Vol. 172, No. 2

JOURNAL OF BACTERIOLOGY, Feb. 1990, p. 942-948 0021-9193/90/020942-07$02.00/0 Copyright © 1990, American Society for Microbiology

Cloning of the Pseudomonas aeruginosa Alkaline Protease Gene and Secretion of the Protease into the Medium by Escherichia coli JEAN GUZZO, MARYSE MURGIER, ALAIN FILLOUX, AND ANDREE LAZDUNSKI* Laboratoire de Chimie Bacterienne, Centre National de la Recherche Scientifique, 31, Chemin Joseph Aiguier, 13402 Marseille Cedex 9, France Received 11 July 1989/Accepted 8 November 1989

Pseudomonas virulence is thought to depend on multiple characteristics, including the production of an extracellular alkaline protease. We report the isolation, from a PAO1 DNA genomic bank, of a cosmid carrying the structural gene coding for alkaline protease. By in vivo mutagenesis using transposon Tnl735, which functions as a transposable promoter, the expression of an 8.8-kilobase DNA fragment under control the tac promoter was obtained. When expressed in Escherichia coli, active alkaline protease was synthesized and secreted to the extracellular medium in the absence of cell lysis. phatase. P. aeruginosa was grown in tryptic soy broth (TSB) and tryptic soy agar (TSA) (Difco). Pseudomonas isolation agar (Difco) supplemented with the appropriate antibiotics was used to select for P. aeruginosa in the matings experiments. The strains were grown at 37°C with aeration. Antibiotics were used in selective media at the following concentrations (in micrograms per milliliter): tetracycline, 20 (E. coli) and 100 (P. aeruginosa); chloramphenicol, 50 (E. coli) and 200 (P. aeruginosa); ampicillin, 50 (E. coli); and kanamycin, 25 (E. coli). Isolation of mutants. Ethyl methanesulfonate mutagenesis of strain PA103 was done by the method of Watson and Holloway (35). Protease-negative mutants were examined for the absence of zones of clearing on skim milk-agar plates (15% [vol/vol] in TSA). Colonies that produced no halo of hydrolysis after 48 h were chosen for further study. Transfer of plasmids from E. coil to P. aeruginosa. Triparental matings were performed to promote transfer of recombinant cosmids from E. coli 1046 to the different PA103 protease-negative mutants. pRK2013 served as the helper plasmid for mobilization (7). Transconjugants were selected on Pseudomonas isolation agar plates containing tetracycline and tested for a protease-positive phenotype on skim milk-agar plates. Identification of strains producing PAO1 alkaline protease. An agar well assay developed by Ohman et al. (28) was used. Colonies were patched onto TSA plates. Small wells were made in the medium approximately 5 mm from each patch with a sterile Pasteur pipette. Each well was filled with approximately 2 RI of anti-alkaline protease antiserum, and the plates were incubated at 37°C for 48 h. Clones producing sufficient amounts of PAO1 alkaline protease had a distinct band of precipitation between the colony and the adjacent antiserum well. Transposon mutagenesis. The transposons used were Tnl735 Cm and Tnl725. Transposon Tnl735 Cm promotes insertion-mediated fusions of the strong tac promoter with genes or operons of interest, their expression being controlled by the lac repressor and inducible by isopropyl,B-D-thiogalactopyranoside (IPTG) (32). This transposon was carried by the large conjugative F'102 plasmid, giving rise to plasmid pRU681 (32). Transposon mutagenesis was based on the method described by Ubben (32), with slight modifications.

Pseudomonas aeruginosa is an opportunistic pathogen that can cause fatal infections in compromised hosts. This virulence is related to the secretion of several extracellular proteins, some of which are virulence factors (20, 36). P. aeruginosa secretes two proteases, elastase and alkaline protease. It has been shown that alkaline protease is required for the establishment of corneal infections (17); it can also inhibit the antiviral and immunomodulatory activities of human gamma interferon (16). The enzyme has been purified, crystallized, and characterized (26, 27), yet very little information is available about its structure. Knowledge of the alkaline protease gene sequence would help clarify questions regarding function, processing, and secretion of the protease. This last point is of particular interest because genetic studies have shown that alkaline protease has its own specific and independent secretion pathway (10, 37). The secretion of proteins into the medium (hereafter referred to simply as secretion) has been extensively studied in gram-positive bacteria, which do not have an outer membrane. The mechanism of protein secretion in gramnegative bacteria, which involves the crossing of both the cytoplasmic and outer membranes, is still poorly understood. Several specific and independent pathways appear to be used for the secretion of different proteins in various gram-negative bacteria (29). We have chosen to study the alkaline protease of P. aeruginosa as an example of a protein secreted by gram-negative bacteria. In this study, we report the cloning of the alkaline protease structural gene (apr) on a cosmid with an insert of approximately 20 kilobase pairs (kb). By in vivo mutagenesis using a TnJ721-derivative transposon that functions as a transposable promoter, we succeeded in obtaining the expression of an 8.8-kb DNA fragment under control of the tac promoter. When expressed in Escherichia coli, active alkaline protease is synthesized and secreted into the medium in the absence of cell lysis and without prior intracellular accumulation. MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used are listed in Table 1. Media and antibiotics. E. coli was grown in LB broth, LB agar, and Proteose Peptone (Difco Laboratories, Detroit, Mich.) glucose medium for the assay of alkaline phos*

Corresponding author. 942

ALKALINE PROTEASE OF P. AERUGINOSA

VOL. 172, 1990

TABLE 1. Bacterial strains and plasmids Strain or plasmid

Strains E. coli 1046 DH1 RR1

Genotype or phenotype

met supE supF hsdS recA thi endA hsdRJ7 recA gyrA96 supE44 relAl (X) leu pro thi rpsL hsdR hsdM

reference

24 13 33

lacZAM15(F' lacIqZAM15 pro+) P. aeruginosa

PAO1

Prototroph Elastase-deficient prototroph Protease-negative mutant

14 28 This study

Cosmid vector derived from pLAFR1; IncPl X cos+ rlx

11

pRK2013 pUC19 pRU681

Tcr ColEl with tra (RK2)+ Kmr ColEl Apr lacI ¢80dlacZ F'102::Tnl735 Cm (ptac/lacIq) Cmr argG+

7 38 32

pRU669

tra' IncF Rtsl::Tnl 725 Cmr Kmr tra+(Ts) IncT

31

PA103 PA103-11 Plasmids pLAFR3

pJA67

pJAT367 pJUK70 pJUE25 pJUEK72

pJUB45

21-kb PA01 DNA insert in pLAFR3 Tn1735 Cm insertion in pJA67 7-kb KpnI insert in pUC19 2.5-kb EcoRI insert in pUC19 8.8-kb KpnI-EcoRI insert in pUC19 4.5-kb KpnI-BamHI insert in pUC19

This study This study

This study This study This study

This study

E. coli DH1(pRU681) was transformed with the target plasmid pJA67 (selection for Tc), and single transformants were grown at 30°C in Luria broth containing chloramphenicol (50 jig/ml) for transposon selection. To isolate recombinant plasmids containing transposon insertions, we used a technique taking advantage of the ability of bacteriophage P1 to package high-molecular-weight plasmids during propagation on E. coli. Overnight cultures of E. coli containing both plasmids were used to prepare lysates of P1; such lysates were used to transduce recombinant plasmids into E. coli 1046. Plasmids that sustained a transposon insertion were isolated by selection for transductants on nutrient agar containing chloramphenicol (50 ,ug/ml) and tetracycline (15 ,ug/ml). They were then tested on skim milk plates containing IPTG (1 mM) and chloramphenicol. Protease-positive clones were purified and cultivated for (i) agar well assays, (ii) quantitative assays of IPTG-induced alkaline protease, and (iii) restriction mapping of the pJA67::TnJ 735 Cm insertions. Mutagenesis with Tnl725 was carried out as described previously (9). Expression of genes under control of the tac promoter. E. coli 1046 containing pJAT367 was grown at 37°C overnight in LB medium containing appropriate antibiotics. Cells were diluted 100-fold in LB medium and grown to an optical density at 600 nm (OD6w) of 0.2. Plasmid-borne genes downstream of the tac promoter were induced by the addition of 1 mM IPTG. When experiments were carried out on solid medium, skim milk-agar plates plus 1 mM IPTG were used. Preparation of cell lysates and supernatants. Cells of E. coli

943

and P. aeruginosa were harvested by centrifugation, and the culture supernatant was rapidly frozen. The cells were washed in 10 mM Tris hydrochloride (pH 8) and then sonicated on ice with four 15-s pulses. After centrifugation at 10,000 x g for 10 min, the cell lysates were collected and frozen. When necessary, cell lysates and supernatants were concentrated by precipitation with 17% trichloroacetic acid overnight at 4°C. Samples were then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) and immunoblotting. SDS-PAGE and immunoblotting. SDS-PAGE was done by the method of Lugtenberg et al. (22). Separating gels contained 11% acrylamide. Purified PAO1 alkaline protease was from Nagase Biochemicals, Japan. Antiserum against this protease was prepared as previously reported (10). Immunoblotting was done as described elsewhere (10). Enzyme assays. P-Galactosidase activity was assayed by the o-nitrophenyl-3-D-galactopyranoside assay as described by Miller (25). Alkaline phosphatase was determined as previously described (8). Alkaline protease was determined by the sensitive assay described by Howe and Iglewski (17), using Hide powder azure (Sigma Chemical Co., St. Louis, Mo.). DNA procedures. Small-scale purifications (1- to 10-ml culture volumes) of plasmid DNA from E. coli were performed by a modification of the rapid-boiling method (15). Large-scale preparations were carried out by the method of Bimboim and Doly (3), followed by cesium chloride gradient centrifugation (24). Restriction endonucleases, T4 DNA ligase, and bovine intestinal phosphatase were used as recommended by the manufacturer (Boehringer GmbH, Mannheim, Federal Republic of Germany). Plasmids were introduced into calcium chloride-treated E. coli (19). Gel electrophoresis in 0.6% agarose was performed in Trisborate-EDTA buffer (24). Construction of recombinant plasmids. Figure 1 shows a restriction map of the 20-kb DNA fragment in pJA67. The 7-kb KpnI2-KpnI3 fragment and the 4.5-kb KpnI2-BamHI3 fragment were prepared and ligated into pUC19 behind the lac promoter to give, respectively, pJUK70 and pJUB45. The subclone pJUEK72 was generated in two steps. First, a 2.5-kb EcoRI4-EcoRI5 fragment was subcloned in pUC19 and deleted from the 500-base-pair EcoRI4-KpnI3 fragment. Second, the 7-kb KpnI2-KpnI3 insert in the proper orientation was ligated into the deleted plasmid to reconstruct the entire KpnI2-EcoRI5 fragment from pJA67 behind the lac promoter on pUC19. Quantitation of alkaline protease antigen. Supernatants and cell lysates were assayed by an enzyme-linked immunosorbent assay (ELISA) specific for detection of the P. aeruginosa alkaline protease antigen. The technique used was based on the indirect competition assay described by Clark et al. (5), with slight modifications. RESULTS Cloning of the apr gene. A genomic library of P. aeruginosa PAO1 DNA had previously been constructed in the broad-host-range cosmid pLAFR3 (1, 9). The recombinant plasmids were maintained in E. coli 1046 as separate clones. To isolate the apr gene from the genomic bank, we needed Pseudomonas mutants specifically deficient in alkaline protease. Unfortunately, P. aeruginosa PA01 produces elastase together with alkaline protease. Because alkaline protease does not possess a unique substrate specificity, it is

944

GUZZO ET AL.

H1 H2

B1

I~ ~

K1 B2 E1 K2E2

II

E3

Name

B3 E4K3 Bg1C5 I II I

E coZi Apr

phenotype

K2

U~

pLAFR3

I

Alkaline protease

C.R.M

pJA67

/\ /\w 2 14

plLAFR3

m

J. BACTERIOL.

+

K3

1U

I__

pJAT367

+

pJUB45

_

pJUEK72

+

_

ltwc

K2

BE (4.5)

1 kb %---

____- I(8.8)

+

K3

K2

pJUK70

(7)

K2

pLAFR3

PA 103-1 1 Apr

phenotype

E3 B3 E4

pJAT767

Tn 1725

FIG. 1. Restriction endonuclease map of the 20-kb chromosomal DNA fragment inserted into the BamHI site of pLAFR3. Plasmid DNA represented by the heavy line. Triangles under the line indicated the sites of TnJ735 Cm insertions, as determined by restriction endonuclease analysis. The numbers of insertions located in a given region are indicated under the triangles. Arrows indicate the direction of transcription from the tac promoter on the transposon. The proteolytic phenotype was determined by halo formation on skim milk-agar plates. Number in parentheses are in kilobases. is

difficult to isolate mutants specifically affected in this protease. To circumvent this problem, we chose to isolate mutants in strain PA103, which is naturally elastase deficient (28). Alkaline protease-negative mutants were obtained after ethyl methanesulfonate mutagenesis. A total of 20,000 colonies were examined. Eighteen presumptive mutants were isolated that produced no zones of clearing on skim milkagar plates. When the mutants were grown in TSB liquid medium, the amount of residual alkaline protease produced was variable and ranged form 0 to 40% of the parental level. Previous studies had shown that mutants isolated on the basis of protease deficiency are often affected in the secretion of exoproteins (37). For this reason, we examined the production of three other extracellular products (phospholipase, alkaline phosphatase, and lipase). Synthesis and secretion of these proteins were not affected (data not shown). Ten mutants were kept for further studies; they may carry mutations in structural or regulatory genes. Some residual production of alkaline protease by strain PA103 was not a significant problem because we could distinguish between

the proteases synthesized by strains PA103 or PAO1. Indeed, our PAO1 anti-alkaline protease antiserum did not react with the protease from strain PA103. This result was confirmed by ELISA and agar well assays (Fig. 2). It was thus possible to recognize in strain PA103 the alkaline protease produced by an apr recombinant plasmid from PAO1. Hybrid plasmids were mobilized from E. coli to the various protease-negative mutants and examined for restoration of the protease-positive phenotype on skim milk-agar plates. Three cosmids that gave a protease-positive phenotype were identified. Only one of them encoded a protease reacting with the PAO1 anti-alkaline protease antiserum (Fig. 2). This result was confirmed by ELISA. It can therefore be considered that this cosmid, pJA67, carried the PAO1 apr gene. Restriction analysis of this plasmid showed a DNA insert of approximately 20 kb (Fig. 1). apr expression in E. coli and localization of the apr gene on plasmid pJA67. To ascertain the cloning of the structural gene for PAO1 alkaline protease, its expression in E. coli was tested. As a control, we checked that E. coli did not

VOL. 172, 1990

ALKALINE PROTEASE OF P. AERUGINOSA 1

945

2 3 4 5 C SC S CS C S

C

FIG. 2. Detection of alkaline protease activity in various P. aeruginosa PAO1 and E. coli strains. Bacterial cells were spotted onto skim milk-agar plates and incubated at 37°C for 24 h. (A) PAO1; (B) PA103; (C) PA103-11(pJA67); (D) E. coli 1046(pJAT367).

express any detectable activity with the protease substrate used nor any cross-reacting material directed against antiPAO1 alkaline protease antiserum (data not shown). When pJA67 was introduced into E. coli 1046, neither protease activity nor cross-reacting material was observed (Table 2, line b; Fig. 3, lanes 5S and SC). This result was not surprising, since many Pseudomonas promoters are very poorly recognized by E. coli RNA polymerase (4, 23). To circumvent this problem, we took advantage of a Tnl 721-derivative transposon that functions as transposable promoter (32). This Tn1735 Cm variant allows insertionmediated fusions of the strong tac promoter with genes or operons of interest, their expression being controlled by the lac repressor and inducible by IPTG owing to the transposon-borne laClq gene (32). Independent insertions of Tnl735 Cm into pJA67 were obtained as described in Materials and Methods. They were then tested for an IPTG-inducible protease phenotype on skim milk-agar plates. Out of 500, we found 16 positive clones, as indicated by a halo of clearing around the E. coli colonies. By agar well assays, we were able to show that all clones produced a detectable precipitin band when anti-PAO1 alkaline protease antiserum was used (Fig. 2). By restriction mapping, we determined the location and TABLE 2. Distribution of protease activity as a function of

growth stagea Protease activity

Strain

a. E. b. E. c. E. d. E. e. E.

coli(pLAFR3) coli(pJA67) coli(pJAT367) coli(pJAT367) coli(pJAT367)

IPTG addition

-

+ +

(/1

D OD6

4.5 4.3 3.9 0.8 4.2

(U/mi) S

C

Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the protease into the medium by Escherichia coli.

Pseudomonas virulence is thought to depend on multiple characteristics, including the production of an extracellular alkaline protease. We report the ...
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