA02003(pVB81) is 20 to 50 times greater in surface area than that surrounding PA02003(pMW79) (data not shown). The levels of enzyme activity from PA02003(pVB81) in P,rich media are 500 to 1,000 times the level of PA02003(pMW79), whereas in Pi-deficient media the levels of enzyme produced by PA02003(pVB81) are 10 to 20 times that found in cultures of PA02003(pMW79) (Table 3; Vasil and Berka, unpublished data). These data are consistent with previously reported results which indicate that phospholipase C production is 20- to 100-fold repressible by Pi, depending on the conditions and methods used for assay
437
to the phospholipase C there are five other proteins which appear to have been cloned along with this enzyme on the 4.1-Mdal piece of DNA. The molecular weights of these proteins are 37,000, 34,000, 31,000, 29,000, and 28,000 and are indicated by arrows on Fig. 4B. The HPLC profile also indicates that there is an increase in extracellular protein from PA02003(pVB81) in this molecular weight range (Fig. 4A). Interestingly, three of these proteins have the same molecular weight (37,000, 34,000, and 31,000) as three Pi-repressible extracellular proteins from P. aeruginosa PAO1 (8, 9). It is possible that one or more of these proteins function in binding or transport or both of Pi. In support of this hypothesis is the observation that the rate of P1 transport by PA02003(pVB81) is fourfold greater than that of PA02003(pMW79) (Vasil and Berka, unpublished data). It should be noted that the conditions used for growth in these experiments were the same as those used for the study of extracellular Pi-repressible proteins in P. aeruginosa (8, 9). A protein band with increased intensity at 78,000 daltons corresponding to the known molecular weight of phospholipase C can be seen in the PAO2003(pVB81) track in Fig. 4B, supporting the HPLC data and indicating that the entire phospholipase C gene has been cloned. While performing some of the above experiments, we noticed that PA02003(pVB81) appeared to be producing a substantially greater amount of a blue-green pigment than did PA02003(pMW79). Further (8, 9). Comparisons were made of the extracellular investigation revealed that this blue-green pigproteins from Ps-deficient cultures of ment was pyocyanin, which is a phenazine pigPA02003(pMW79) and PA02003(pVB81), using ment produced by P. aeruginosa. Interestingly, HPLC and SDS-PAGE (Fig. 4A and B). The it has been previously reported that the producHPLC chromatogram reveals that there is a tion of pyocyanin is repressed by Pi in the substantial increase in protein in the 75,000- to medium (14). We found that the production of 80,000-dalton range from the extracellular pro- pyocyanin by PA02003(pVB81) was 10- to 14-fold teins of PA02003(pVB81) compared with increased compared with PA02003(pMW79) and PA02003(pMW79) (Fig. 4A). Assay of the frac- that production of pyocyanin was only repressed tions collected from the HPLC indicated that the 2-fold by >10 mM Pi (Berka and Vasil, unpubmajority of the phospholipase C activity from lished data). Because the synthesis of pyocyanin both chromatograms eluted between 11 and 13 involves an extensive pathway, it is unlikely that min. When the total enzyme activity from frac- we have cloned genes directly involved in the tions eluting between 10 and 16 min were as- synthesis of pyocyanin, but rather it seems sayed, it was found that the activity from the likely that we have cloned a gene involved in the PA02003(pVB81) fractions was 143 U/ml (1 U = regulation of pyocyanin production (2a). 1 ,mol of p-nitrophenylphosphorylcholine hyUsing 135 as an average molecular weight for drolyzed per min), whereas that from the an amino acid, the amount of DNA necessary to PA02003(pMW79) chromatography fractions code for all of the proteins identified above was 7 U/ml. These data taken together indicate would be 6.2 kilobases (4.1 Mdal), which is that PA02003(pVB81) is synthesizing approxi- essentially the same size of cloned DNA carried mately 20 times more enzyme than by pGV26 or pVB81. PA02003(pMW79) and that the gene for phosDISCUSSION pholipase C was cloned intact. Examination of SDS-PAGE profiles (Fig. 4B) In this report we have described the construcof extracellular proteins of PA02003(pVB81) and PA02003(pMW79) revealed that in addition tion of hybrid plasmids carrying several chromo-
438
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VASIL ET AL.
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FIG. 4. HPLC and SDS-PAGE of extracellular protein produced in low-Pi medium (tryptose minimal medium) by PA02003(pMW79) and PA02003(pVB81). (A) HPLC chromatogram of extraceliular protein. Retention times for major protein peaks are designated by numbers above the peaks, and elution positions for the molecular weight standards are designated by the molecular weights with arrows. Protein samples were prepared as described in the text, and 1.0 ml of each of the samples was injected onto the gel permeation column TSK2000. (B) SDS-PAGE of the same samples of extracellular proteins; 10 1l of sample was applied to each lane. The relative mobility of the molecular weight standards is designated on the left side, and the positions of the proteins which have been proposed to be coded for by the 4.1-Mdal BamHI fragment of PA01 genomic DNA are indicated by arrows. (Lane 1) Extracellular proteins of PA02003(pMW79); (lane 2) extracellular proteins of
PAO2003(pVB81). somal genes from P. aeruginosa, including one which directs the synthesis of the heat-labile hemolysin (phospholipase C). Data have been presented that indicate that the entire phospholipase C gene was cloned intact and that the synthesis of the enzyme from the recombinant plasmids is not regulated by Pi in E. coli or P. aeruginosa as is the chromosomal gene in P. aeruginosa. At least one likely reason for the lack of P1 control of the cloned gene expression is that synthesis is directed from the Tcr gene promoter in pBR322 or pMW79. In support of this hypothesis is the observation that the plasmid-directed synthesis of phospholipase C is
partially inducible by autoclaved chlortetracycine, a known inducer of the Tcr gene. Despite the fact that the plc gene was on a multicopy plasmid, the level of activity of the phospholipase C from E. coli was only a little more than twice that of the P. aeruginosa chromosomal gene-directed synthesis under repressed conditions. However, it is not possible to tell at the present time whether this is a result of reduced expression of a Pseudomonas gene in E. coli, as has been previously reported (11, 12), or whether the activity of the synthesized enzyme is reduced, or both. Because the synthesis of plasmid phospholipase C is most likely directed from
VOL. 152, 1982
CLONING OF A HEMOLYSIN GENE FROM P. AERUGINOSA
the Tcr promoter and not from its own Piregulated promoter, it may be that the phospholipase C protein in E. coli is a fusion product with the Tcr protein. Alternatively, because phospholipase C is an extracellular protein in P. aeruginosa, it is also possible that the enzyme synthesized in E. coli has a leader sequence that is not cleaved by E. coli proteases, thereby reducing its potential enzyme activity (e.g., proenzyme). It is interesting to note at this point that the enzyme from E. coli has both hemolytic activity and phospholipase C activity. If, in fact, the phospholipase C has a leader sequence and is fused with a Tcr protein, it seems remarkable that P. aeruginosa is capable of handling the increased synthesis from the multicopy plasmid and is capable of efficiently processing and secreting relatively large amounts of the protein in its native form. Based on the data presented in Fig. 4B, three genes of particular interest, in addition to the phospholipase C gene, which code for proteins with molecular weights of 37,000, 34,000, and 31,000 have also been cloned. The interesting aspect of these proteins is that their molecular weights are identical to three previously described Pi-repressible proteins from P. aeruginosa (8, 9; R. E. W. Hancock, personal communication). The conditions for production of these extracellular proteins were identical in all cases. Thus, it is tempting to speculate that phospholipase C is a part of a Pi regulon similar to that reported in E. coli (32) and that the gene for this hemolytic enzyme shares a common promoter with several other Pi-regulated genes, some of which were cloned on the 4.1-Mdal piece of DNA. The function for these other Pi-regulated proteins is not totally clear; however, it appears that we have cloned a gene which regulates the production of pyocyanin, a secondary metabolite which has been shown to be regulated by Pi (14), and that we have cloned a gene which encodes a Pi transport or Pi-binding protein (Hancock, personal communications; Vasil and Berka, unpublished data). It is clear that the alkaline phosphatase gene of P. aeruginosa was probably not cloned on the 4.1-Mdal insert because no alkaline phosphatase activity could be detected in Pi-rich cultures of PA02003(pVB81) (Vasil and Berka, unpublished data). In this regard, it has recently been shown that the synthesis of alkaline phosphatase and phospholipase C are coordinately regulated by a single genetic locus in P. aeruginosa (8, 9). Therefore, if both alkaline phosphatase and phospholipase C are part of the same operon in P. aeruginosa, it may be that alkaline phosphatase is more proximal to the promoter in relation to phospholipase C. The availability of recombinant plasmids car-
439
rying the phospholipase C gene of P. aeruginosa should facilitate studies on the role of this hemolytic enzyme in the pathogenesis of Pseudomonas infections and its function in the Pi-scavenging system of P. aeruginosa. Further studies which could be of interest, using the recombinant plasmid described herein, are as follows. (i) With regard to the study of the role of the hemolysin in the virulence of P. aeruginosa, it should be possible to: (a) induce, in vitro or in vivo, an inactivating mutation in the structural gene for hemolysin (16, 28); (b) transfer this mutant gene to a variety of P. aeruginosa clinical strains from different sources; and (c) isolate stable recombinants which are altered only in hemolysin (phospholipase C) activity (16). It would then be possible to more precisely assess the role of heat-labile hemolysin in Pseudomonas pathogenesis by comparing virulence of such recombinant strains with their parental strains in several well-characterized experimental animal models (5, 25, 31, 37). (ii) With the cloned hemolysin gene from P. aeruginosa as a probe, it is now possible to do comparative homology studies on the various hemolysins of gram-negative bacteria at the molecular level. (iii) Because of the Pi-repressible nature of phospholipase C synthesis, it will be of interest to use the cloned plc gene as well as the genes for the other Pi-repressible proteins and a series of recently well-characterized Pi regulatory mutants (2, 8, 9) to examine the genetic and physiological mechanisms of the regulation of Pi-repressible proteins in P. aeruginosa. The restriction map shown in Fig. 3 should be useful for subcloning the phospholipase C gene and insertional inactivation of this gene and any other gene of interest found on this DNA. This map also is probably sufficient to sequence at least the phospholipase C (Hly+) gene of P. aeruginosa. Finally, because the phospholipase C has several unusual properties, including a significant number of ornithine and hydroxyproline residues, unusually avid hydrophobicity, and a strong preference for phospholipids with substituted ammonium groups (2a), it will be of interest to obtain a DNA sequence of the gene for studies of the structure-function relationships of this enzyme as well as of other similar hemolysins. ACKNOWLEDGMENTS This study was supported by Public Health Service grant AI15940 from the National Institute of Allergy and Infectious Diseases to M.L.V. We thank Peter Melby for excellent technical assistance. LITERATURE CITED 1. Bachmann, B. J., and K. B. Low. 1980. Linkage map of Escherichia coli K-12, edition 6. Microbiol. Rev. 44:1-56. 2. Berka, R. M., G. L. Gray, and M. L. Vasil. 1981. Studies
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