©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Molecular Characterization of Pyocin S3, a Novel S-type Pyocin from Pseudomonas aeruginosa(*)

Catherine Duport , Christine Baysse , Yvon Michel-Briand

From the (1) Laboratoire de Bactériologie, Faculté de Médecine, 25 030 Besanon, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The genetic determinant for the soluble pyocin S3 was isolated from a genomic library constructed in the plasmid pGV1122, of Pseudomonas aeruginosa strain P12 isolated from a cystic fibrosis patient. The nucleotide sequence of a 3270-base pair DNA fragment was determined, and the two structural genes, pyoS3A and pyoS3I, and the 3`- and 5`-flanking regions were localized. Transcription (Northern blot) analysis showed that the two genes were co-transcribed. The genes pyoS3A and pyoS3I code for polypeptides of 767 and 154 amino acids, respectively, with calculated molecular weights of 81,385 and 17,047. Pyocin S3 was produced in Escherichia coli from a plasmid and purified as a complex of two components (S3A and S3I) corresponding to the pyoS3A and pyoS3I gene products, respectively. The S3A component, like pyocin S3, had a killing effect involving DNase activity and was inhibited by the S3I protein. Comparisons of the predicted amino acid sequence of the two components of pyocin S3 to those of pyocins S1, S2, and AP41 indicate that pyocin S3 is a new type of S-type pyocin.


INTRODUCTION

Pyocins are proteins produced by Pseudomonas aeruginosa that have killing activity against strains of the same species (Jacob, 1954; Hamon, 1956; Hamon, 1961; Patterson, 1965). The bactericidal properties of pyocins have been extensively studied and used for typing P. aeruginosa strains (Govan, 1978; Fyfe et al., 1984).

Pyocin synthesis is regulated such that only a few cells in a population actively produce them. Production can be increased by treatments that cause DNA damage such as ultraviolet irradiation (Higerd et al., 1967) and mitomycin C treatment (Kageyama, 1964). The killing activity is initiated by absorption of pyocin onto specific outer membrane receptors on susceptible cells. Broadly classified as bacteriocins (Govan, 1986), a particular feature of the pyocins is that their genetic determinants are all chromosomal (Sano et al., 1990).

Most P. aeruginosa strains produce various types of pyocins, namely R, F, and S. Particular (R and F) type pyocins have structures similar to bacteriophage tails. The former has a contractile sheath, and the latter a flexible rod-like structure (Ito et al., 1970; Kageyama, 1964; Kuroda and Kageyama, 1979). S-type pyocins are smaller proteins which are soluble and sensitive to proteases (Ito et al., 1970). To date, three S-type pyocins have been extensively studied: S1, S2, and AP41 (Sano and Kageyama, 1981, 1984, 1993; Ohkawa et al., 1980; Sano et al., 1993b). Each pyocin is a complex of two proteins. The large protein ( Mof 65,000, 74,000, and 84,000 for S1, S2, and AP41, respectively) is responsible for the killing activity, degrading chromosomal DNA due to its intrinsic DNase activity. The small protein ( Maround 10,000) has an immunity function, protecting in an equivalent molar ratio the pyocin-producing strain against the DNase activity of the large protein by an as yet uncharacterized mechanism (Sano, 1993). The structural genes of these three pyocins have been cloned and sequenced and have been found to be organized in a similar manner (Sano et al., 1990).

P. aeruginosa regularly colonizes the lungs of cystic fibrosis patients, and the persistence of these bacteria is a major cause of morbidity and mortality. Interestingly, a single or a predominant pyocin-producing strain is generally involved in this chronic infection (Fegan et al., 1991; Godard et al., 1993). The synthesis of an S-type pyocin with particular bactericidal properties or with a wide killing activity spectrum may contribute to the establishment of such a strain. We report here the selection of the pyocin-producing strain P. aeruginosa P12 which was isolated from a cystic fibrosis patient. The structural genes from P12 encoding a new S-type pyocin were cloned in Escherichia coli and sequenced. Expression of these genes resulted in the synthesis of a protein complex of two components (which we named pyocin S3) with a molecular structure different from those of the three known S-type pyocins.


EXPERIMENTAL PROCEDURES

Materials

Reagents and enzymes used for sequencing and restriction were purchased from Boehringer Mannheim (Meylan, France) or Pharmacia SA (Saint Quentin en Yvelines, France) and used as recommended. [-S]dATP and [-P]ATP were obtained from Du Pont Nemours (Les Ulis, France). Antibiotics and supplements for bacterial growth were purchased from Difco (OSI, Paris, France) and Sigma (l'Isle d'Abeau Chesnes, France). All other chemicals were obtained from standard suppliers and were of reagent grade.

Bacterial Strains and Plasmids

P. aeruginosa strains used were P12 (this study), and the 13 indicator strains were labeled 1-8 and A-E (Govan, 1978). The hosts and vectors used for gene cloning were E. coli HB101 (Boyer and Roulland-Dussoix, 1969), DH5 (Life Technologies, Inc., Eragny, France), XL1-blue (Stratagene), and vector plasmids pGV1122 (Leemans et al., 1982) and pUC18 and pUC19 (Yanisch-Perron et al., 1985), and pBluescript KS(Stratagene). Plasmid pYK211 carries the AP41 pyocin genes (Sano and Kageyama, 1993), pYMSS1 the S1 pyocin genes (Sano et al., 1993b), and pYMPS1 the S2 pyocin genes. E. coli strains were routinely grown aerobically at 37 °C in rich (YT or LB) media (Sambrook et al., 1989) with the following antibiotics: ampicillin, 50 µg/ml; tetracycline, 20 µg/ml; spectinomycin, 100 µg/ml. Isopropyl-1-thio-- D-galactopyranoside and 5-bromo-4-chloro-3-indolyl-- D-galactopyranoside were used at 100 and 50 µg/ml, respectively. P. aeruginosa strains were grown aerobically at 37 °C in Mueller-Hinton medium (MH, Diagnostics Pasteur, Marnes-la-Coquette, France) or in minimal G medium (Ito et al., 1970).

DNA and RNA Manipulations

Standard procedures were used for plasmid preparations, DNA digestions, agarose gel electrophoresis, and cloning (Sambrook et al., 1989). Electroporation of E. coli was done in a 0.2-cm cuvette with a Bio-Rad apparatus. DNA fragments were extracted from agarose gels using the PrepA-gene kit (Bio-Rad, Ivry-sur-Seine, France). Partially deleted subclones were constructed using the double strand Nested Deletion kit (Pharmacia SA), purified as DNA templates by the Qiagen procedure (Coger, Paris, France), and sequenced by European Sequencing Gene Service (Montigny le Bretonneux, France). Polymerase chain reaction (PCR)() was carried out in automated thermal cycler (Crocodile II, Appligene, Illkirch, France) for 35 cycles, each consisting of 1 min at 95 °C, 2 min at 45 °C, and 3 min at 72 °C, followed by a 7-min incubation at 72 °C. The primers used for PCR had the following sequences: 5`-gggggatcCTCATGGTCGTGTGGAGGG-3` and 5`-GCGACTGACGttcAGGC-3`. Bases complementary to the pPYS3-3 template are in capital letters. The PCR product was cleaved with BamHI and EcoRV and introduced into the corresponding sites of pUC18. Total in vivo-synthesized RNA was isolated from a 100-ml culture of P. aeruginosa P12 cells grown in G medium with or without mitomycin C (1 µg/ml). RNA was extracted 90 min after induction by a hot phenol method (Schatt et al., 1989). Restriction fragments used as probes for Northern analysis were P-labeled by random-primed DNA labeling (Multiprime DNA labeling system, Amersham, les Ulis, France). The pyoS3A-specific probe was a 1788-base pair (bp) SmaI- BglII fragment, and the pyoS3I-specific probe was a 300-bp Sau3A- EcoRI fragment from pPYS3-3.

Construction and Screening of a P. aeruginosa P12 Library

Total genomic DNA from P12 was prepared by the procedure of Marmur (1961). The gene bank was constructed with Sau3A DNA fragments approximately 10-20 kilobase pairs (kb) long. These fragments were obtained by partial digestion of total DNA and purified on a 5-25% linear sucrose gradient. They were cloned into the 10.8-kb broad host range plasmid vector pGV1122. Ligation and electrotransformation into E. coli HB101 were performed as described by Kieffer (1991). Transformants were selected on LB plates containing spectinomycin and tested for tetracycline sensitivity. A total of 4,000 tetracycline-sensitive transformants were stored individually at -70 °C in 96-well microtiter plates. To screen for pyocin-producing clones, each storage plate was replicated on MH agar using a 96-prong replicator. The plates were incubated at 37 °C overnight, exposed to chloroform vapor (20 min) to prevent residual growth, and then overlaid with G medium soft agar containing an indicator strain (3 10cells in 5 ml). Plates were examined after overnight incubation at 37 °C for clear zones around pyocin-producing colonies. P aeruginosa P12 and E. coli HB101 harboring pGV1122 were used as positive and negative controls, respectively.

Purification of Pyocin S3 from E. coli

A culture (1 liter) of strain DH5 carrying pPYS3-3 was grown overnight at 37 °C in YT medium containing 50 µg/ml ampicillin. The cells were harvested by centrifugation, washed once with 25 m M Tris-HCl, pH 7.5, centrifuged again, and the pellet was collected and resuspended in 25 m M Tris, pH 7.5, containing 1 m M EDTA and 120 µg/ml lysozyme. The cells were incubated at 37 °C for 30 min and centrifuged (10,000 g) for 15 min at 4 °C. The resulting cell-free extract, designated as pyocin lysate, was diluted 2.5-fold in water and applied to a 50-ml DEAE-cellulose column (DE52, Whatman) equilibrated with 10 m M Tris, pH 7.5 (Tris buffer). The proteins were eluted with a linear gradient from 0 to 0.25 M NaCl in Tris buffer (2 250 ml). Peak fractions with pyocin activity were collected and concentrated to less than 2 ml by ultrafiltration. The pyocin was further purified by gel filtration chromatography. The concentrated protein fraction was applied to a column (1 100 cm) of Sephacryl S-200 equilibrated with 25 m M Tris, pH 7.5, containing 0.25 M NaCl. The column was run at a flow rate of 1.5 ml/h.

The 2 components of pyocin complex were separated by SDS-polyacrylamide gel electrophoresis. The purified pyocin complex was applied to a 15% acrylamide denaturing gel. After electrophoresis, the gel was stained with CuCl(Lee et al., 1987), and the two colorless protein bands corresponding to pyocin components were excised. The slices were transferred to microcentrifuge tubes and washed with 0.25 M Tris, pH 9, 0.25 M EDTA for 1 h. The proteins were then eluted overnight at 4 °C by diffusion in 10 m M phosphate buffer, pH 6.8, 5 m M nitrilotriacetic acid, 0.5 m M EDTA.

Analytical Methods

Protein samples were analyzed by 15% SDS-polyacrylamide gel electrophoresis with a 4.5% acrylamide stacking gel using a small slab gel unit (Bio-Rad). The gels were stained with Coomassie Blue or silver (Wray et al., 1981). UV-visible spectra were recorded on a Beckman DU650 spectrophotometer. For N-terminal sequencing, purified pyocin samples were subjected to preparative electrophoresis and then electrotransferred to a prewetted polyvinylidene difluoride membrane (Immobilon PVDP, Millipore, Saint Quentin en Yvelines, France) in 50 m M Tris base, 50 m M boric acid for 18 h at 30 V. Following electrophoretic transfer, the positions of the two pyocin components were identified by staining the membrane briefly with Coomassie Blue. The strips containing the proteins were excised, extensively washed with water, and dried at room temperature. The N-terminal amino acid sequences from these samples were determined using an Applied Biosystems 470 A gas phase protein sequenator at the Institut Pasteur (Paris, France). Protein concentrations were determined using the BCA protein assay reagent (Pierce) with bovine serum albumin as standard. The isoelectric points were measured using the Pharmacia Phast Gel System, and the proteins were visualized by Coomassie Blue staining.

Pyocin Activity Measurement

Pyocin typing of P. aeruginosa strains was performed, with or without trypsin (100 µg/ml) added to soft agar, as described by Fyfe et al. (1984). Pyocin activity in lysates or purified fractions was detected using the spot testing method (Govan, 1978). To estimate the pyocin activity, serially diluted samples were spotted on a indicator strain 1 lawn on MH plates. One unit of activity was defined as the reciprocal of the highest dilution which showed inhibition of growth (Govan, 1974). To measure the killing activity more precisely, the effect of pyocin on colony formation was determined. The surviving bacteria were counted as described by Ohkawa et al. (1980) with the following modifications: aliquots of 10 µl were withdrawn after the indicated periods at 37 °C, diluted with G medium, and plated on MH agar medium. Colonies were counted after a 24-h incubation at 37 °C.

DNase Activity Detection

The DNase activity associated with the pyocin was determined using the nuclease gel assay consisting of SDS-polyacrylamide gels containing heat-denatured salmon sperm DNA (20 µg/ml) in the separating gel following a previously described procedure (Seo and Galloway, 1990). In vitro, DNase activity was determined with phage DNA or chromosomal DNA isolated from indicator strain 1 as substrate (Sano et al., 1993b). In vivo, DNA breakdown was detected in pyocin-treated indicator strain 1 cells as described by Sano et al. (1993a). In both cases, DNA was examined after electrophoresis in 1% agarose gels.


RESULTS

Isolation of the Pyocin-producing Strain P. aeruginosa P12

A collection of 77 wild-type P. aeruginosa strains selected from clinical isolates of cystic fibrosis patients were pyocin-typed. Twelve of the strains produced pyocin pattern type 10/a that corresponds to the growth inhibition of the 13 standard indicator strains, 1-8 and A-E (Govan, 1978). These 12 wild-type P. aeruginosa strains were then assayed for their pyocin activities against 170 different clinical isolates of P. aeruginosa. One of them, strain P12, exhibited pyocin activity against 87% of the tested strains. We therefore used strain P12 for further analysis and determined the structural class(es) of the produced pyocin(s).

Strain P12 produced growth inhibition zones of 7-9 mm in diameter with indicator strains 1, 2, 3, 4, 5, A, B, C and 4 mm with the 5 other indicators on MH agar plates. However, when trypsin was included in the medium, the inhibition zone size was 4 mm for all 13 indicator strains. These results suggest that P12 produces two types of pyocin activity (Fyfe et al., 1984). One of these activities was readily diffusible and sensitive to trypsin and thus corresponds to an S-type pyocin(s). The other activity was less diffusible and resistant to trypsin and is therefore due to particular pyocin(s). The S-type pattern of P12, designated as S1, -2, -3, -4, -5, -A, -B, and -C, is uncommon (Holloway, 1960; Fyfe et al., 1984) and is different from those obtained with the three previously cloned S-type pyocins S1, S2, and AP41 (78/O, S6, E, 22/J, respectively).

Cloning of the Genes Encoding Pyocin S3

A genomic library of P. aeruginosa P12 DNA consisting of Sau3A fragments of approximately 10-20 kb cloned into pGV1122 was screened for pyocin-producing clones using each of the 8 indicator strains sensitive to P12 S-type pyocin(s). Two pyocin producer clones (13E4 and 24B7) showing the same S-type pattern as the P12 strain were isolated. Restriction analyses of p13E4 and p24B7 revealed the presence of 10.4- and 20-kb inserts with a common 9-kb DNA, presumably carrying the pyocin genetic determinant (Fig. 1 A). These results suggest that P12 produces only one S-type pyocin, which we call S3. To localize the pyocin S3 genetic determinant in the 9-kb genomic DNA fragment, a series of DNA fragments were subcloned into pUC plasmids using EcoRI, PstI, and HindIII restriction enzymes. A 6.5-kb HindIII fragment in pPYS3-1 expressed the pyocin activity, and a 4.2-kb Sau3A fragment of this plasmid was subcloned into pBluescript KSto give pPYS3-2. A nested set of 200-250-bp deletions were constructed from pPYS3-2 with exonuclease III. Plasmid pPYS3-3 carrying a 3270-bp fragment was the smallest construct that contained all the components required for pyocin S3 expression. A restriction map of the 3270-bp fragment of pPYS3-3, with the location of the pyocin S3 genes indicated, is shown in Fig. 1 B.


Figure 1: Localization of the genes encoding pyocin S3 and sequencing strategy. A, partial restriction maps of p24B7, p13E4, and their pyocin-producing subclones. Restriction enzymes used were: B, BamHI; E, EcoRI; Ev, EcoRV; H, HindIII; P, PstI. B, the 3.27-kb insert of pPYS3-3 is shown with the location of the pyoS3A and pyoS3I genes indicated. Serial deletion plasmids generated from pPYS3-3 with exonuclease III were used as DNA templates. The plasmids were sequenced using either the T3 primer or synthetic oligonucleotides. Arrows indicate the extent of each sequence determination.



Nucleotide Sequence Analysis

The entire 3270-bp DNA fragment from pPYS3-3 was sequenced (Fig. 1 B). It contains two open reading frames (ORFs) designated pyoS3A and pyoS3I, respectively. The nucleotide sequences and the deduced translation products of the predicted coding sequences are shown in Fig. 2 . pyoS3A is 2.3-kb in length and is predicted to encode a 767-amino acid protein with a molecular weight of 81,385 and a pI of 8.54. pyoS3I (462 bp) appears to be translationally coupled to pyoS3A, as the predicted start codon of pyoS3I (ATG) and the stop codon of pyoS3A (TGA) overlap. The nucleotide sequence of pyoS3I predicts a protein of 154 amino acids with a molecular weight of 17,047 and a pI of 5.83. Both of these ORFs are preceded by potential ribosomal binding sites located 8 bases upstream of the methionine start codons. Although these proposed ribosome binding sites are somewhat different from the E. coli consensus Shine-Dalgarno sequence (Rosenberg and Court, 1979), many P. aeruginosa genes have been reported to have similar ribosome binding sites. The G + C contents of the 2 ORFs, 56.5 and 42.7%, respectively, are somewhat lower than the average G + C content of P. aeruginosa genes (60.1 to 69.5%; West and Iglewski (1988)). Two regions with limited similarity to the canonical -35 (TTccag at nucleotide 93) and -10 (TATttT at nucleotide 114) regions of E. coli 70-type promoter were identified in the region upstream of pyoS3A. The region upstream of these two putative promoter signals contains a 50-bp sequence in which the ATTG nn( n)GT nn( n) motif is repeated four times (Fig. 2). This sequence is remarkably similar to the P box consensus sequence involved in the regulation of pyocin promoters (Matsui et al., 1993). Downstream of the stop codon TGA of pyoS3I, there is an inverted repeat followed by 4 thymidine residues (nucleotides 2921 to 2941) which may act as a -independent transcriptional terminator (Carlomagno et al., 1985).


Figure 2: Nucleotide sequences of pyoS3A and pyoS3I and deduced amino acid sequences. The putative P box and 70-like promoter sequences are singly and doubly underlined, respectively. Potential ribosomal binding sites are in bold italics upstream of the ATG start codons. The two regions of dyad symmetry possibly involved in transcription termination are indicated by converging arrows.



Transcription Analysis of the Pyocin S3 Genes

In plasmid pPYS3-3, the pyocin S3 genes were in the same orientation as the lacZ` of the vector pBluescript KS. This provides an inducible promoter (P lac) for the genes and may allow expression of the P. aeruginosa DNA in E. coli. Plasmid pPYS3-3 was used to transform the lacIstrain XL1-blue. The same pyocin activity was detected both in the presence and absence of isopropyl-1-thio-- D-galactopyranoside suggesting that the expression of the pyocin S3 genes were not under lac promoter control (data not shown). A DNA fragment encompassing the region upstream pyoS3A, extending to the EcoRV site (Fig. 1 B), was generated by PCR (see ``Experimental Procedures''). A BamHI restriction site was introduced at the 3` end of the fragment. The PCR product was subcloned into the BamHI and SmaI sites of pUC18 such that the pyocin S3 genes were oriented in the opposite direction with respect to P lac and thus were not under lac promoter control. This plasmid was called pPYS3-4. The E. coli strain DH5 carrying plasmid pPYS3-4 exhibited the same pyocin activity as DH5 (pPYS3-3). Thus, pyocin S3 genes on the cloned fragment are expressed in E. coli from their native promoter.

Northern blots of total RNA from P12 cells treated with mitomycin C ( lanes 1 and 3) or not treated ( lanes 2 and 4) are shown in Fig. 3. Restriction fragments specific for pyoS3A ( lanes 1 and 2) and pyoS3I ( lanes 3 and 4) were used as probes in hybridizations. In both cases, a single transcript of approximately 2900 bases was revealed in mitomycin C-induced cells. The size of the transcript was only slightly greater than the 2 coding regions suggesting that the two genes are co-transcribed. RNA from untreated cells gave no detectable signal with either DNA probe. Thus, transcription of pyocin S3 genes can be induced by addition of mitomycin C.


Figure 3: Northern hybridization analyses of pyoS3A and pyoS3I. Total RNA (20 µg) from P12 treated with mitomycin C ( lanes 1 and 3) or not treated ( lanes 2 and 4) was subjected to Northern blot analysis. Lanes 1 and 2 show the hybridization pattern with the 1788-bp pyoS3A-specific SmaI- BglII probe. Lanes 3 and 4 show hybridization of a 300-bp pyoS3I-specific Sau3A- EcoRI probe. RNA molecular size markers are indicated on left in kilobases.



Purification and Properties of the Pyocin S3 Synthesized in E. coli

Pyocin S3 was purified from DH5(pPYS3-3) as described under ``Experimental Procedures.'' The specific activity of the purified sample was 1 10units/mg. The purified pyocin S3 appeared as a single protein upon Sephacryl S-200 gel filtration ( Mof 90,000) and as a single polypeptide in nondenaturing gel electrophoresis (data not shown). However, two protein bands were resolved by SDS-polyacrylamide gel electrophoresis (Fig. 4 A). As the two proteins were dissociated only under denaturing conditions, pyocin S3 is presumably a complex of two components with M= 80,000 (large component) and 17,000 (small component). These sizes agree with the predicted sizes of the products encoded by pyoS3A and pyoS3I. Isoelectric focusing of the purified complex gave a pI of 6.5, and those of the large and small components were 8.25 and 6, respectively. The UV absorption curve of pyocin showed a typical protein spectrum. The ratio of absorption at 280 nm to that at 260 nm was 0.8. The N-terminal amino acid sequences of the two proteins were determined to verify that they were the same as those predicted from the nucleotide sequences. Ten amino acids of the large component were determined and corresponded exactly to the deduced amino acid sequence (Fig. 2). The N-terminal methionine was absent, indicating that the protein was processed in E. coli. The 10 N-terminal residues of the small component agreed with those predicted from pyoS3I. Unlike the large component, the N-terminal methionine was present in the small component. We conclude that the two pyocin S3 components synthesized in E. coli are the products of the genes pyoS3A and pyoS3I. The large and small components of pyocin S3 are designated S3A and S3I, respectively.


Figure 4: Identification of the pyocin produced in E. coli and detection of its DNase activity. Lysates and samples were prepared, and electrophoresis was performed as described under ``Experimental Procedures.'' A, SDS-polyacrylamide gel electrophoresis (15%) analysis and Coomassie Blue staining; lane 1, molecular weight markers ( 10); lane 2, pyocin lysate; lane 3, pyocin-containing fraction after DEAE-chromatography; lane 4, both large (80,000) and small (17,000) components of purified pyocin after Sephacryl S-200 gel filtration. B, DNase activity revealed by ethidium bromide staining (1 µg/ml, 30 min); SDS-polyacrylamide gel electrophoresis (15%) containing heat-denatured salmon sperm DNA (20 µg/ml) was examined after a 24-h incubation in 40 m M Tris, pH 7.5, 2 m M CaCl, 2 m M MgCl; lane 1, the molecular weight markers as a negative control; lanes 2 and 3, purified pyocin S3 (2 and 1 µg, respectively).



The biological activity of the purified pyocin S3 was measured by testing its killing efficiency on indicator 1 cells grown in G medium. The number of viable cells in culture decreased exponentially with time and was less than 2% after 30 min of treatment with 3 µg of pyocin (Fig. 5). However, a small proportion of the cells survived even in the presence of a large excess of pyocin (data not shown). Treatment by trypsin completely inactivated the killing activity of pyocin S3 (Fig. 5). The killing activity of S3A was very similar to that of the complex S3, whereas S3I had no activity (Fig. 5). Thus, the S3A component carries the pyocin S3 killing activity.


Figure 5: Analysis of the killing activity of purified pyocin S3. Aliquots of indicator 1 cell suspension treated with pyocin S3 () or proteins S3A () or S3I () were removed at the indicated times, and the survival rate was calculated by comparison to the control without pyocin (). Effect of trypsin on the purified S3 pyocin was assayed (X).



Chromosomal DNA extracted from indicator 1 cells which had been exposed to the pyocin S3 showed extensive degradation (Fig. 6). This result suggests that killing activity of pyocin S3 is associated with a DNase activity. Using the nuclease gel assay (Seo and Galloway, 1990), this DNase activity was co-localized with the 80,000 protein band, whereas no DNase activity was associated with the 17,000 protein band (Fig. 4 B). We therefore conclude that the large component, S3A, kills the cell by damaging host cell DNA. The purified S3 complex (which includes the S3I protein) had no DNase activity in vitro, whereas the isolated S3A component degraded chromosomal DNA of both phage or indicator strain 1 (Fig. 7). These results suggest that the activity of S3A is inhibited by the S3I component.

Comparison of Pyocin S3 with the S-type Pyocins S1, S2, and AP41

The predicted amino acid sequences of the pyocin S3 components were compared with those of the 3 other cloned and sequenced S-type pyocins (Sano et al., 1993b; Sano and Kageyama, 1993). The sequence of S3I could not be aligned by computer with those of the small components of pyocins S1, S2, or AP41. However, the amino acid sequence of S3A has significant similarities with those of the other large components (Fig. 8). The similarity is highest between S3 and S2 (27.8% sequence identity and 57% sequence similarity if conservative amino acid substitutions are included) and lower between S3 and S1 (24.5% identity and 46.2% similarity) and S3 and AP41 (23.7% identity 51.2% similarity). The N-terminal sequence of the S3 large component was very different from the others, whereas the C-terminal halves were much more similar (Fig. 8).


Figure 8: Alignment of the deduced amino acid sequence of S3A with those of the large components of pyocins S1, S2, and AP41. Numbers refer to positions of the amino acids residues in the sequence of each protein. Regions with amino acid identity with the S3A sequence are boxed. Putative structural domains ( I to IV) of S3A are indicated. The Lasergeneprogram in the DNASTAR software package was used for alignment.




DISCUSSION

This study brings to four the number of S-type pyocins from P. aeruginosa whose genetic determinants have been sequenced. They are the pyocins S1 from P. aeruginosa NIH (Sano et al., 1993b), S2 from P. aeruginosa PAO (Sano et al., 1993b), AP41 from P. aeruginosa PAF (Sano and Kageyama, 1993), and S3 from P. aeruginosa P12. The expression of P. aeruginosa pyocin genes in E. coli facilitated the cloning and sequencing of the pyocin S3 genetic determinant. Indeed, unlike many genes of P. aeruginosa (Vliegenthart et al., 1991; Savioz et al., 1993), the S-type pyocin genes are efficiently expressed in E. coli. In addition, plasmid-encoded pyocin was secreted into the extracellular medium and can therefore be detected easily by testing bactericidal activity of the E. coli host strain against P. aeruginosa indicator strains (Govan, 1978).

The pyocin S3 genetic determinant comprises two structural genes, pyoS3A and pyoS3I, which encode the killing protein (S3A) and the immunity protein (S3I), respectively. Four lines of evidence suggest that pyoS3A and pyoS3I are transcribed as a two-gene operon. (i) Northern blot analysis indicated that pyoS3A and pyoS3I were co-transcribed as a polycistronic mRNA of approximately 2900 bases (Fig. 3). (ii) DNA sequence analysis suggests that the two genes are translationally coupled. Indeed, a sequence similar to the Shine-Dalgarno sequence is located at the 3` end of the pyoS3A gene. It is separated by only 6 nucleotides from the stop codon TGA and by 8 nucleotides from the initiation codon of the pyoS3I gene. With this structure, ribosomes should remain in simultaneous contact with the termination codon of the first gene and the initiation codon of the second, since they physically span about 35 bases of mRNA. The presence of the Shine-Dalgarno sequence should prevent dissociation from the mRNA of the 30 S subunit of the terminating ribosome and thereby enable interrupted translation of pyoS3I. (iii) A putative terminator sequence is located immediately downstream pyoS3I (Fig. 2). This sequence is centered 30 nucleotides from the TGA stop codon and may give a stem and loop structure with a G = -81.9 kJ/mol. (iv) The two genes are transcribed in E. coli and P. aeruginosa from their native promoter located within the region between the ATG start codon of pyoS3A and position -146.

The activity of these genes depends on a nucleotide motif, resembling E. coli 70 promoter, in the region upstream of the pyoS3A gene. E. coli 70-like promoter sequences have previously been observed in P. aeruginosa, and they have been classified in the functional group CEC70 (Deretic et al., 1989). This group of P. aeruginosa promoters is either constitutively expressed or recognized by RNA polymerase in E. coli. However, some of them may be regulated and expressed in E. coli as is probably the case for the promoter of the pyocin S3 genes. Pyocin S3 is constitutively expressed in E. coli (data not shown), whereas it is inducible by treatments that cause damage to DNA (UV, mitomycin C) in P. aeruginosa. In addition, we identified a P box-like sequence upstream from the putative CEC70 promoter that may act as binding site for a positive regulator (Matsui et al., 1993). Thus, we can assume that expression of pyocin S3 is regulated in a manner similar to that reported for the other S-type pyocins. Constitutive expression in E. coli may be due to the absence of appropriate transcriptional regulatory proteins.

Detection of an S-type pyocin activity in P. aeruginosa P12, identification of the nucleotide sequence, and pyoS3A and pyoS3I specific transcripts proved that pyocin S3 is indeed synthesized by this strain. Pyocin S3 is a complex of M= 81,000 (S3A) and 17,000 (S31) proteins which are functionally similar to the two components of pyocins S1, S2, and AP41 (Sano et al., 1993b; Sano and Kageyama, 1993). Large components of S-type pyocins were proposed to comprise three (S1) or four (AP41, S2) functional domains arranged from the N terminus to the C terminus as follows (Matsui et al., 1993): receptor-binding domain (I), translocation domain (III), and DNase domain (IV). The function of domain II which lies between domains I and III in AP41 and S2 is unknown and is not required for the killing activity. On the basis of amino acid sequence similarities, S3A seems to be composed of four domains like AP41 and S2 (Fig. 8). The putative S3 translocation domain is particularly similar to those of the three other pyocins, most markedly at the C terminus of the polypeptide (residues 615 to 633) which is required for the killing activity (Matsui et al., 1993). The DNase domain is different from those conserved of pyocins S1, S2, and AP41. Interestingly, the small component S3I ( M= 17,000), which is assumed to interact with the DNase domain of large component S3A is dissimilar to the highly conserved small components of S1, S2, and AP41 ( M= 10,000). This might suggest that the region responsible for the DNase activity of S3A and the S3I protein have evolved together. In addition, we can assume that the S3I protein, unlike the small components S1, S2, and AP41, originated from an ancestor different from that of the E2 group colicins (Sano and Kageyama, 1993), according to the low GC% content of pyoS3I. All these arguments indicate that pyocin S3 is a new type of soluble pyocin. Tredgett et al. (1990) showed the existence of a P. aeruginosa subpopulation involved in chronic pulmonary colonization that could be associated with pyocin production. Since the S3-producing strain was isolated from cystic fibrosis patients, we could speculate that the basis for this P. aeruginosa subpopulation may include uncommon S-type pyocins.

The receptor binding domain of S3A was assigned to the N-terminal region from residues 1 to 275 (Fig. 8). As expected, it shows relatively little similarity with the other pyocins. The difference between the S3 pyocin type pattern and those of pyocins S1, S2, and AP41 may possibly therefore be due to receptor specificities. Furthermore, cross-immunity between S3 and other S-type pyocins seems impossible, like what has been demonstrated for S1, S2, and AP41 (Sano et al., 1990). The iron concentration in the growth medium affects the susceptibility of an indicator strain to pyocin S3 (data not shown). This suggests that the pyocin S3 receptor is probably an iron-regulated outer membrane protein as reported for pyocins S2 and Sa (Ohkawa et al., 1980; Govan, 1986; Smith et al., 1992). The receptor is currently being investigated.

The broad range activity spectrum of strain P12 is due not only to the production of pyocin S3 but also to particular pyocin(s). This is not surprising because most P. aeruginosa strains produce more than one pyocin type (Fyfe et al., 1984). The activity spectrum of particular pyocin(s) produced by P12, and their types (R or F) were not determined.


FOOTNOTES

*
This work was supported by grants from the Association Franaise de Lutte contre la Mucoviscidose. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) X77996.

The abbreviations used are: PCR, polymerase chain reaction; bp, base pair(s); kb, kilobase pair(s).


ACKNOWLEDGEMENTS

We thank Prof. Kageyama for kindly providing plasmids pYK211, pYMSS1, and pYMPSS1. The skillful assistance of Christiane Bailly and Thierry Nicolas for bacteriological screening is acknowledged.


REFERENCES
  1. Boyer, H. W., and Roulland-Dussoix, D. (1969) J. Mol. Biol. 41, 459-472 [Medline] [Order article via Infotrieve]
  2. Carlomagno, M. S., Riccio, A., and Bruni, C. B. (1985) J. Bacteriol. 163, 362-368 [Medline] [Order article via Infotrieve]
  3. Deretic, V., Konyecsnic, W. M., Mohr, C. D., Martin, D. W., and Hibler, N. S. (1989) Biotechnology 7, 1249-1254
  4. Fegan, M., Francis, M. P., Hayward, A. C., and Fuerst, J. A. (1991) J. Clin. Microbiol. 29, 2151-2157 [Medline] [Order article via Infotrieve]
  5. Fyfe, J. A. M., Harris, G., and Gowan, J. R. W. (1984) J. Clin. Microbiol. 20, 47-50 [Medline] [Order article via Infotrieve]
  6. Godard, C., Plesiat, P., and Michel-Briand, Y. (1993) Eur. J. Med. 2, 117-120 [Medline] [Order article via Infotrieve]
  7. Govan, J. R. W. (1974) J. Gen. Microbiol. 80, 1-15 [Medline] [Order article via Infotrieve]
  8. Govan, J. R. W. (1978) Pyocin Typing of Pseudomonas aeruginosa (Bergan, T., and Norris, J. R., eds) Vol. 10, pp. 61-91, Academic Press Inc., London
  9. Govan, J. R. W. (1986) Scand. J. Infect. Dis. Suppl. 49, 31-37 [Medline] [Order article via Infotrieve]
  10. Hamon, Y. (1956) Ann. Inst. Pasteur 91, 82-90 [Medline] [Order article via Infotrieve]
  11. Hamon, Y. (1961) Ann. Inst. Pasteur 88, 193-204
  12. Higerd, T. B., Baechler, C. A., and Berk, S. (1967) J. Bacteriol. 93, 1976-1986 [Medline] [Order article via Infotrieve]
  13. Holloway, B. W. (1960) J. Pathol. Bacteriol. 80, 448-450 [Medline] [Order article via Infotrieve]
  14. Ito, S., Kageyama, M., and Egami, F. (1970) J. Gen. Appl. Microbiol. 16, 205-214
  15. Jacob, F. (1954) Ann. Inst. Pasteur 86, 149-160 [Medline] [Order article via Infotrieve]
  16. Kageyama, M. (1964) J. Biochem. (Tokyo) 55, 49-53 [Medline] [Order article via Infotrieve]
  17. Kieffer, B. L. (1991) Gene (Amst.) 115-119
  18. Kuroda, K., and Kageyama, M. (1979) J. Biochem. (Tokyo) 78, 7-19
  19. Lee, C., Levin, A., and Branton, D. (1987) Anal. Biochem. 166, 308-312 [Medline] [Order article via Infotrieve]
  20. Leemans, J., Langenakens, J., De Greve, H., Deblaere, R., Van Montagu, M., and Schell, J. (1982) Gene (Amst.) 19, 361-364 [CrossRef][Medline] [Order article via Infotrieve]
  21. Marmur, J. (1961) J. Mol. Biol. 3, 208-218
  22. Matsui, H., Sano, Y., Ishihara, H., and Shinomiya, T. (1993) J. Bacteriol. 175, 1257-1263 [Abstract]
  23. Ohkawa, I., Shiga, S., and Kageyama, M. (1980) J. Biochem. (Tokyo) 87, 323-331 [Abstract]
  24. Patterson, A. C. (1965) J. Gen. Microbiol. 39, 295-303 [Medline] [Order article via Infotrieve]
  25. Rosenberg, M., and Court, D. (1979) Annu. Rev. Genet. 13, 319-353 [CrossRef][Medline] [Order article via Infotrieve]
  26. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
  27. Sano, Y. (1993) J. Bacteriol. 175, 912-915 [Abstract]
  28. Sano, Y., and Kageyama, M. (1981) J. Bacteriol. 146, 733-739 [Medline] [Order article via Infotrieve]
  29. Sano, Y., and Kageyama, M. (1984) J. Bacteriol. 158, 562-570 [Medline] [Order article via Infotrieve]
  30. Sano, Y., and Kageyama, M. (1993) Mol. & Gen. Genet. 237, 161-170
  31. Sano, Y., Matsui, H., Kobayashi, M., and Kageyama, M. (1990) in Pseudomonas Biotransformations Pathogenesis and Evolving Biotechnology (Silver, S., Chakrabarty, A. M., Iglewski, B., and Kaplan, S., eds) pp. 352-358, American Society for Microbiology, Washington D. C.
  32. Sano, Y., Kobayashi, M., and Kageyama, M. (1993a) J. Bacteriol. 175, 6179-6185 [Abstract]
  33. Sano, Y., Matsui, H., Kobayashi, M., and Kageyama, M. (1993b) J. Bacteriol. 175, 2907-2916 [Abstract]
  34. Savioz, A., Zimmerman, A., and Haas, D. (1993) Mol. & Gen. Genet. 238, 74-80
  35. Schatt, E., Jouanneau, Y., and Vignais, P. M. (1989) J. Bacteriol. 171, 6218-6226 [Medline] [Order article via Infotrieve]
  36. Seo, Y., and Galloway, D. R. (1990) Biochem. Biophys. Res. Commun. 172, 455-461 [Medline] [Order article via Infotrieve]
  37. Smith, A. W., Hirst, P. H., Hugues, K., Gensberg, K., and Govan, J. R. W. (1992) J. Bacteriol. 174, 4847-4849 [Abstract]
  38. Tredgett, M. W., Doherty, C., and Govan, J. R. W. (1990) J. Med. Microbiol. 32, 169-172 [Abstract]
  39. Vliegenthart, J. S., Ketelaar-van Gaalen, P. A. G., and Van de Klundert, J. A. M. (1991) Antimicrob. Agents Chemother. 35, 892-897 [Medline] [Order article via Infotrieve]
  40. West, S. E. H., and Iglewski, H. (1988) Nucleic Acids Res. 16, 9323-9335 [Abstract]
  41. Wray, W., Boulikas, T., Wray, V. P., and Hancock, R. (1981) Anal. Biochem. 118, 197-203 [Medline] [Order article via Infotrieve]
  42. Yanisch-Perron, C., Viera, J., and Messing, J. (1985) Gene (Amst.) 33, 103-119 [CrossRef][Medline] [Order article via Infotrieve]

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