From the
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.
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 ( M
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.
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
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).
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.
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
The
activity of these genes depends on a nucleotide motif, resembling
E. coli
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
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.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank/EMBL Data Bank with accession number(s) X77996.
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
of 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 ( M
around 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).
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 10
cells 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.
(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.
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).
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 lacI
strain 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.
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
10
units/mg.
The purified pyocin S3 appeared as a single protein upon Sephacryl
S-200 gel filtration ( M
of 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.
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.
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.
= 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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.